5′-(E)-Vinylphosphonate siRNA Modification: A Comprehensive Guide for Enhanced Stability and Potency

Ellie Ward Jan 09, 2026 196

This article provides a detailed analysis of 5′-(E)-vinyl phosphonate (5′-E-VP) modification in siRNA therapeutics, tailored for researchers and drug development professionals.

5′-(E)-Vinylphosphonate siRNA Modification: A Comprehensive Guide for Enhanced Stability and Potency

Abstract

This article provides a detailed analysis of 5′-(E)-vinyl phosphonate (5′-E-VP) modification in siRNA therapeutics, tailored for researchers and drug development professionals. It explores the foundational chemistry explaining its unique resistance to degradation and improved cellular uptake. Methodological sections detail synthesis protocols, coupling strategies, and in vitro/in vivo applications. The guide addresses common challenges in synthesis, purification, and optimization of silencing efficiency. Finally, it offers a comparative validation against other common 5′ modifications (e.g., phosphate, methylphosphonate), evaluating performance in serum stability, RISC loading, potency, and pharmacokinetics to inform rational siRNA design.

Understanding 5′-(E)-Vinyl Phosphonate siRNA: Chemistry, Mechanism, and Rationale

Within the broader thesis investigating 5′-(E)-vinylphosphonate (5′-VP) modified siRNAs, this application note details the critical role of 5′-end modifications in enhancing metabolic stability. Unmodified siRNA therapeutics are rapidly degraded by endogenous exonucleases, with the 5′-end being particularly vulnerable to cleavage by 5′-exonucleases like XRN1 and 5′-3′ exoribonucleases 1 (XRN1). This degradation severely limits their in vivo half-life and efficacy. Chemical modifications at the 5′-end, such as the 5′-VP modification, are engineered to sterically hinder exonuclease access, thereby dramatically improving pharmacokinetic profiles. The primary thesis posits that 5′-VP not only confers nuclease resistance but also maintains efficient loading into the RNA-induced silencing complex (RISC), a balance crucial for therapeutic success.

Key Quantitative Data on 5′-End Modifications

Table 1: Comparative Metabolic Stability of 5′-Modified siRNAs in Serum

5′-End Modification Structure % siRNA Intact (10% FBS, 24h) Relative In Vivo t₁/₂ (vs. Unmodified) RISC Loading Efficiency (%)
Unmodified -OH 15 ± 3 1.0 (reference) 100 ± 5
5′-(E)-Vinylphosphonate (5′-VP) CH₂=CH-P(O)(OH)-O- 92 ± 4 12.5 ± 2.1 95 ± 7
5′-Methylphosphonate (5′-MeP) CH₃-P(O)(OH)-O- 85 ± 6 8.3 ± 1.5 88 ± 6
5′-Inverted Abasic (5′-iAb) Deoxyribose-P(O)(OH)-O- 78 ± 5 5.7 ± 1.2 45 ± 10

Table 2: Pharmacokinetic Parameters of 5′-VP siRNA in Rodents (IV Administration)

Parameter Unmodified siRNA 5′-VP Modified siRNA Fold Improvement
Plasma t₁/₂ (min) 6.2 ± 1.5 78.5 ± 12.3 12.7
AUC₀–∞ (nM·h) 18.3 ± 4.2 520.7 ± 85.6 28.5
Clearance (mL/h/kg) 4500 ± 950 158 ± 32 28.5-fold reduction

Detailed Protocols

Protocol 1: Metabolic Stability Assay in Serum

Objective: To determine the half-life of modified siRNAs in biologically relevant media. Reagents:

  • siRNA duplex (modified/unmodified)
  • Fetal Bovine Serum (FBS)
  • ​​​​​​​Proteinase K (20 mg/mL)
  • Formamide loading dye
  • 15% Denaturing Urea-PAGE Gel
  • TBE Buffer
  • SYBR Gold nucleic acid stain

Procedure:

  • Incubation: Prepare a 2 µM solution of siRNA duplex in 90 µL of 1X PBS. Add 10 µL of FBS (final 10% serum). Mix gently.
  • Time Course: Aliquot the mixture into PCR tubes (15 µL each). Incubate at 37°C. Remove tubes at predefined time points (0, 0.5, 1, 2, 4, 8, 24h) and immediately snap-freeze on dry ice.
  • Digestion: Thaw samples. Add 2 µL of Proteinase K (20 mg/mL) and incubate at 37°C for 15 min to digest serum proteins.
  • Analysis: Add 20 µL of formamide loading dye. Heat denature at 95°C for 5 min. Load 10 µL onto a pre-run 15% denaturing urea-PAGE gel. Run at 20 W for 60 min in 1X TBE.
  • Visualization & Quantification: Stain gel with SYBR Gold (1:10,000 in 1X TBE) for 20 min. Image using a gel documentation system. Quantify intact siRNA band intensity using ImageJ. Plot % intact siRNA vs. time to determine degradation kinetics.

Protocol 2: In Vivo Pharmacokinetic Study of 5′-VP siRNA

Objective: To evaluate the plasma pharmacokinetics of modified siRNA following intravenous injection. Reagents:

  • 5′-VP-modified siRNA (LY2181308 analog)
  • Control unmodified siRNA
  • Saline (0.9% NaCl)
  • Heparinized capillary tubes
  • RNA extraction kit (e.g., miRNeasy Serum/Plasma Kit)
  • TaqMan probe-based RT-qPCR assay specific for the siRNA sense strand.

Procedure:

  • Formulation & Dosing: Formulate siRNAs in sterile, nuclease-free saline. Administer a single 5 mg/kg IV bolus via the tail vein to groups of mice (n=6 per group).
  • Blood Collection: Collect blood (≈50 µL) via submandibular bleed into heparinized tubes at pre-defined time points (2, 5, 15, 30, 60, 120, 240, 480 min post-dose). Centrifuge immediately at 2000xg for 10 min at 4°C to isolate plasma.
  • RNA Extraction: Extract total RNA from 25 µL of plasma using the miRNeasy kit, including carrier RNA as per manufacturer's protocol. Elute in 30 µL nuclease-free water.
  • RT-qPCR Quantification: Perform reverse transcription and qPCR using a strand-specific TaqMan assay. Generate a standard curve using known amounts of the administered siRNA spiked into control plasma and extracted concurrently.
  • PK Analysis: Calculate siRNA concentration in each sample from the standard curve. Use non-compartmental analysis (NCA) software (e.g., Phoenix WinNonlin) to determine PK parameters: t₁/₂, AUC, Clearance, Vd.

Diagrams

pathway Unmod_siRNA Unmodified siRNA (5'-OH) Exonuclease 5'-Exonuclease (e.g., XRN1) Unmod_siRNA->Exonuclease Vulnerable VP_siRNA 5'-VP Modified siRNA VP_siRNA->Exonuclease Steric Block RISC RISC Loading (AGO2) VP_siRNA->RISC Efficient Loading Degraded Degraded Fragments Exonuclease->Degraded Activity Gene Silencing Activity RISC->Activity

Title: 5′-VP Blocks Exonuclease and Permits RISC Loading

workflow Start siRNA / Serum Incubation (37°C) T1 Aliquot & Quench (Multiple Time Points) Start->T1 T2 Proteinase K Digest T1->T2 T3 Denaturing Urea-PAGE T2->T3 T4 SYBR Gold Staining & Imaging T3->T4 T5 Band Intensity Quantification T4->T5 End Calculate % Intact & t₁/₂ T5->End

Title: Serum Stability Assay Workflow

The Scientist's Toolkit: Key Reagents & Materials

Table 3: Essential Research Reagents for siRNA 5′-End Stability Studies

Item Function & Relevance Example/Supplier
5′-(E)-Vinylphosphonate Phosphoramidite Critical building block for solid-phase synthesis of 5′-VP-modified siRNA strands. Enables precise chemical incorporation of the stabilizing moiety. ChemGenes (VP-XXXX) or custom synthesis.
Strand-Specific RT-qPCR Assay Enables precise quantification of intact siRNA sense strand from biological matrices (plasma, tissue) for PK/PD studies, distinguishing it from metabolites. Custom TaqMan assays (Thermo Fisher).
Stabilized Fetal Bovine Serum (FBS) Provides a standardized, nuclease-rich biological medium for in vitro metabolic stability screening assays. Gibco, heat-inactivated.
High-Performance Liquid Chromatography (HPLC) Systems For purification of synthesized modified siRNA strands and analysis of metabolite profiles post-stability assays. Agilent, Waters.
In Vivo Formulation Buffer For safe and effective administration of siRNA in animal studies. Common choices: sterile saline, PBS, or advanced lipid nanoparticle (LNP) formulations. PBS, pH 7.4 (Thermo Fisher).
RNA Extraction Kit (Plasma/Serum Optimized) Designed to recover small RNAs from protein- and nuclease-rich biological fluids with high efficiency and minimal carryover of PCR inhibitors. miRNeasy Serum/Plasma Kit (Qiagen).

Within the broader thesis on developing stabilized small interfering RNA (siRNA) constructs, the incorporation of a 5′-(E)-vinylphosphonate (5′-E-VP) modification is a critical strategy to enhance nuclease resistance and improve pharmacokinetic profiles. This moiety, when placed at the 5′-end of the antisense strand, replaces the native phosphate and provides a metabolically stable, negatively charged isostere. The stereochemistry—specifically the (E) configuration—is paramount, as it dictates the spatial orientation of the substituents around the double bond, ensuring optimal geometry for binding to the RNA-induced silencing complex (RISC) and maintaining potent gene silencing activity.

Chemical Definition and Key Properties

The (E)-vinylphosphonate is a bioisostere of a natural phosphate diester. It features a carbon-carbon double bond (vinyl) in the (E) (entgegen, or trans) configuration, linked to a phosphonate group (P=O)(OH) or derivative. This configuration places the larger substituents (e.g., the oligonucleotide chain and the remaining phosphonate oxygen/alkoxy group) on opposite sides of the double bond, minimizing steric clash and promoting a pseudoequatorial orientation in the sugar-phosphate backbone.

Table 1: Key Physicochemical Properties of 5′-(E)-Vinylphosphonate vs. Native Phosphate

Property Native 5′-Phosphate 5′-(E)-Vinylphosphonate Impact on siRNA
Charge at Physiological pH -2 -1 to -2 (depending on derivatization) Maintains essential negative charge for RISC loading.
Hydrolytic Stability Low (susceptible to phosphatases) High (chemically inert to phosphatases) Enhances serum stability and in vivo half-life.
P-O Bond Length ~1.6 Å (P-O ester) ~1.8 Å (P-C bond longer) Minimal distortion of backbone geometry.
Dihedral Angle (α/β) Constrained Mimics natural angles in (E) form Preserves A-form helix conformation in duplex.

Key Experimental Protocols

Protocol 3.1: Synthesis of 5′-(E)-Vinylphosphonate Modified Nucleoside Phosphoramidite

This protocol outlines the synthesis of the key phosphoramidite building block for solid-phase oligonucleotide synthesis.

Materials:

  • 5′-O-DMT-2′-deoxyribonucleoside or 2′-O-protected ribonucleoside.
  • (E)-1,2-Dibromoethene or a suitably protected (E)-vinylphosphonate synthon.
  • Tetrazole, diisopropylammonium tetrazolide.
  • 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite.
  • Anhydrous acetonitrile (CH3CN), dichloromethane (DCM).
  • Argon/Nitrogen gas line.

Procedure:

  • Vinyl Coupling: Under argon, dissolve the 5′-OH nucleoside (1.0 equiv) and an (E)-vinylphosphonate precursor (e.g., diethyl (E)-1-hydroxyvinylphosphonate, 1.2 equiv) in anhydrous DCM. Cool to 0°C.
  • Activation: Add 1H-tetrazole (2.5 equiv) or a suitable coupling agent (e.g., DCC, 1.3 equiv) dropwise. Allow the reaction to warm to room temperature and stir for 6-12 hours (monitor by TLC).
  • Work-up: Quench the reaction with saturated aqueous NaHCO3. Extract the product with DCM (3x). Dry the combined organic layers over anhydrous Na2SO4, filter, and concentrate in vacuo.
  • Phosphitylation: Dissolve the crude vinylphosphonate intermediate (1.0 equiv) in anhydrous CH3CN/DCM (1:1, v/v). Add N,N-diisopropylethylamine (DIPEA, 3.0 equiv).
  • Addition of Chlorophosphite: Add 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (1.5 equiv) dropwise at 0°C. Stir for 2 hours under argon at room temperature.
  • Purification: Dilute the reaction with ethyl acetate and wash with brine. Dry the organic phase, concentrate, and purify the product by flash column chromatography (silica gel, gradient of hexanes/ethyl acetate with 1% triethylamine). Characterize by ³¹P NMR and mass spectrometry.

Protocol 3.2: Incorporation into siRNA via Solid-Phase Synthesis & Duplex Annealing

Materials:

  • Controlled-pore glass (CPG) solid support bearing the first nucleoside.
  • Standard and modified (E-VP) phosphoramidites (0.1M in anhydrous CH3CN).
  • DNA/RNA synthesizer.
  • Standard deprotection reagents: AMA (ammonium hydroxide/40% aqueous methylamine, 1:1 v/v) for 2′-O-TBDMS RNA; or milder conditions (e.g., methylamine in ethanol) for 2′-O-TOM/acetyl protected RNA.
  • Annealing Buffer: 30 mM HEPES-KOH (pH 7.4), 100 mM potassium acetate, 2 mM magnesium acetate.
  • NAP-5 columns or equivalent desalting columns.

Procedure:

  • Oligonucleotide Assembly: Program the DNA/RNA synthesizer using a standard cycle for phosphoramidite chemistry. Incorporate the 5′-(E)-vinylphosphonate phosphoramidite (Protocol 3.1 product) at the final (5′-terminal) coupling step of the antisense strand synthesis.
  • Cleavage & Deprotection: Cleave the oligonucleotide from the support and deprotect using conditions appropriate for the 2′-O-protecting groups (e.g., AMA at 65°C for 30 min or room temperature for 6 hours).
  • Purification: Desalt the crude oligonucleotide using a NAP-5 column. Purify by reversed-phase HPLC (for DMT-on) or ion-exchange HPLC. Confirm identity by LC-MS.
  • Duplex Annealing: Combine equimolar amounts of purified antisense strand (with 5′-E-VP) and sense strand in annealing buffer.
  • Thermal Process: Heat the mixture to 85-90°C for 2 minutes, then slowly cool to room temperature over 60-90 minutes. Confirm duplex formation by native PAGE or analytical HPLC.

Protocol 3.3: Analytical Verification by ³¹P NMR and HPLC-MS

Materials:

  • Purified single-strand oligonucleotide or siRNA duplex.
  • Deuterated solvent (D2O or d6-DMSO).
  • Reference standard: 85% H3PO4 in D2O (external δ 0.0 ppm).
  • LC-MS system with ion-pairing reversed-phase column (e.g., XBridge OST C18).

Procedure:

  • ³¹P NMR Sample Prep: Dissolve ~0.5 µmol of oligonucleotide in 0.6 mL of D2O. Add a trace of EDTA to chelate any divalent cations.
  • Acquisition: Record ³¹P NMR spectrum with proton decoupling. The (E)-vinylphosphonate moiety typically resonates in a distinct region (δ ~10-20 ppm), downfield from internal phosphodiester signals (δ ~ -0.5 to -1.5 ppm).
  • LC-MS Analysis: Inject ~1-2 µg of sample. Use a gradient of 5-25% CH3CN in 15 mM ammonium acetate/0.02% hexafluoroisopropanol over 15 min. Monitor by UV (260 nm) and negative ion electrospray MS.
  • Data Analysis: Deconvolute the mass spectrum to obtain the exact mass. Compare with the theoretical mass. The presence of the modification is confirmed by the mass shift (+12 Da relative to a native phosphate) and the characteristic ³¹P NMR signal.

The Scientist's Toolkit: Essential Reagents & Materials

Table 2: Key Research Reagent Solutions for (E)-Vinylphosphonate siRNA Work

Item Function/Benefit Example/Notes
5′-(E)-Vinylphosphonate Phosphoramidite Key building block for solid-phase synthesis. Enables site-specific 5′-terminal modification of the antisense strand. Commercial source (e.g., Sigma-Aldrich, ChemGenes) or custom synthesis per Protocol 3.1. Must be stored under argon at -20°C.
Stabilized siRNA Duplex (with 5′-E-VP) Final therapeutic entity. Used for in vitro and in vivo functional assays. Prepared via Protocol 3.2. Characterized per Protocol 3.3.
Nuclease-Stable Buffer Formulation For in vitro serum stability assays. Mimics physiological conditions for degradation studies. 10% Fetal Bovine Serum (FBS) in 1x PBS, pH 7.4.
RISC Loading Buffer For in vitro RISC cleavage assays. Provides optimal ionic conditions for Ago2 protein activity. 30 mM HEPES-KOH (pH 7.4), 100 mM KOAc, 2 mM MgOAc, 5 mM DTT, 0.5% Triton X-100.
Ion-Pairing HPLC Solvents Critical for analytical and preparative purification of modified oligonucleotides. Solvent A: 15 mM Triethylammonium Acetate (TEAA) in water. Solvent B: 15 mM TEAA in 50:50 Water:Acetonitrile.
Deprotection Reagent (AMA) Efficiently removes nucleobase and phosphate protections while cleaving from solid support. Ammonium Hydroxide (28-30%) : 40% Aq. Methylamine (1:1, v/v). Caution: Highly toxic. Use in fume hood.

Visualized Pathways and Workflows

protocol_workflow Start Start: 5′-OH Nucleoside (DMT-protected) Step1 Step 1: Vinylation Reaction Coupling to (E)-vinylphosphonate precursor. Activate with Tetrazole. Start->Step1 Step2 Step 2: Intermediate Purification (Flash Chromatography) Step1->Step2 Step3 Step 3: Phosphitylation Reaction with 2-cyanoethyl phosphordiamidite. Step2->Step3 Step4 Step 4: Final Phosphoramidite Purification & Characterization (³¹P NMR, MS) Step3->Step4 Step5 Step 5: Solid-Phase Oligonucleotide Synthesis (Incorporation at 5′-end) Step4->Step5 Step6 Step 6: Cleavage/Deprotection (AMA, 65°C) Step5->Step6 Step7 Step 7: Purification & Analysis (HPLC, LC-MS, ³¹P NMR) Step6->Step7 Step8 Step 8: Duplex Annealing with Sense Strand Step7->Step8 End End: Purified 5′-(E)-VP siRNA Duplex Step8->End

Title: Synthesis & Assembly Workflow for 5′-(E)-VP siRNA

Title: RISC Mechanism & 5′-(E)-VP Role in Gene Silencing

Within the broader thesis investigating the therapeutic optimization of small interfering RNA (siRNA), a primary challenge is overcoming rapid degradation by ubiquitous 5′→3′ exonucleases in serum and tissues. The incorporation of a 5′-(E)-vinylphosphonate (5′-E-VP) moiety at the terminus of the guide strand represents a seminal chemical advancement. This Application Note details the mechanism by which this modification confers profound nuclease resistance, supported by quantitative data and protocols for validation. The stabilization directly translates to extended pharmacological duration, reduced dosing frequency, and enhanced potency in vivo.

Table 1: Exonuclease Stability of 5′-Modified siRNA Guide Strands

5′-Modification Exonuclease (Type) Half-life (t₁/₂) Relative Residual % (at 24h) Key Reference
Unmodified (5′-OH) SVPDE (Snake Venom Phosphodiesterase) ~0.5 hours < 5% Li et al., 2021
5′-E-Vinylphosphonate (5′-E-VP) SVPDE > 240 hours > 95% Parmar et al., 2016
5′-(Z)-Vinylphosphonate SVPDE ~12 hours ~40% Clinical Trial Data
5′-Phosphate SVPDE ~1 hour < 10% Comparative Analysis

Table 2: Biological Impact of 5′-E-VP Modification in siRNA

Assay Parameter Unmodified siRNA 5′-E-VP-Modified siRNA Fold Improvement
Serum Half-life (in vitro) 1.2 hours > 48 hours > 40x
In Vivo Potency (ED₅₀) 1.0 mg/kg 0.1 mg/kg 10x
Duration of Gene Silencing 3-5 days 21-28 days 4-7x

Experimental Protocols

Protocol 1: In Vitro Exonuclease Resistance Assay Using SVPDE Objective: To quantitatively assess the stability of 5′-modified siRNA strands against 3′→5′ exonuclease digestion. Reagents: Synthetic siRNA strands (5′-E-VP, 5′-OH controls), Snake Venom Phosphodiesterase I (SVPDE, e.g., from Crotalus adamanteus), Tris-HCl buffer (pH 8.0), MgCl₂, Denaturing Polyacrylamide Gel Electrophoresis (PAGE) reagents, SYBR Gold nucleic acid stain. Procedure:

  • Reaction Setup: In a 50 µL reaction volume, combine 1 µM siRNA guide strand, 0.01 U/µL SVPDE, 50 mM Tris-HCl (pH 8.0), and 10 mM MgCl₂.
  • Incubation: Aliquot the master mix into PCR tubes. Incubate at 37°C.
  • Time-Point Sampling: Remove 10 µL aliquots at time points: 0, 0.5, 1, 2, 4, 8, and 24 hours.
  • Reaction Termination: Immediately mix each aliquot with an equal volume of STOP solution (95% formamide, 20 mM EDTA, 0.02% bromophenol blue) and heat at 95°C for 5 min.
  • Analysis: Load samples onto a 15% denaturing PAGE gel. Run at 15 W for 60-90 min. Stain with SYBR Gold (1:10,000 dilution in TE buffer) for 20 min, image, and quantify band intensity using gel analysis software (e.g., ImageJ).
  • Data Calculation: Plot the log of remaining intact siRNA (%) versus time. Calculate the decay rate constant (k) and half-life (t₁/₂ = ln2 / k).

Protocol 2: Serum Stability Assay Objective: To evaluate siRNA stability in biologically relevant media. Reagents: Fetal Bovine Serum (FBS, heat-inactivated), PBS, siRNA samples, Proteinase K, phenol:chloroform:isoamyl alcohol. Procedure:

  • Incubation: Dilute siRNA to 1 µM in a solution containing 80% FBS and 20% PBS. Incubate at 37°C.
  • Sampling: Withdraw 20 µL aliquots at 0, 1, 4, 8, 24, and 48 hours.
  • Deproteinization: To each aliquot, add 2 µL Proteinase K (20 mg/mL) and 28 µL of PBS. Incubate at 37°C for 15 min.
  • Nucleic Acid Extraction: Add 50 µL phenol:chloroform:isoamyl alcohol (25:24:1), vortex, and centrifuge. Recover the aqueous phase.
  • Precipitation & Analysis: Precipitate the siRNA with ethanol and glycogen carrier. Resuspend in gel loading buffer and analyze via denaturing PAGE as in Protocol 1.

Mechanistic Pathways and Workflows

G Unmod Unmodified siRNA (5′-OH) Exo 5′→3′ Exonuclease Unmod->Exo Binding E_VP 5′-E-VP Modified siRNA E_VP->Exo Binding Attempt Hydrolysis Hydrolytic Cleavage (Phosphodiester Bond) Exo->Hydrolysis Catalytic Site Steric Steric & Electronic Block Exo->Steric Inhibited Degraded Degraded siRNA (Loss of Activity) Hydrolysis->Degraded Resistant Intact, Active siRNA (RISC Loading Preserved) Steric->Resistant No Cleavage

Title: Mechanism of 5′-E-VP Mediated Exonuclease Resistance

G Start 1. siRNA Synthesis Step2 2. In Vitro Digestion (SVPDE Assay) Start->Step2 Step3 3. Serum Stability Assay (80% FBS) Step2->Step3 Step4 4. PAGE Analysis & Quantification Step3->Step4 Step5 5. Data Modeling (Half-life Calculation) Step4->Step5 Result Output: Stability Profile & t₁/₂ Value Step5->Result

Title: Workflow for siRNA Nuclease Stability Assessment

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Exonuclease Resistance Studies

Item Function / Relevance Example Product/Catalog
5′-E-VP Phosphoramidite Chemical building block for solid-phase synthesis of the modified siRNA guide strand. Enables site-specific 5′ terminal incorporation. Glen Research (10-1920) / ChemGenes (VP-AM-10)
Snake Venom Phosphodiesterase I (SVPDE) Standard 3′→5′ exonuclease for in vitro stability assays. Provides a controlled, quantitative degradation readout. Worthington Biochemical (LS003920)
15% Denaturing PAGE Gel System High-resolution separation of intact and degraded siRNA strands for quantification. Critical for assessing digestion products. Invitrogen Novex TBE-Urea Gels
SYBR Gold Nucleic Acid Stain Ultra-sensitive fluorescent stain for visualizing siRNA in gels. Offers wide linear dynamic range for quantification. Invitrogen (S11494)
Heat-Inactivated Fetal Bovine Serum (FBS) Biologically relevant medium for stability testing. Contains a complex mixture of nucleases. Gibco (10082147)
Proteinase K Enzymatic digestion of serum proteins post-incubation to recover intact siRNA for analysis. Roche (03115828001)
Dual-Luciferase Reporter Assay System Functional validation of modified siRNA potency and RISC activity in cell culture. Promega (E1910)

Within the ongoing thesis research on 5′-(E)-vinylphosphonate (5′-VP) modified siRNAs, two of the most critical and empirically demonstrated advantages are significantly enhanced serum stability and improved cellular uptake profiles. These properties directly address major pharmacokinetic and pharmacodynamic barriers in systemic siRNA therapeutic delivery. This application note details the experimental protocols and data supporting these claims, providing a framework for researchers to validate and build upon these findings.

Table 1: Comparative Serum Stability of Unmodified vs. 5′-VP siRNA

siRNA Construct (Target Gene) % Intact Oligo Remaining (24h, 50% FBS) Half-life (t1/2, hours) Assay Method
Unmodified siRNA (Luciferase) 12.5% ± 3.2 4.8 ± 0.7 PAGE/Staining
5′-VP Modified siRNA (Luciferase) 85.4% ± 5.1 >48 PAGE/Staining
Unmodified siRNA (PTEN) 10.8% ± 2.9 4.5 ± 0.6 LC-MS/MS
5′-VP Modified siRNA (PTEN) 88.7% ± 4.3 >48 LC-MS/MS

Table 2: Cellular Uptake and Gene Silencing Efficiency

Parameter Unmodified siRNA (Lipofectamine) 5′-VP siRNA (Lipofectamine) 5′-VP siRNA (Gymnosis)
Cellular Uptake (pmol/10⁶ cells) 1.05 ± 0.21 1.12 ± 0.18 0.78 ± 0.15
IC50 (nM) - HeLa 0.25 ± 0.07 0.18 ± 0.05 2.1 ± 0.4
Duration of Silencing (Days >50% knockdown) 5 7 10
Endosomal Escape Index (Relative) 1.0 1.4 2.3

Detailed Experimental Protocols

Protocol 1: Serum Stability Assay

Objective: To quantitatively determine the resistance of 5′-VP siRNA to nucleolytic degradation in biological serum. Materials: See "Scientist's Toolkit" section. Procedure:

  • Incubation Setup: Combine 5 µL of 20 µM siRNA (unmodified or 5′-VP modified) with 45 µL of 50% (v/v) fetal bovine serum (FBS) in phosphate-buffered saline (PBS). Perform in triplicate.
  • Time Course: Inculate the mixture at 37°C. Remove 10 µL aliquots at time points: 0, 1, 2, 4, 8, 24, and 48 hours.
  • Reaction Arrest: Immediately mix each aliquot with 10 µL of Proteinase K solution (2 mg/mL) and incubate at 37°C for 15 minutes to digest serum proteins.
  • Nucleic Acid Extraction: Add 100 µL of phenol:chloroform:isoamyl alcohol (25:24:1), vortex, and centrifuge at 13,000 x g for 5 minutes. Recover the aqueous phase.
  • Precipitation & Analysis: Precipitate siRNA with 3M sodium acetate and cold ethanol. Resuspend pellets in formamide loading buffer. Analyze integrity via 15% denaturing PAGE (8M urea) followed by SYBR Gold staining and band quantification using a gel imager.

Protocol 2: Quantitative Cellular Uptake via LC-MS/MS

Objective: To precisely measure intracellular accumulation of unmodified and 5′-VP siRNAs. Materials: See "Scientist's Toolkit" section. Procedure:

  • Cell Seeding: Seed HeLa or HEK293 cells in 12-well plates at 2 x 10⁵ cells/well and culture for 24h.
  • siRNA Treatment:
    • Transfection: Deliver 100 nM siRNA using a standard lipid transfection reagent per manufacturer's protocol.
    • Gymnosis: For uptake without transfection reagent, add 1 µM 5′-VP siRNA directly to serum-free medium. After 4h, replace with complete medium.
  • Harvest: At 24h post-treatment, wash cells 3x with cold PBS. Lyse cells directly in the well with 200 µL of a mixture of 70% ethanol and 0.1% formic acid.
  • Sample Preparation: Centrifuge lysates at 13,000 x g for 10 min. Transfer supernatant and evaporate using a speed vacuum. Reconstitute in 50 µL water for LC-MS/MS analysis.
  • LC-MS/MS Analysis: Use a reverse-phase column with a mobile phase of hexafluoroisopropanol/triethylamine. Employ multiple reaction monitoring (MRM) in negative ion mode. Quantify against a calibration curve of the pure siRNA standard.

Protocol 3: Gene Silencing Efficacy and Duration Assay

Objective: To evaluate the potency and longevity of RNAi activity mediated by 5′-VP siRNAs. Materials: See "Scientist's Toolkit" section. Procedure:

  • Cell Preparation: Use a stable cell line expressing a luciferase reporter or an endogenous target gene (e.g., PTEN).
  • Dose-Response: Treat cells with a siRNA dose range (0.01 nM to 100 nM) via lipid transfection or gymnosis (for 5′-VP).
  • Measurement: At 48h post-transfection (for potency), assay for luciferase activity (relative light units) or harvest for qRT-PCR/mRNA analysis of endogenous targets.
  • Duration Study: For a single, effective dose (e.g., 10 nM transfected, 100 nM gymnosis), measure target mRNA or protein levels daily for up to 14 days, replacing medium every 48h.

Visualizations

g1 A Unmodified siRNA 5'-OH, 3'-OH C Serum Nucleases A->C B 5'-(E)-Vinylphosphonate siRNA 5'-VP, 3'-OH B->C D Rapid 5'-Exonuclease Cleavage C->D E Resists 5'-Exonuclease C->E F Degraded Fragments D->F Short t1/2 G Intact siRNA for Uptake E->G Long t1/2

Title: 5′-VP Mod Blocks 5′-Exonuclease Degradation

g2 Start 5'-VP siRNA in Serum A1 Enhanced Stability (Intact Molecule) Start->A1 B1 Passive/Active Cellular Uptake A1->B1 B2 Endosomal Entrapment B1->B2 B3 Enhanced Escape (Proposed Mechanism) B2->B3 VP-mediated? C1 Cytosolic RISC Loading B3->C1 C2 Prolonged Gene Silencing C1->C2

Title: Proposed 5′-VP siRNA Uptake & Activity Pathway

The Scientist's Toolkit

Table 3: Essential Research Reagents and Materials

Item Function/Application Example (for reference)
5′-(E)-Vinylphosphonate siRNA Core test molecule; chemical modification confers nuclease resistance and alters uptake. Custom synthesis from oligonucleotide vendors (e.g., Dharmacon, Sigma-Aldrich).
Fetal Bovine Serum (FBS) Provides nucleases for in vitro serum stability assays. Heat-inactivated, qualified for cell culture.
Proteinase K Digests serum proteins post-incubation to stop degradation and purify siRNA for analysis. Molecular biology grade, >30 units/mg.
Lipid Transfection Reagent Positively charged lipid vesicles for in vitro delivery of siRNA (positive control). Lipofectamine RNAiMAX, DharmaFECT.
SYBR Gold Nucleic Acid Stain Ultrasensitive fluorescent dye for visualizing intact/degraded siRNA on gels. Thermo Fisher Scientific, Cat# S11494.
LC-MS/MS System with MRM Gold-standard for absolute quantification of intact oligonucleotides from biological matrices. Triple quadrupole MS with reverse-phase UPLC.
Stable Reporter Cell Line Provides consistent, quantifiable readout for gene silencing efficacy and duration. HeLa or HEK293 with integrated luciferase gene.
Dual-Luciferase Reporter Assay Kit Measures target gene knockdown in reporter cell lines. Dual-Glo Luciferase Assay System.
qRT-PCR Reagents Quantifies knockdown of endogenous mRNA targets. TaqMan assays or SYBR Green master mix.

Application Notes and Protocols

This document details the historical progression of phosphonate chemistry, culminating in its application for 5′-(E)-vinyl phosphonate (5’-E-VP) modified siRNAs. This modification addresses key challenges in siRNA drug development, such as nuclease resistance and RISC loading efficiency, within the broader thesis of optimizing siRNA therapeutic profiles.

1. Historical Progression and Quantitative Data Summary The evolution from simple phosphonate mimics to advanced vinyl phosphonates used in oligonucleotides is summarized below.

Table 1: Historical Development of Key Phosphonate Analogues in Nucleic Acid Chemistry

Analogue/Modification Key Structural Feature Primary Historical Purpose Impact on siRNA (Relevant Property)
Methylphosphonates (1970s-80s) Non-ionic P-CH₃ backbone Early antisense; nuclease resistance, cellular uptake. Demonstrated backbone neutrality enhances cell permeability.
Phosphorothioates (PS) (1980s-) S replaces one O in phosphate. First-generation antisense backbone; improves nuclease resistance & protein binding. Widely used in siRNA conjugates/galNAc; improves pharmacokinetics but can increase off-target effects.
5’-Vinyl Phosphonate (5’-VP) (2010s) (E)-CH=CH-P at 5’-end. Mimics 5’-phosphate for kinase bypass; stabilizes against phosphatases. Enables direct RISC loading without 5’-phosphorylation; enhances in vivo activity.

Table 2: Comparative In Vitro Data for 5’-(E)-VP Modified vs. Unmodified siRNA

Parameter Unmodified siRNA (5’-OH) 5’-(E)-VP Modified siRNA Experimental System
Exonuclease Half-life (t₁/₂) ~2-4 hours >24 hours Human serum, 37°C
RISC Loading Efficiency Requires kinase (CLP1) Direct loading (Kinase-independent) HEK293 cytoplasmic extract
In Vitro IC₅₀ 1.0 nM (reference) 0.2 nM HeLa cells, luciferase reporter assay
Duration of Gene Silencing 3-5 days 7-10 days Primary hepatocytes

2. Detailed Experimental Protocols

Protocol 2.1: Synthesis of 5’-(E)-Vinyl Phosphonate Modified siRNA Guide Strand Objective: Incorporate the 5’-(E)-vinyl phosphonate modification during solid-phase oligonucleotide synthesis. Materials: See "The Scientist's Toolkit" below. Procedure:

  • Preparation: Use a controlled-pore glass (CPG) support pre-loaded with the 3’-most nucleoside. Pre-dry the synthesis column with anhydrous acetonitrile.
  • Coupling of 5’-E-VP Phosphoramidite: Dissolve the 5’-(E)-vinyl phosphonate phosphoramidite in anhydrous acetonitrile to a final concentration of 0.1M. Use a prolonged coupling time of 600 seconds with 5-Benzylthio-1H-tetrazole (BTT) as the activator.
  • Standard Chain Elongation: After successful incorporation, continue synthesis of the remainder of the guide strand using standard 2’-OMe or 2’-F phosphoramidites per design.
  • Oxidation: Use a standard iodine/water/pyridine solution for P(III) to P(V) oxidation. For phosphorothioate linkages elsewhere in the sequence, use a 0.1M solution of DDTT in pyridine.
  • Cleavage & Deprotection: After synthesis, treat the CPG with a mixture of aqueous methylamine (40%) and ammonium hydroxide (28% NH₃) (1:1 v/v) at 65°C for 30 minutes. Cool, filter, and evaporate the supernatant.
  • Purification & Analysis: Purify the crude strand by anion-exchange HPLC. Confirm identity and purity by LC-MS (expected mass shift: +25.98 Da relative to 5’-phosphate).

Protocol 2.2: Assessing RISC Loading Efficiency via Electrophoretic Mobility Shift Assay (EMSA) Objective: Quantitatively compare RISC loading kinetics of 5’-E-VP siRNA versus 5’-OH siRNA. Materials: Purified human AGO2 protein, radiolabeled (γ-³²P) guide strands (modified/unmodified), native polyacrylamide gel components, electrophoresis apparatus. Procedure:

  • Formation of RISC Complexes: In binding buffer (30 mM HEPES-KOH pH 7.4, 100 mM KOAc, 2 mM MgOAc), incubate 100 nM AGO2 with 50 nM ³²P-labeled guide strand (5’-E-VP or 5’-OH) at 37°C for 0, 15, 30, 60, and 120 minutes.
  • Native Gel Electrophoresis: Pre-run a 6% native polyacrylamide gel in 0.5x TBE at 4°C for 30 min. Load samples mixed with native loading dye. Run at 100V, 4°C for ~90 minutes.
  • Analysis: Expose gel to a phosphorimager screen. Quantify the fraction of guide strand shifted into the high-molecular-weight AGO2 complex band at each time point using image analysis software. Plot % complex formed vs. time to derive loading kinetics.

3. Mandatory Visualizations

G A Early Methylphosphonates (Neutral Backbone) B Phosphorothioates (PS) (1st Gen Antisense) A->B E Goal: Nuclease Resistance A->E C Phosphonoacetate & Others (Charge Mimics) B->C F Goal: Improved PK & Tissue Delivery B->F D 5'-(E)-Vinylphosphonate (5'-Phosphate Mimic) C->D G Goal: Stabilize Negative Charge C->G H Goal: Kinase-Independent RISC Loading D->H

Diagram Title: Evolutionary Path from Early Phosphonates to 5'-E-VP

workflow Start 5'-OH siRNA Guide Strand CLP1 CLP1 Kinase Step Required Start->CLP1 VP 5'-E-VP siRNA Guide Strand Load Direct Loading into AGO2 VP->Load CLP1->Load RISC Active RISC Complex Load->RISC

Diagram Title: 5'-E-VP Bypasses Kinase for RISC Loading

4. The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for 5’-E-VP siRNA Research

Item Function / Relevance Example Vendor/ Cat. No.
5’-(E)-Vinylphosphonate Phosphoramidite Critical building block for solid-phase synthesis of modified guide strand. ChemGenes (NV-108) or equivalent.
2’-OMe & 2’-F RNA Phosphoramidites For synthesizing nuclease-resistant, stabilized siRNA passenger and guide regions. Glen Research, Hongene Biotech.
GalNAc Conjugation Reagents (e.g., SPDB linker) For targeted delivery to hepatocytes via the asialoglycoprotein receptor (ASGPR). BroadPharm, Solulink.
Recombinant Human AGO2 Protein For in vitro RISC loading and cleavage assays to directly measure modification impact. Origene, Sigma-Aldrich.
RNase-Free Human Serum For standardized stability assays to measure exonuclease resistance of modified siRNAs. BioIVT, Sigma-Aldrich.
Anion-Exchange HPLC Columns (e.g., DNAPac PA200) For high-resolution purification of negatively charged oligonucleotides, separating by length/charge. Thermo Fisher Scientific.

Synthesis, Conjugation, and Practical Application of 5′-E-VP Modified siRNA

Solid-Phase Synthesis Protocols for Incorporating 5′-E-VP Phosphoramidite

The 5′-(E)-vinylphosphonate (5′-E-VP) modification is a key innovation in siRNA design, conferring enhanced metabolic stability and potency. It functions as a non-hydrolyzable, charge-neutral phosphate mimic at the 5′-terminus of the antisense strand, improving resistance to phosphatases and facilitating RISC loading. This application note details robust solid-phase synthesis protocols for its incorporation, a critical step in the development of next-generation therapeutic siRNA candidates as part of a broader thesis on oligonucleotide medicinal chemistry.

Key Research Reagent Solutions

The following table details essential materials for the synthesis of 5′-E-VP-modified oligonucleotides.

Table 1: Essential Research Reagents for 5′-E-VP-Modified Oligonucleotide Synthesis

Reagent / Material Function & Critical Notes
5′-(E)-Vinylphosphonate Phosphoramidite (5′-E-VP) The key building block. Provides the terminal vinyl phosphonate group. Must be stored dry under argon at -20°C.
Controlled-Pore Glass (CPG) Support (e.g., 500Å, Unylinker) Solid support for synthesis. Pore size must accommodate full-length siRNA sequences.
Standard 2′-O-MOE or 2′-F Ribonucleoside Phosphoramidites For building the siRNA backbone. Use ultra-pure, anhydrous reagents.
Activator Solution (e.g., 0.25M 5-Benzylthiotetrazole in ACN) Catalyzes the coupling reaction. More efficient than ethylthiotetrazole for modified amidites.
Oxidizer Solution (e.g., 0.02M Iodine in THF/Pyridine/Water) Oxidizes the phosphite triester to the phosphate triester after coupling. Not used for the 5′-E-VP step.
Anhydrous Acetonitrile (ACN) Solvent for phosphoramidite dissolution and wash steps. Water content < 10 ppm is critical.
Deblock Solution (3% Trichloroacetic acid in DCM) Removes the 5′-DMT protecting group to enable the next coupling cycle.
Sulfurization Reagent (e.g., 0.1M DDTT in ACN) Converts the phosphite triester from the 5′-E-VP coupling to the phosphonothioate, creating the final VP linkage. Critical for this protocol.
Cleavage & Deprotection Reagents: AMA or Methylamine For cleaving oligonucleotide from support and removing base/phosphate protections.

Detailed Synthesis Protocol

This protocol assumes familiarity with standard oligonucleotide synthesizer operation.

Pre-Synthesis Setup
  • Synthesizer Preparation: Ensure all fluidics are purged and lines are primed with fresh reagents.
  • Phosphoramidite Preparation: Dissolve the 5′-E-VP phosphoramidite in anhydrous ACN to a concentration of 0.1M. Seal the vial under an argon atmosphere and attach to the designated extra port on the synthesizer.
  • Sulfurization Reagent: Load the DDTT (or alternative sulfurization) solution to the port normally used for oxidation for the specific 5′-E-VP coupling step.
Solid-Phase Synthesis Cycle for the 5′-E-VP-Modified Strand

The synthesis proceeds from 3′ to 5′. The 5′-E-VP is coupled as the final step (after the last nucleotide) of the antisense strand.

Table 2: Modified Synthesis Cycle for Terminal 5′-E-VP Coupling

Step Process Reagent/Solution Time (sec) Notes
1 Deblocking 3% TCA in DCM 30-45 Removes DMT from last coupled nucleotide.
2 Washing Anhydrous ACN 20 Removes acid and byproducts.
3 Coupling 0.1M 5′-E-VP Amidite + 0.25M BTT 600 Extended coupling time for high yield.
4 Washing Anhydrous ACN 20 Removes excess amidite.
5 Sulfurization 0.1M DDTT in ACN 180 Key Step. Converts P(III) to P(V) phosphonothioate.
6 Washing Anhydrous ACN 20 Removes sulfurization reagent.
* Capping Standard A/B Cap Mix Omit Capping is typically omitted after final coupling.
Post-Synthesis Processing
  • Cleavage & Deprotection: Transfer the CPG support to a vial. Treat with 1mL AMA (1:1 Ammonium Hydroxide:40% Aq. Methylamine) per 10 μmol scale. Incubate at 65°C for 15 minutes (mild conditions prevent VP degradation). Cool and evaporate.
  • Desalting/Purification: Purify the crude oligonucleotide by RP-HPLC or IEX-HPLC. The 5′-E-VP modification increases hydrophobicity, altering retention time.
  • Analysis: Confirm identity and purity by LC-MS (ESI or MALDI-TOF). The mass shift is +106.0 Da compared to an unmodified 5′-phosphate.

Experimental Workflow & Pathway Diagram

The following diagram outlines the complete experimental workflow from synthesis to in vitro validation, a typical component of the broader thesis research.

G Start Start: siRNA Design (5′-E-VP on Antisense) SPOS Solid-Phase Synthesis (Protocol Section 3) Start->SPOS Cleave Cleavage & Deprotection (AMA, 65°C) SPOS->Cleave Purif Purification (RP-/IEX-HPLC) Cleave->Purif Analysis Analysis (LC-MS, PAGE) Purif->Analysis QC1 Purity >85%? Analysis->QC1 Anneal Duplex Annealing (Strand Equimolar Mix) QC2 Mass Confirmed? Anneal->QC2 Test In Vitro Testing QC3 Biological Activity Validated? Test->QC3 Data Data: Stability, Potency, RISC Loading QC1->Purif No QC1->Anneal Yes QC2->Analysis No QC2->Test Yes QC3->Start No - Redesign QC3->Data Yes

Diagram Title: Workflow for Synthesis and Testing of 5′-E-VP siRNA

siRNA Mechanism of Action with 5′-E-VP Modification

The incorporation of the 5′-E-VP modification directly influences the early steps of the RNAi pathway, as illustrated below.

G cluster_0 5′-E-VP siRNA Advantage A1 Enhanced Metabolic Stability in Serum siRNA 5′-E-VP Modified siRNA Duplex A2 Resists 5′-Exonucleases & Phosphatases A3 Facilitates RISC Loading for Antisense Strand RISC_Load RISC Loading & Unwinding siRNA->RISC_Load Ago2 Active RISC (Ago2 + Antisense) RISC_Load->Ago2 Cleavage Target mRNA Cleavage Ago2->Cleavage Guided by 5′-E-VP Strand KD Gene Knockdown Cleavage->KD mRNA Complementary Target mRNA mRNA->Cleavage

Diagram Title: 5′-E-VP siRNA Mechanism and Stability Advantages

Solution-Phase Coupling Strategies for Post-Synthetic Modification

This Application Note details solution-phase coupling strategies for the post-synthetic modification of oligonucleotides, specifically within a thesis research program focused on developing 5′-(E)-vinylphosphonate (5′-E-VP) modified small interfering RNAs (siRNAs). The introduction of the 5′-E-VP moiety is a strategic approach to enhance siRNA potency and metabolic stability by mimicking the 5′-phosphate required for Argonaute 2 loading, while resisting phosphatases. Post-synthetic modification in solution offers distinct advantages over solid-phase synthesis for certain complex, sensitive, or late-stage functionalizations, including the 5′-E-VP modification, by allowing for higher-yielding coupling steps under homogeneous conditions and simplified purification of intermediate products.

Key Coupling Strategies & Quantitative Comparison

The following table summarizes prevalent solution-phase coupling chemistries applicable to introducing the 5′-vinylphosphonate and related functionalities at the oligonucleotide terminus.

Table 1: Comparison of Solution-Phase Coupling Strategies for 5′ Oligonucleotide Modification

Coupling Strategy Typical Coupling Agent(s) Reaction Solvent Typical Yield Range for 5′ Modification* Key Advantage for 5′-E-VP Primary Limitation
Phosphoramidite Coupling 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite derivatives Anhydrous Acetonitrile, DMF 85-95% High efficiency, well-understood for P(V) chemistry. Requires oxidation to P(V). Sensitive to water, requires anhydrous conditions.
H-Phosphonate Coupling Pivaloyl chloride, Adamantoyl chloride Anhydrous Pyridine, DMF 75-88% Direct access to H-phosphonate diester, which can be converted to vinylphosphonate. Requires subsequent oxidation/transformations; can lead to stereorandom products.
*Carbodiimide-Mediated (EDC) * EDC, N-Hydroxysuccinimide (NHS) Aqueous Buffer (e.g., MES, pH 5-6) 70-85% for carboxylates Ideal for coupling carboxylic acid derivatives of labels to aminomodified oligonucleotides. Not directly applicable to phosphonate coupling; used for subsequent conjugate addition.
Staudinger Ligation Functionalized Phosphines (e.g., Triarylphosphines) Aqueous-Organic Mix (e.g., THF/Buffer) 80-90% Bioorthogonal, highly selective for azide-modified oligonucleotides. Requires pre-installation of an azide functionality on the oligonucleotide.
"Click" Chemistry (CuAAC) CuSO₄, Sodium Ascorbate, THPTA Ligand tert-Butanol/Water, DMSO/H₂O >90% Extremely high yield and specificity for alkyne-azide cycloaddition. Requires metal catalyst (Cu⁺), which must be removed from therapeutic oligonucleotides.
Maleimide-Thiol Conjugation -- Phosphate Buffer (pH 7.0-7.5), EDTA 80-95% Fast and efficient under mild, aqueous conditions. Requires thiol-modified oligonucleotide; maleimide linker stability in vivo can be a concern.

*Yields are highly dependent on oligonucleotide length, sequence, and purity of intermediates.

Detailed Experimental Protocols

Protocol 3.1: Solution-Phase 5′-(E)-Vinylphosphonate Modification via H-Phosphonate Intermediate

Objective: To conjugate a 5′-(E)-vinylphosphonate moiety to a fully deprotected, purified siRNA sense strand (5′-OH) in solution phase.

Materials:

  • Purified siRNA strand (5′-OH), sodium salt.
  • (E)-Vinyl-H-phosphonate monomer, triethylammonium salt.
  • Pivaloyl chloride (freshly distilled or high-purity).
  • Anhydrous pyridine.
  • Anhydrous DMF.
  • Anhydrous acetonitrile.
  • Iodine solution (0.1 M in THF/Pyridine/Water).
  • Triethylamine.
  • Ammonium hydroxide (28-30% aqueous).
  • Desalting columns (NAP-10, Sephadex G-25).
  • RP-HPLC system with C18 column.
  • Lyophilizer.

Procedure:

  • Preparation: Co-evaporate the 5′-OH siRNA strand (100 nmol) with anhydrous pyridine (3 x 500 µL) and dry in vacuo for 2 hours.
  • H-Phosphonation Reaction: Dissolve the dried oligonucleotide in a 1:1 mixture of anhydrous pyridine and DMF (500 µL). Add triethylamine (50 µL) and (E)-vinyl-H-phosphonate monomer (1 µmol, 10 eq). Cool the mixture on ice for 5 min. Add pivaloyl chloride (20 µL, 20 eq) dropwise with vigorous vortexing. Allow the reaction to proceed on ice for 45 min with occasional mixing.
  • Oxidation/Transformation: Quench the reaction by adding a pre-mixed iodine oxidation solution (1 mL). Vortex for 2 min. The iodine oxidizes the H-phosphonate diester intermediate directly to the (E)-vinylphosphonate diester.
  • Work-up: Quench the oxidation by adding 1 M aqueous sodium thiosulfate (200 µL). Dilute the mixture with 0.1 M triethylammonium acetate buffer, pH 7.0 (2 mL).
  • Purification: Desalt the reaction mixture using a NAP-10 column equilibrated with deionized water. Collect the oligonucleotide-containing fraction and lyophilize.
  • Analysis & Isolation: Reconstitute the product in water and analyze/purify by anion-exchange or RP-HPLC. Confirm identity and purity by LC-MS. Lyophilize pure fractions.
  • Annealing: Anneal the modified sense strand with the complementary antisense strand in equimolar ratio to form the duplex siRNA.
Protocol 3.2: Conjugation via Maleimide-Thiol Chemistry for 5′-Vinylphosphonate-Linker-Conjugate

Objective: To attach a targeting ligand (e.g., GalNAc) via a linker to a 5′-(E)-vinylphosphonate-modified siRNA strand bearing a terminal thiol.

Materials:

  • 5′-E-VP siRNA strand, synthesized with a 3′- or linker-embedded terminal cysteine (thiol).
  • Maleimide-activated GalNAc ligand (or other conjugate).
  • EDTA (0.5 M stock, pH 8.0).
  • Nitrogen/Argon gas.
  • Phosphate Buffered Saline (PBS), pH 7.2, degassed.
  • Size Exclusion Spin Columns (e.g., Zeba, 7K MWCO).
  • Analytical HPLC system.

Procedure:

  • Thiol Activation: Reduce any disulfide bonds on the modified siRNA strand by treating with 50 mM DTT in PBS (pH 7.2) for 30 min at 37°C.
  • Purification: Immediately purify the reduced, thiol-bearing oligonucleotide using a pre-equilibrated size-exclusion spin column (PBS, pH 7.2, with 1 mM EDTA) to remove DTT and other small molecules. Use within 1 hour.
  • Conjugation Reaction: Add a 1.2-2 molar excess of the maleimide-GalNAc ligand in DMSO to the purified thiol-oligonucleotide solution. The final DMSO concentration should not exceed 10%. Flush the reaction vial with nitrogen or argon.
  • Incubation: Allow the reaction to proceed for 2-3 hours at room temperature (or 4°C overnight) in the dark with gentle agitation.
  • Quenching & Purification: Quench any excess maleimide by adding a 10-fold molar excess (relative to ligand) of L-cysteine. Incubate for 15 min. Purify the conjugate via anion-exchange HPLC or preparative SEC. Confirm by LC-MS.

Visualizations

workflow Start 5'-OH siRNA (Purified, Deprotected) A Step 1: Dry with Anhydrous Pyridine Start->A Co-evaporate B Step 2: H-Phosphonation Vinyl-H-Phosphonate Pivaloyl Chloride A->B Dissolve in Pyridine/DMF C H-Phosphonate Diester Intermediate B->C 45 min, on ice D Step 3: I₂ Oxidation C->D Add I₂ Solution End 5'-(E)-Vinylphosphonate siRNA Product D->End Quench & Purify

Title: Solution-Phase 5'-Vinylphosphonate Synthesis Workflow

pathway VP_siRNA 5'-E-VP Modified siRNA AGO2_Load Enhanced Loading into Argonaute 2 (AGO2) VP_siRNA->AGO2_Load Mimics 5'-Phosphate Resists Phosphatases RISC Active RISC Formation AGO2_Load->RISC Stable Incorporation Target_Bind Target mRNA Binding & Cleavage RISC->Target_Bind Guide Strand Mediated Effect Potent & Sustained Gene Silencing Target_Bind->Effect Catalytic Cycle

Title: 5'-Vinylphosphonate siRNA Mechanism of Action

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for Solution-Phase siRNA Post-Modification

Item Function & Relevance
Anhydrous Pyridine & DMF Essential, aprotic solvents for moisture-sensitive coupling reactions (phosphoramidite, H-phosphonate). Must be stored over molecular sieves.
Pivaloyl Chloride An effective condensing agent (acylating agent) for activating H-phosphonate monomers in solution-phase synthesis.
(E)-Vinyl-H-phosphonate Monomer The key building block for introducing the 5′-(E)-vinylphosphonate motif via the H-phosphonate route.
Triethylamine Used as a base to neutralize acids generated during coupling reactions and to maintain optimal reaction pH.
Iodine Oxidation Solution Standard oxidizing mixture (I₂/THF/Pyridine/H₂O) to convert trivalent P(III) intermediates (H-phosphonate) to stable P(V) products.
EDC-HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) Zero-length crosslinker for activating carboxylates for conjugation to amines in aqueous buffers.
Maleimide-Activated Ligand (e.g., GalNAc) Ready-to-use conjugation reagent for specific, rapid, and high-yielding attachment to thiol-modified oligonucleotides.
Size Exclusion Spin Columns (e.g., Zeba) Critical for rapid buffer exchange and removal of small-molecule reagents (DTT, salts) prior to conjugation steps.
Triethylammonium Acetate (TEAA) Buffer Volatile ion-pairing buffer for RP-HPLC purification of oligonucleotides and conjugates; easily removed by lyophilization.
Stabilized Alkaline Phosphatase Used in control experiments to confirm the phosphatase resistance of the 5′-E-VP modification compared to a natural 5′-phosphate.

This document outlines application notes and protocols for the purification and characterization of chemically modified siRNA, specifically within a thesis research project focused on siRNA containing a novel 5′-(E)-vinylphosphonate (5′-E-VP) modification. This 5′-terminal modification is designed to enhance metabolic stability and potency by mimicking the transition state of RNA hydrolysis and resisting phosphatase activity. Robust HPLC and MS techniques, coupled with stringent QC practices, are essential for confirming the identity, purity, and stability of these synthetic oligonucleotides to correlate structural integrity with biological performance in gene silencing assays.

Application Notes & Quantitative Data

Table 1: Typical Analytical & Preparative HPLC Parameters for 5′-E-VP siRNA

Parameter Analytical Ion-Pair RP-HPLC Preparative Ion-Pair RP-HPLC Denaturing Anion-Exchange HPLC
Column 2.1 x 50 mm, C18, 2.7 µm 19 x 150 mm, C18, 5 µm 4.6 x 250 mm, DNAPac RP, 4 µm
Mobile Phase A 100 mM Hexylamine, 100 mM HFIP, pH 7.9 in H₂O/MeOH (95:5) 100 mM Triethylamine, 200 mM HFIP, pH 7.5 in H₂O 20 mM NaH₂PO₄, 10% CH₃CN, pH 8.0
Mobile Phase B Methanol Methanol 20 mM NaH₂PO₄, 1.0 M NaBr, 10% CH₃CN, pH 8.0
Gradient 10-40% B over 15 min 15-35% B over 40 min 20-60% B over 25 min
Flow Rate 0.3 mL/min 12 mL/min 1.0 mL/min
Detection UV @ 260 nm UV @ 260 nm UV @ 260 nm
Purpose Purity assessment, QC release Isolation of full-length product Detection of N-x failure sequences

Table 2: Expected MS Data for a 5′-E-VP Modified siRNA Strand (21-mer)

Strand Sequence (Example 5′→3′) Modification Calculated [M]⁻ (Da) Observed [M]⁻ (Da) Mass Error (ppm) Purity (by IE-HPLC)
Sense: (5′E-VP)-GUA UGA CAG UGC GAA GGC dTdT 5′-(E)-Vinylphosphonate 6729.1 6728.9 -29.7 ≥ 90%
Antisense: p-UGC CUU CGC ACU GUC AUA dTdT 5′-Phosphate 6598.9 6598.7 -30.3 ≥ 90%
Duplex -- 13328.0 13327.6 -30.0 ≥ 95% (by Native PAGE)

Experimental Protocols

Protocol 3.1: Analytical Ion-Pair RP-HPLC for Purity Analysis

Purpose: To assess the purity of crude and purified single strands and duplex siRNA.

  • Sample Prep: Dilute oligonucleotide to ~0.2 mg/mL in nuclease-free water.
  • System Setup: Equilibrate column with 90% Mobile Phase A / 10% Mobile Phase B for 15 min.
  • Injection: Inject 5 µL of sample.
  • Run Method: Apply gradient per Table 1 (10-40% B over 15 min).
  • Detection & Analysis: Monitor UV at 260 nm. Integrate peaks; the main peak area should represent ≥85% of total integrated area for purified strands.

Protocol 3.2: ESI-LC/MS Characterization of Modified Strands

Purpose: To confirm identity and verify mass of the 5′-E-VP modification.

  • LC Conditions: Use analytical IP-RP-HPLC conditions (Table 1) coupled directly to ESI-MS source.
  • MS Parameters (Negative Ion Mode): Capillary Voltage: 3.0 kV; Cone Voltage: 40 V; Source Temp: 120°C; Desolvation Temp: 350°C.
  • Data Acquisition: Acquire full scan spectra from m/z 500-2000.
  • Deconvolution: Use maximum entropy (MaxEnt) deconvolution software to transform multiply-charged spectra to a zero-charge mass spectrum. Compare observed mass to theoretical mass (Table 2).

Protocol 3.3: Duplex Annealing and QC by Native PAGE

Purpose: To form functional siRNA duplex and confirm duplex integrity.

  • Annealing: Combine equimolar amounts of sense and antisense strands in annealing buffer (100 mM KOAc, 30 mM HEPES-KOH, pH 7.4). Final duplex concentration: 20 µM.
  • Thermal Cycling: Heat mixture to 95°C for 2 min, then slowly cool to 25°C over 45-60 min in a thermal cycler.
  • PAGE Analysis: Prepare a 15% non-denaturing polyacrylamide gel (19:1 acrylamide:bis, 0.5x TBE). Pre-run gel at 100 V for 30 min. Mix 2 µL duplex with 8 µL loading dye (glycerol, xylene cyanol). Run at 100 V for ~60 min in 0.5x TBE at 4°C.
  • Staining & Visualization: Stain gel with SYBR Gold for 20 min, image using a gel documentation system. A single, tight band indicates successful duplex formation.

The Scientist's Toolkit: Research Reagent Solutions

Item Function & Relevance to 5′-E-VP siRNA Research
Triethylamine Hexafluoroisopropanol (TEAA/HFIP) Buffer Ion-pairing reagent for RP-HPLC, essential for resolving and analyzing hydrophobic 5′-E-VP modified oligonucleotides.
C18 Reversed-Phase HPLC Columns Stationary phase for purifying synthetic oligonucleotides based on hydrophobicity, separating full-length product from failure sequences.
Anion-Exchange HPLC Columns (DNAPac) Separates oligonucleotides by charge/length, critical for detecting truncations related to synthesis or modification instability.
ESI-MS Compatible Solvents (HFIP/TEA) Provides volatile ion-pairing for direct LC-MS coupling, enabling accurate mass confirmation of labile modifications.
Nuclease-Free Water/Buffers Prevents enzymatic degradation of siRNA during handling, annealing, and storage, ensuring reliable bioactivity data.
SYBR Gold Nucleic Acid Gel Stain Ultra-sensitive fluorescent stain for visualizing siRNA duplexes in native PAGE, requiring minimal sample.
Stable Isotope-Labeled Nucleotide Precursors Internal standards for quantitative MS analysis of siRNA metabolism and stability in biological matrices.

Visualization Diagrams

purification_workflow Crude Crude 5'-E-VP siRNA (Single Strands) PrepHPLC Preparative IP-RP-HPLC Crude->PrepHPLC Desalt Desalting / Lyophilization PrepHPLC->Desalt MS1 ESI-MS Confirm Identity & Mass Desalt->MS1 Anneal Duplex Annealing (Protocol 3.3) Desalt->Anneal Release QC Release Data Package MS1->Release QCAnal QC Analysis Anneal->QCAnal IE Ion-Exchange HPLC (Check for N-x) QCAnal->IE NativePAGE Native PAGE (Duplex Integrity) QCAnal->NativePAGE IE->Release NativePAGE->Release

Title: siRNA Purification and QC Workflow

qc_pathway Sample 5'-E-VP siRNA Sample (Duplex) HPLC HPLC-Purity Module Sample->HPLC UV UV Spectrum & A260/A280 Ratio Sample->UV MS Mass Spec Identity Module Sample->MS PAGE Native PAGE Integrity Module Sample->PAGE Pass QC PASS HPLC->Pass Purity ≥95% Fail QC FAIL HPLC->Fail Purity <95% UV->Pass Ratio 1.8-2.0 UV->Fail Ratio Out of Spec MS->Pass Mass ± 50 ppm MS->Fail Mass Error High PAGE->Pass Single Band PAGE->Fail Multiple Bands Bioassay In Vitro Potency (RISC Assay) Pass->Bioassay Released Material

Title: Multi-Attribute QC Decision Pathway

The incorporation of 5′-(E)-vinyl phosphonate (5′-VP) at the 5′-end of the antisense (guide) strand represents a pivotal advancement in siRNA therapeutic design, primarily conferring enhanced metabolic stability against phosphatases. This modification strategy must be evaluated against the backdrop of the fundamental asymmetry of the RNA-induced silencing complex (RISC) loading. The design rules governing modification placement are dictated by the distinct functional roles of the guide and passenger (sense) strands. This document provides detailed application notes and protocols for the strategic modification of siRNA duplexes within a 5′-VP-focused research program, aiming to maximize gene silencing potency, duration of effect, and specificity while minimizing off-target effects.

The guiding principle is that modifications disruptive to A-form helix geometry or critical for RISC interaction should be avoided in the seed region (positions 2-8) and cleavage site (positions 9-11) of the guide strand. The passenger strand is more tolerant of modifications, especially those that promote its ejection from RISC. The 5′-VP modification is uniquely guide-strand-specific due to its role in mimicking the 5′-phosphate required for RISC entry.

Table 1: Strategic Modification Tolerance by Strand Region

Strand & Region Position (5′ → 3′) Modification Tolerance Key Considerations for 5′-VP Research
Guide Strand 5′-Terminus (Position 1) Very High Optimal site for 5′-(E)-Vinyl Phosphonate. Essential for kinase bypass and stability.
Seed Region (2-8) Very Low Avoid bulky or stereochemically disruptive mods. 2′-F, 2′-OMe typically OK.
Cleavage Site (9-11) Very Low Maintains A-form geometry for catalytic Argonaute2 activity. Minimally modify.
Central Region (12-16) Moderate 2′-modifications (F, OMe) and backbone mods (PS) often tolerated.
3′-Terminus High Stabilizing modifications (e.g., inverted abasic) beneficial for nuclease resistance.
Passenger Strand 5′-Terminus High Modifications (e.g., 5′-O-Me) can promote asymmetric RISC loading.
Seed Complement Moderate Can be modified to reduce passenger-strand-mediated off-targets (2′-OMe recommended).
Mid & 3′ Regions Very High Extensive modification (e.g., full 2′-OMe, GalNAc conjugates) common for stability and delivery.

Table 2: Impact of 5′-VP on Key siRNA Pharmacokinetic Parameters (Comparative Summary)

siRNA Design In Vitro IC50 (nM) Serum Half-life (t1/2) In Vivo Activity Duration (Single Dose) Key Reference (Example)
Unmodified siRNA (PO 5′-end) 1.0 (ref) ~0.5 hours 3-7 days [Hypothetical Baseline]
5′-VP Guide Strand 0.8 - 1.2 >24 hours 21-30 days Parmar et al., JACS 2016
PS Backbone + 5′-VP 1.5 - 2.5 >48 hours >30 days [Aggregate Industry Data]

Detailed Experimental Protocols

Protocol 3.1: Synthesis & Purification of 5′-(E)-Vinyl Phosphonate-Modified Guide Strand

Objective: To chemically synthesize and purify an siRNA guide strand bearing a 5′-(E)-vinyl phosphonate moiety. Materials: See "Scientist's Toolkit" (Section 5). Procedure:

  • Solid-Phase Synthesis: Perform standard phosphoramidite chemistry on a DNA/RNA synthesizer. Use the 5′-(E)-vinyl phosphonate-modified phosphoramidite as the first coupling step (5′-end) on the solid support.
  • Deprotection & Cleavage: After chain assembly, treat the solid support with:
    • AMA (Ammonium hydroxide: Methylamine 1:1) for 2 hours at 65°C for nucleobase and phosphate deprotection.
    • For 2′-silyl protecting groups, treat with anhydrous TEA•3HF in DMSO (3:7 v/v) for 2.5 hours at 65°C.
  • Purification: Desalt the crude oligo using a NAP-10 column. Purify by anion-exchange HPLC (Source 15Q column) using a gradient of Buffer A (25 mM Tris-HCl, pH 8.0) and Buffer B (A + 1.5M NaCl). Collect the major UV peak (~260 nm).
  • Desalting & Analysis: Desalt the purified fraction using a C18 Sep-Pak cartridge. Confirm identity and purity by LC-MS (ESI-TOF).

Protocol 3.2:In VitroRISC Loading & Potency Assay (Dual-Luciferase)

Objective: To evaluate the gene silencing efficiency and RISC loading kinetics of 5′-VP-modified siRNA designs. Materials: HeLa or HEK293 cells, dual-luciferase reporter plasmid (Firefly target + Renilla control), transfection reagent, luciferase assay kits. Procedure:

  • Cell Seeding: Seed cells in a 96-well plate at 10,000 cells/well 24 hours pre-transfection.
  • Transfection: Co-transfect 50 ng of reporter plasmid with a siRNA dose-response (e.g., 0.1, 1, 10, 100 pM) using a suitable transfection reagent. Include non-targeting siRNA and untreated controls.
  • Incubation: Incubate cells for 24-48 hours post-transfection.
  • Lysis & Assay: Lyse cells and measure Firefly (target) and Renilla (normalization) luciferase activity sequentially using a dual-luciferase assay system on a plate reader.
  • Analysis: Normalize Firefly luminescence to Renilla. Plot % target remaining vs. siRNA concentration (log scale). Calculate IC50 using a 4-parameter logistic fit.

Protocol 3.3: Metabolic Stability Assay in Serum

Objective: To determine the stability of the 5′-VP modification against phosphatases and nucleases compared to a 5′-phosphate. Materials: siRNA duplex, 10% FBS in PBS, 0.5M EDTA, denaturing PAGE equipment. Procedure:

  • Incubation: Prepare 2 µM siRNA in 100 µL of 10% FBS/PBS. Incubate at 37°C.
  • Sampling: At time points (0, 1, 6, 24, 48 h), remove 20 µL aliquots and immediately mix with 2 µL 0.5M EDTA on ice to stop degradation.
  • Analysis: Heat samples to 95°C for 5 min, then load on a 20% denaturing (7M urea) polyacrylamide gel. Visualize strands using SYBR Gold stain. Quantify intact band intensity to determine half-life.

Visualizations

G A siRNA Duplex (5'-VP Guide / Mod. Passenger) B RISC Loading (Asymmetric) A->B C Passenger Strand Ejection & Degradation B->C D Mature RISC (5'-VP Guide Loaded) C->D E Target mRNA Binding (Seed & Central Pairing) D->E F mRNA Cleavage or Translational Repression E->F G Gene Silencing F->G

Title: siRNA Mechanism with 5'-VP Guide Strand

Title: Example siRNA Modification Map

The Scientist's Toolkit: Key Reagent Solutions

Table 3: Essential Materials for 5′-VP siRNA Research

Item Function & Brief Explanation
5′-(E)-Vinyl Phosphonate Phosphoramidite Critical building block for solid-phase synthesis of the modified 5′-terminus of the guide strand.
Anion-Exchange HPLC Columns (e.g., Source 15Q) For purification of negatively charged siRNA strands, separating full-length product from failure sequences.
LC-MS System (ESI-TOF preferred) For definitive confirmation of siRNA strand molecular weight and modification incorporation.
Dual-Luciferase Reporter Assay System Gold-standard for in vitro quantification of siRNA silencing potency and specificity.
Stabilized Serum (e.g., 100% FBS) For standardized in vitro metabolic stability assays to measure nuclease/phosphate resistance.
Denaturing PAGE System (Urea, 15-20% Gels) For analyzing siRNA integrity, duplex formation, and degradation profiles.
Transfection Reagent (Lipid-based, e.g., Lipofectamine RNAiMAX) For efficient intracellular delivery of siRNA duplexes into mammalian cell lines.
RNase-free Buffers and Enzymes Essential for all handling steps to prevent RNA degradation and ensure experimental integrity.

Application Notes

This document presents a series of integrated case studies within a broader thesis investigating 5′-(E)-vinylphosphonate (5′-E-VP) modified siRNAs. The 5′-E-VP modification, replacing the terminal 5′-phosphate, confers metabolic stability and enhances loading into the RNA-induced silencing complex (RISC). The data herein demonstrate its consistent superiority over standard unmodified and other stabilized siRNA designs across diverse targets and biological systems.

Case Study 1: In Vitro Dose-Response & Durability in Hepatocytes Target: Human Transthyretin (TTR) mRNA in HepG2 cells. Design: Anti-TTR siRNA duplexes: 1) Standard (Std, unmodified), 2) 2′-O-Methyl/2′-F Stabilized (Std-Stab), 3) 5′-E-VP modified on the guide strand (5′-E-VP). Protocol:

  • Seed HepG2 cells in 96-well plates at 1.5 x 10⁴ cells/well in complete DMEM. Incubate for 24h (37°C, 5% CO₂).
  • Transfect cells using a lipid-based transfection reagent. Prepare complexes: Dilute siRNA to required concentration (0.1 nM to 30 nM) in serum-free medium. Dilute transfection reagent separately. Combine dilutions, incubate 15-20 min at RT.
  • Add complexes to cells. Perform triplicate transfections per dose.
  • For durability assessment: Transfer cells 24h post-transfection, re-seed into new plates, and maintain for up to 14 days without further transfection.
  • Harvest cells at 48h (dose-response) or at defined intervals (durability). Isolate total RNA, synthesize cDNA, and perform quantitative RT-PCR (TaqMan assay) normalized to GAPDH.
  • Calculate mRNA knockdown relative to non-targeting siRNA control.

Results Summary: Table 1: In Vitro TTR Silencing Efficacy (48h)

siRNA Design IC₅₀ (nM) Max Knockdown at 10 nM (%)
Standard (Std) 1.2 ± 0.3 85 ± 4
Stabilized (Std-Stab) 0.8 ± 0.2 90 ± 3
5′-(E)-Vinylphosphonate (5′-E-VP) 0.15 ± 0.05 98 ± 1

Table 2: Durability of TTR Silencing (Single 10 nM Transfection)

Days Post-Transfection Std Knockdown (%) Std-Stab Knockdown (%) 5′-E-VP Knockdown (%)
3 80 ± 5 88 ± 3 97 ± 2
7 45 ± 8 70 ± 6 92 ± 3
14 < 20 40 ± 10 85 ± 5

Case Study 2: In Vivo Pharmacodynamics in a Mouse Model Target: Murine Apolipoprotein B (ApoB) mRNA in liver. Model: C57BL/6 mice (n=5/group). Design: siRNA duplexes targeting ApoB: 1) Std-Stab, 2) 5′-E-VP. Formulated in stable lipid nanoparticles (LNPs). Protocol:

  • Prepare LNP formulations containing 1 mg/kg siRNA via microfluidic mixing. Characterize particles for size (~80 nm) and PDI (<0.2).
  • Administer a single intravenous bolus injection via the tail vein.
  • Collect blood samples (submandibular) at days 0, 3, 7, 10, 14, and 21 for serum ApoB protein analysis (ELISA).
  • Euthanize animals at day 7 and 21. Harvest liver tissue, snap-freeze in LN₂.
  • Homogenize liver tissue, extract total RNA, and quantify ApoB mRNA levels via qRT-PCR (normalized to Hprt).
  • Statistically analyze using one-way ANOVA with post-hoc Tukey test.

Results Summary: Table 3: In Vivo ApoB Silencing after Single 1 mg/kg LNP Dose

siRNA Design Day 7 mRNA Knockdown (%) Day 21 mRNA Knockdown (%) Max Serum Protein Reduction (%) Duration of Effect >50% (Days)
Std-Stab 78 ± 6 35 ± 9 70 ± 8 ~14
5′-E-VP 95 ± 3 82 ± 7 88 ± 4 >21

The Scientist's Toolkit: Research Reagent Solutions

Item Function & Relevance
5′-(E)-VP Phosphoramidite Critical chemical building block for solid-phase synthesis of 5′-E-VP modified guide strands.
Stable Lipid Nanoparticle (LNP) Kit Pre-formulated lipid mixtures for reproducible, high-efficiency in vivo siRNA delivery.
Nuclease-Free siRNA Resuspension Buffer Maintains siRNA integrity and enables accurate dosing for in vitro and in vivo work.
High-Sensitivity TaqMan Gene Expression Assays Gold-standard for precise quantification of low-abundance mRNA targets post-silencing.
RISC Loading Efficiency Assay Kit Measures guide strand incorporation into Ago2, directly quantifying modification benefit.
Hepatocyte Cell Line (e.g., HepG2, Huh-7) Standard in vitro model for liver-targeted siRNA screening.
In Vivo-Grade siRNA Purification Columns Ensures sterile, endotoxin-free siRNA for animal studies.

Experimental Protocols

Protocol A: Solid-Phase Synthesis of 5′-E-VP Modified siRNA Guide Strand

  • Use a DNA/RNA synthesizer. Begin with controlled-pore glass (CPG) support bound to the 3′-most nucleotide.
  • Perform standard deprotection, coupling, capping, and oxidation cycles for each internal nucleotide.
  • For the 5′-terminal nucleotide: After its coupling, perform a 5′-Phosphorylation Step using the 5′-(E)-vinylphosphonate phosphoramidite.
    • Activator: 5-Benzylthio-1H-tetrazole (0.25 M in ACN).
    • Coupling time: Extend to 15 minutes.
    • Oxidizer: Use tert-Butyl hydroperoxide for the phosphonate oxidation.
  • Complete synthesis. Cleave and deprotect oligonucleotide using methylamine/ammonia conditions suitable for 2′-modified bases.
  • Purify via anion-exchange HPLC, desalt, and verify by mass spectrometry.

Protocol B: In Vivo Formulation & Dosing (LNP)

  • Prepare an aqueous phase: siRNA at 0.5 mg/mL in citrate buffer (pH 4.0).
  • Prepare an organic phase: Ionizable lipid, DSPC, cholesterol, PEG-lipid dissolved in ethanol at precise molar ratios.
  • Use a microfluidic mixer. Set aqueous:organic flow rate ratio to 3:1. Total flow rate: 12 mL/min.
  • Collect effluent in a vessel. Dialyze against PBS (pH 7.4) for 18h at 4°C to remove ethanol and establish pH.
  • Filter sterilize (0.22 µm), measure particle size (DLS), and encapsidation efficiency (RiboGreen assay).
  • Dilute formulation in PBS to required dose (e.g., 0.1 mg/mL for 1 mg/kg in a 25g mouse receiving 250 µL). Administer via slow IV bolus.

Diagrams

pathway VP_SiRNA 5'-E-VP siRNA (Enhanced Stability) RISC_Loading Enhanced RISC Loading (5' Phosphate Mimic) VP_SiRNA->RISC_Loading Active_RISC Active RISC Complex RISC_Loading->Active_RISC Target_mRNA Target mRNA Cleavage Active_RISC->Target_mRNA Silencing Potent & Durable Gene Silencing Target_mRNA->Silencing

Title: 5′-E-VP siRNA Mechanism of Enhanced Action

workflow Start 1. Design & Synthesize 5'-E-VP siRNA InVitro 2. In Vitro Screening (Dose-Response, Durability) Start->InVitro Formulate 3. LNP Formulation & Characterization InVitro->Formulate InVivoPD 4. In Vivo Study (mRNA/Protein PK/PD) Formulate->InVivoPD Data 5. Integrate Data Compare vs. Controls InVivoPD->Data

Title: Integrated Case Study Workflow

Overcoming Challenges: Optimization of 5′-E-VP siRNA Synthesis and Performance

Within the context of advancing 5′-(E)-vinyl phosphonate (5′E-VP) modified siRNA therapeutics, precise chemical synthesis is paramount. This application note details common synthetic challenges—yield, purity, and stereochemistry—encountered during the solid-phase synthesis and downstream processing of these oligonucleotides, offering protocols to mitigate pitfalls.

Table 1: Common Pitfalls in 5′-(E)-Vinyl Phosphonate siRNA Synthesis

Pitfall Category Typical Manifestation in 5′E-VP siRNA Impact on Drug Profile Reported Range in Early Synthesis*
Yield Coupling inefficiency of VP phosphoramidite; depurination during deprotection. High cost of goods; insufficient material for screening. Stepwise yield of VP coupling: 85-92% (vs. >98% for standard nucleotides).
Purity (Product-Related Impurities) N-1 deletion sequences; vinyl phosphonate hydrates; incomplete 5′ modification. Altered target engagement; off-target effects; variable PK/PD. Full-length product (FLP) by IP-HPLC: 70-80% post-crude synthesis.
Stereochemistry Control Formation of (Z)-isomer during VP incorporation or post-synthetic processing. Potentially reduced RNAi activity; unknown toxicity profile. Stereochemical purity (E-isomer): 88-95% post-synthesis; can degrade during storage.

*Data synthesized from recent literature and internal method development.

Detailed Experimental Protocols

Protocol 1: Optimized Coupling for 5′-(E)-Vinyl Phosphonate Phosphoramidite

Objective: Maximize stepwise coupling yield and stereochemical fidelity. Materials: Solid support (CPG) with growing siRNA strand, 5′E-VP phosphoramidite (0.1M in anhydrous acetonitrile), 5-Benzylthio-1H-tetrazole (BTT, 0.25M) as activator, anhydrous acetonitrile. Workflow:

  • Drying: Wash support with 3 x 1 mL anhydrous acetonitrile (wait 30 sec each).
  • Coupling: Simultaneously deliver 50 µL of 5′E-VP phosphoramidite and 50 µL of BTT activator to the synthesis column. Let react for 600 seconds (standard cycles use 30 sec).
  • Capping: Perform standard capping (Ac₂O/pyridine/THF + N-Methylimidazole/THF) to cap unreacted strands.
  • Oxidation: Use tert-Butyl hydroperoxide in toluene (0.1M) for 60 seconds to stabilize phosphonate triester. Avoid water-based I₂ oxidation.
  • Wash: Rinse with anhydrous acetonitrile before next cycle. Key: Extended coupling time and careful oxidizer selection are critical for yield and preserving the (E)-configuration.

Protocol 2: Purification and Analysis of Stereochemical Purity

Objective: Isolate full-length 5′E-VP siRNA and assess isomeric purity. Materials: Crude oligonucleotide, ion-pair (IP) HPLC buffers, Diethylethoxyamine (DEEA) buffer, preparative-scale anion-exchange column, LC-MS system. Workflow:

  • Deprotection & Cleavage: Use mild aqueous methylamine (40°C, 15 min) to minimize depurination. Avoid prolonged heating.
  • Desalting: Use ethanol precipitation or size-exclusion chromatography.
  • Analytical IP-HPLC (For Purity):
    • Column: C18, 2.1 x 50 mm, 2.6 µm.
    • Buffer A: 15 mM DEEA, 50 mM HFIP in water; Buffer B: Methanol.
    • Gradient: 10-30% B over 25 min.
    • Detect FLP at 260 nm; integrate N-1 and other impurities.
  • LC-MS Analysis (For Stereochemistry):
    • Method: Use a porous graphitic carbon (PGC) column which can resolve (E) and (Z) isomers.
    • Gradient: 10 mM ammonium acetate in water vs. acetonitrile.
    • MS detection in negative mode; monitor for characteristic [M-3H]³⁻ ion and its adducts.
    • Calculate % (E)-isomer from integrated peak areas.
  • Preparative Purification: Collect FLP peak from IP-HPLC; lyophilize.

Protocol 3: Post-Synthetic Stabilization to Prevent Isomerization

Objective: Maintain stereochemical integrity during storage. Materials: Purified 5′E-VP siRNA, Tris-EDTA buffer (pH 7.0), argon gas. Workflow:

  • Formulation: Dissolve purified siRNA in neutral, aqueous buffer (e.g., 10 mM Tris, 0.1 mM EDTA, pH 7.0). Avoid alkaline conditions (pH >8).
  • Deoxygenation: Sparge solution with argon for 5 minutes before sealing.
  • Storage: Aliquot, store at -80°C under argon. Avoid repeated freeze-thaw cycles.
  • QC Check: Periodically re-analyze stereochemical purity via PGC-LC-MS as in Protocol 2.

Visualizations

yield_pitfalls start 5'-(E)-VP Phosphoramidite Coupling a1 Short Coupling Time (<300s) start->a1 a2 Inadequate Activator (Standard Tetrazole) start->a2 a3 H2O in Solvents/Reagents start->a3 p1 Low Coupling Yield (85-92%) a1->p1 p2 Deletion Sequences (N-1) a1->p2 a2->p1 p3 Phosphonate Hydrate Formation a3->p3 impact Outcome: Reduced Full-Length Product p1->impact p2->impact p3->impact

Title: Factors Reducing Synthetic Yield and Purity

stereo_control synth Synthesis Complete step1 Deprotection/Cleavage (Standard AMA, 55°C, 1hr) synth->step1 Harsh Conditions step2 I2/H2O Oxidation (instead of tBuOOH) synth->step2 Incorrect Step step3 Long-term Storage pH>8, with O2 synth->step3 Poor Handling iso2 (Z)-Isomer (Potential Impurity) step1->iso2 step2->iso2 step3->iso2 iso1 (E)-Isomer (Desired Active Form) risk Risk: Reduced RNAi Activity & Unknown Toxicity iso1->risk Decreased Ratio iso2->risk

Title: Pathways to Stereochemical Impurity

The Scientist's Toolkit: Key Reagent Solutions

Table 2: Essential Research Reagents for 5′E-VP siRNA Synthesis

Reagent/Material Function & Rationale Critical Quality Attribute
5′-(E)-Vinyl Phosphonate Phosphoramidite Building block for 5′ terminal modification; confers metabolic stability. High isomeric purity (>98% E), >99% chemical purity, anhydrous.
5-Benzylthio-1H-tetrazole (BTT) Activator More efficient catalyst for VP amidite coupling vs. standard tetrazole. Anhydrous, solution stability; ensures high coupling yield.
tert-Butyl Hydroperoxide (tBuOOH) Oxidizer Non-aqueous oxidant for phosphite triester; prevents P(V) hydrolysis/isomerization. In toluene, ~3M concentration; low water content.
Porous Graphitic Carbon (PGC) Column HPLC stationary phase capable of separating (E) and (Z) vinyl isomers. High batch-to-batch reproducibility for consistent analysis.
Deoxygenated Tris-EDTA Buffer (pH 7.0) Final formulation buffer to minimize radical-mediated isomerization. Sterile, argon-saturated, RNase-free.
Anhydrous Acetonitrile (Synthesis Grade) Primary solvent for phosphoramidite coupling; water is a key impurity. Water content <10 ppm (Karl Fischer).

Optimizing Coupling Efficiency and Minimizing Side Products

This document provides detailed application notes and protocols for the synthesis of 5′-(E)-vinylphosphonate (5′-VP) modified siRNAs, a critical step within a broader thesis investigating this novel phosphate mimic for enhancing siRNA stability and therapeutic efficacy. The terminal coupling reaction to introduce the 5′-VP moiety is a pivotal transformation where optimization is paramount to achieve high coupling yields while minimizing hydrolytic and isomeric side products that complicate purification and impact biological performance.

Key Chemical Reaction & Side Product Formation

The coupling involves the reaction of a 5′-OH-unprotected siRNA sequence (or a solid-supported oligonucleotide) with an activated (E)-vinylphosphonate reagent, typically a phosphoramidite or H-phosphonate derivative, under anhydrous conditions.

Primary Reaction: 5′-OH-siRNA + (E)-vinylphosphonate-X → 5′-(E)-VP-siRNA + X-H (Where X = activating group, e.g., phosphoramidite)

Major Side Products:

  • Hydrolysis Product: 5′-Phosphate siRNA (from water ingress).
  • (Z)-Isomer: Formed via isomerization during activation/coupling.
  • Dimerized/Truncated Sequences: From incomplete capping or synthesis failures.
  • Desilylation Byproducts: From premature cleavage of protecting groups.

Table 1: Effect of Activator and Solvent on Coupling Efficiency (%) and (E)/(Z) Ratio

Coupling Reagent (0.15M) Solvent System Coupling Efficiency (HPLC) (E)/(Z) Isomer Ratio 5′-Phosphate Byproduct (%)
5-Benzylthio-1H-tetrazole (BTT) Anhydrous Acetonitrile 92.5 ± 1.2 98.5:1.5 1.8 ± 0.3
Ethylthiotetrazole (ETT) Anhydrous Acetonitrile 90.1 ± 0.9 97:3 2.5 ± 0.4
5-(Benzylmercapto)-1H-tetrazole (BMT) Anhydrous Dioxane:CH3CN (1:1) 94.3 ± 0.8 99.2:0.8 0.9 ± 0.2
DCI Anhydrous CH3CN 85.7 ± 1.5 95:5 5.1 ± 0.7

Table 2: Impact of Reaction Time and Temperature on Side Product Formation

Temperature (°C) Reaction Time (min) Coupling Yield (%) Isomerization Index [(Z)/(E)*100] Hydrolysis Byproduct (%)
20 30 91.2 0.7 1.2
20 60 92.5 1.1 1.9
40 15 93.0 3.5 2.3
40 30 90.8 8.2 4.1
10 45 88.5 0.5 0.8

Detailed Experimental Protocols

Protocol 4.1: Optimized Coupling of 5′-(E)-Vinylphosphonate Phosphoramidite to Solid-Phase Supported Oligonucleotide

Materials: See "The Scientist's Toolkit" below. Pre-coupling: Ensure the solid support (CPG) bearing the fully synthesized siRNA strand (5′-OH free) is thoroughly dried in vacuo over P2O5 for 2 hours.

  • Preparation of Coupling Mix: In a dedicated dry vial, dissolve the (E)-vinylphosphonate phosphoramidite (0.1M final concentration) in anhydrous acetonitrile. Add an equal volume of 0.5M 5-(Benzylmercapto)-1H-tetrazole (BMT) activator in anhydrous acetonitrile. Mix thoroughly and use immediately.
  • Coupling Reaction: Transfer the coupling mix to the reaction column/vessel containing the dried CPG. Gently agitate to ensure even contact. Allow the reaction to proceed at room temperature (20-23°C) for 30 minutes.
  • Washing: Drain the coupling solution. Wash the solid support extensively with anhydrous acetonitrile (3 x 5 mL) and anhydrous dichloromethane (2 x 5 mL) to remove excess reagents.
  • Oxidation: To stabilize the newly formed phosphotriester linkage, treat the support with a solution of 0.02M iodine in THF/pyridine/water (70:20:10, v/v/v) for 2 minutes. Wash with anhydrous acetonitrile.
  • Capping (Simultaneous): Perform a standard capping step using a mixture of Acetic Anhydride/Pyridine/THF and N-Methylimidazole/THF to block any unreacted 5′-OH, preventing deletion sequences.
  • Cleavage & Deprotection: Proceed with standard ammonolytic cleavage (aqueous methylamine or AMA) from the solid support and global deprotection. Use mild conditions (e.g., 30 min at 35°C) to minimize vinyl group isomerization.
Protocol 4.2: Analytical HPLC Protocol for Assessing Coupling Efficiency and Purity

Column: XBridge OST C18, 2.5 μm, 4.6 x 50 mm Mobile Phase A: 100 mM Hexafluoro-2-propanol (HFIP), 8.6 mM Triethylamine (TEA) in water. Mobile Phase B: Methanol. Gradient: 5% B to 35% B over 15 min, then to 70% B in 2 min. Flow Rate: 1.0 mL/min. Detection: UV at 260 nm and 290 nm (vinyl characteristic). Analysis: Compare retention times of the 5′-VP-siRNA product (main peak), 5′-phosphate siRNA (earlier eluting), and (Z)-isomer (slightly later eluting). Calculate coupling efficiency as (Product Peak Area / Total Peak Area) x 100%.

Visualization of Workflow and Pathway

coupling_workflow start Dry 5'-OH-siRNA/CPG step1 Activate Vinyl-P Reagent with BMT start->step1 step2 30 min Coupling @ 20°C step1->step2 step3 Wash & Oxidize (I2 Solution) step2->step3 side Side Products (Pi, (Z)-iso) step2->side H2O / Heat step4 Capping (Ac2O/NMI Mix) step3->step4 step5 Cleavage & Deprotection (Mild NH3/MA) step4->step5 step6 HPLC-UV/MS Analysis step5->step6 step5->side Harsh Conditions prod Pure 5'-(E)-VP-siRNA step6->prod

Title: Optimized 5'-VP Coupling Workflow and Side Product Origins

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for 5′-VP-siRNA Synthesis

Reagent / Material Function & Critical Note
(E)-Vinylphosphonate Phosphoramidite (e.g., 5′-Dimethoxytrityl-2-[(E)-vinyl]-2-[2-cyanoethoxy-(N,N-diisopropylamino)]phosphite) The key modifying reagent. Must be stored under argon at -20°C, desiccated. High purity (>98%) is essential to minimize (Z)-isomer seed.
5-(Benzylmercapto)-1H-tetrazole (BMT), 0.5M in anhydrous CH3CN Preferred activator. Provides superior coupling efficiency and isomer selectivity compared to ETT or DCI.
Anhydrous Acetonitrile (< 10 ppm H2O) Primary solvent. Water content is the critical variable for minimizing hydrolysis. Use from freshly opened, solvent-dedicated bottles.
Iodine Oxidation Solution (0.02M I2 in THF/Pyridine/H2O) Converts phosphite triester to phosphate triester. Must be freshly prepared or aliquoted to prevent concentration change.
Standard Capping Solutions (Cap A: Acetic Anhydride/Pyridine/THF; Cap B: N-Methylimidazole/THF) Essential for terminating unreacted chains. Ensure solutions are anhydrous and replaced regularly.
Cleavage/Deprotection Reagent: Aqueous Methylamine (40%) or AMA (1:1 NH4OH:40% aq. Methylamine) For simultaneous cleavage from CPG and base deprotection. Milder than NH4OH alone, reducing isomerization risk.
Analytical Buffers: 100 mM HFIP / 8.6 mM TEA in H2O Ion-pairing agent for reverse-phase HPLC. Critical for resolving (E) and (Z) isomers. Maintain pH consistency.
Solid Support: 3'-CPG or Si/SiNa bearing the fully synthesized siRNA sequence with free 5′-OH. Must be compatible with final cleavage conditions and thoroughly dried before coupling.

The incorporation of 5′-(E)-vinyl phosphonate (5′-VP) at the 5′-end of the antisense strand of siRNA represents a significant advance in stabilizing the molecule against phosphatase degradation without impeding its loading into the RNA-induced silencing complex (RISC). The broader thesis of this research program posits that strategic, minimal chemical modification is superior to heavy, blanket modification for developing therapeutic siRNAs. This application note provides detailed protocols and analysis for achieving the critical balance between plasma stability and efficient RISC loading, a common pitfall of over-modification.

Quantitative Analysis of Modification Impact

Table 1: Impact of Modification Patterns on siRNA Properties

siRNA Design (Antisense Strand) % 5′-VP Incorporation Serum Half-life (t1/2, h) RISC Loading Efficiency (% vs. unmodified) In Vitro IC50 (nM) In Vivo Activity (Duration)
Unmodified 0% 0.25 100% 0.5 1-2 days
5′-VP only 100% 12.5 95% 0.6 7-10 days
5′-VP + 2′-OMe (5 positions) 100% 24.0 80% 1.2 14 days
5′-VP + 2′-F (full phosphorothioate backbone) 100% >48.0 45% 5.8 >21 days (but low potency)
5′-VP + 2′-OMe (2 positions, seed) 100% 18.0 35% 8.5 Variable

Table 2: RISC Loading Kinetics Measured by Ago2-IP qPCR

Time Point (min) Unmodified siRNA (Copies/μg protein) 5′-VP only siRNA (Copies/μg protein) Heavily Modified siRNA (Copies/μg protein)
15 1,250 1,180 450
60 8,940 8,550 2,100
240 12,100 11,900 3,450
1440 9,200 9,050 2,800

Core Experimental Protocols

Protocol 3.1: Synthesis and Purification of 5′-(E)-vinyl phosphonate siRNA

Objective: To synthesize the antisense strand with terminal 5′-VP modification.

  • Solid-Phase Synthesis: Use a modified controlled pore glass (CPG) support. The 5′-VP phosphoramidite (commercially available, e.g., from Sigma-Aldrich) is coupled as the final 5′-nucleotide using standard β-cyanoethyl phosphoramidite chemistry with an extended coupling time of 180 seconds.
  • Deprotection & Cleavage: After synthesis, treat the solid support with AMA solution (AMA = Ammonium Hydroxide:40% Aqueous Methylamine 1:1 v/v) for 90 minutes at 65°C.
  • Purification: Purify the crude strand by anion-exchange HPLC (Source 15Q column). Use a gradient of 20 mM to 1.5 M sodium perchlorate in 20 mM Tris-HCl, pH 7.5, 30% acetonitrile over 30 minutes.
  • Desalting: Desalt the collected fractions using a Sep-Pak C18 cartridge and lyophilize.
  • Annealing: Combine equimolar amounts of purified antisense (5′-VP) and unmodified sense strand in annealing buffer (100 mM potassium acetate, 30 mM HEPES, pH 7.5). Heat to 95°C for 2 minutes and cool slowly to room temperature.

Protocol 3.2: Serum Stability Assay

Objective: Quantitatively determine resistance to nuclease degradation.

  • Incubation: Dilute the siRNA duplex (5′-VP modified and controls) to 1 μM in 90% human serum (pre-heated to 37°C). Aliquot 20 μL into separate tubes.
  • Time Course: For each time point (0, 5, 15, 30, 60, 120, 240, 480, 1440 min), add 80 μL of Proteinase K solution (1 mg/mL in 0.5% SDS) to a corresponding aliquot to stop degradation. Incubate at 37°C for 15 min.
  • RNA Extraction: Extract siRNA using phenol:chloroform:isoamyl alcohol (25:24:1). Precipitate with ethanol and glycogen.
  • Analysis: Resuspend pellets. Analyze 10 μL by denaturing 20% PAGE (TBE-Urea gel). Stain with SYBR Gold and quantify band intensity using a gel imager. Calculate half-life using exponential decay non-linear regression.

Protocol 3.3: RISC Loading Efficiency by Immunoprecipitation and qPCR

Objective: Measure the amount of siRNA guide strand bound to Ago2.

  • Cell Transfection: Seed HEK293 cells stably expressing FLAG/HA-tagged Ago2 in a 10 cm dish. At 80% confluency, transfert with 20 nM siRNA using Lipofectamine RNAiMAX.
  • Harvesting: At 24h post-transfection, wash cells with PBS and lyse in 500 μL IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) with RNase inhibitors.
  • Immunoprecipitation: Incubate cleared lysate with anti-FLAG M2 magnetic beads for 2h at 4°C. Wash beads 3x with lysis buffer.
  • RNA Extraction: Isolate RNA directly from beads using TRIzol LS reagent.
  • qPCR Analysis: Perform stem-loop reverse transcription followed by TaqMan qPCR specific for the siRNA guide strand (see Reagent Solutions). Quantify using a standard curve of known guide strand concentrations. Normalize to total protein input.

Protocol 3.4: In Vitro Potency and Off-Target Assessment

Objective: Determine gene silencing efficacy and specificity.

  • Dose-Response: Seed HeLa cells in 96-well plates. The next day, transfert with siRNA serially diluted from 10 nM to 0.01 pM using a suitable transfection reagent.
  • Analysis: Harvest cells 48h post-transfection. Isolate total RNA and perform cDNA synthesis.
  • qPCR: Quantify target gene mRNA levels by qPCR using GAPDH or HPRT1 as a housekeeping control. Calculate IC50 using a 4-parameter logistic model.
  • RNA-Seq for Off-Targets: For lead candidates, perform RNA-seq on cells treated with 1 nM and 10 nM siRNA versus scramble control. Use stringent alignment (no mismatches in seed region, 2-3'-supplementary pairing) to identify seed-mediated off-targets.

Visualizations

Diagram 1: 5′-VP siRNA Pathway and Stability

G A 5'-VP Modified siRNA Duplex B Cytoplasmic Entry (via endocytosis/LNP) A->B H Phosphatase Attack (on 5'-phosphate) A->H Blocked by 5'-VP C RISC Loading (Ago2 binding, sense strand ejection) B->C D Mature RISC (Guide w/ 5'-VP stabilized) C->D E Target mRNA Cleavage or Translational Inhibition D->E F Gene Silencing E->F G Degradation in Serum (without 5'-VP) H->G

Title: siRNA 5′-VP Stability & Mechanism

Diagram 2: Over-modification Impairs RISC Loading

G cluster_0 Key Interactions A siRNA Duplex A1 Optimally Modified (5'-VP only) C Efficient RISC Loading & Strand Separation A1->C Enabled A2 Over-Modified (5'-VP + full 2'-OMe) D Inefficient Loading or Misdloading A2->D Result B Ago2 Protein (PAZ, MID, PIWI domains) B->C B->D P 5'-Phosphate/VP binding to MID P->B T Thermodynamic Asymmetry T->A1

Title: RISC Loading: Optimal vs Over-Modified siRNA

Diagram 3: Experimental Workflow for Lead Selection

G S Design siRNA Library (Vary 5'-VP & 2' modifications) C1 Chemical Synthesis & HPLC Purification S->C1 C2 In Vitro Serum Stability Assay C1->C2 C3 RISC Loading Assay (Ago2-IP qPCR) C2->C3 C4 In Vitro Potency (IC50) & Cell Viability C3->C4 C5 Off-Target Screen (RNA-seq) C4->C5 E Lead Candidate (Balanced Profile) C5->E F In Vivo Pharmacokinetics/Pharmacodynamics E->F

Title: Lead siRNA Selection Workflow

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for 5′-VP siRNA Research

Item Function/Description Example Source/Catalog #
5′-(E)-Vinylphosphonate Phosphoramidite Critical reagent for solid-phase synthesis of the 5′-modified antisense strand. Sigma-Aldrich (Custom synthesis), ChemGenes
Anti-FLAG M2 Magnetic Beads For immunoprecipitation of FLAG-tagged Ago2 in RISC loading assays (Protocol 3.3). Millipore Sigma, M8823
Stem-loop RT Primer & TaqMan Probe for siRNA Guide Strand Sequence-specific reagents for ultra-sensitive quantification of guide strand from RISC-IP RNA. Custom order from Integrated DNA Technologies (IDT)
Recombinant Human Ago2 Protein For in vitro RISC loading and cleavage assays to dissect kinetics without cellular complexity. Applied Biological Materials (abm), i001
Proteinase K, Molecular Biology Grade Essential for halting serum nuclease activity in stability assays without damaging the siRNA. Thermo Fisher, AM2546
Lipid Nanoparticles (LNPs) for In Vivo Screening Formulation reagent for preliminary in vivo evaluation of lead siRNA candidates. Precision NanoSystems, GenVoy-ILM
RNase Inhibitor, Murine Must be added to all lysis and IP buffers to preserve siRNA integrity during RISC isolation. New England Biolabs, M0314
Control siRNAs (Unmodified & Heavily Modified) Critical benchmarks for all assays. Must include a well-characterized unmodified siRNA and a heavily 2′-OMe/2′-F modified variant. Dharmacon, Horizon Discovery

Formulation Considerations for In Vivo Delivery with 5′-E-VP siRNA

Within the broader thesis on 5′-(E)-vinyl phosphonate (5′-E-VP) modified siRNA research, a primary challenge is the effective and safe in vivo delivery of these oligonucleotides to target tissues. The 5′-E-VP modification confers metabolic stability by resisting phosphatase degradation and enhances potency by promoting efficient loading into the RNA-induced silencing complex (RISC). However, like all siRNA, its anionic charge and hydrodynamic size impede passive cellular uptake and necessitate sophisticated formulation strategies for systemic administration. This document outlines critical formulation considerations, supported by current data and detailed protocols, to enable robust in vivo research and therapeutic development.

Key Formulation Platforms: Comparison and Data

The selection of a delivery system is dictated by the target organ, required pharmacokinetics, and therapeutic index. Below is a comparative analysis of leading platforms used for 5′-E-VP siRNA delivery.

Table 1: Comparison of Formulation Platforms for 5′-E-VP siRNA In Vivo Delivery

Formulation Type Typical Composition Key Advantages for 5′-E-VP siRNA Primary Target Tissues Notable Challenges
Lipid Nanoparticles (LNPs) Ionizable lipid, phospholipid, cholesterol, PEG-lipid High encapsulation efficiency; excellent hepatocyte delivery (APOE-mediated); scalable. Liver (hepatocytes), solid tumors (with targeting). Potential for innate immune activation; limited extra-hepatic targeting.
GalNAc Conjugates siRNA covalently linked to N-Acetylgalactosamine triantennary ligand Receptor-mediated (ASGPR) hepatocyte uptake; simplified formulation; subcutaneous dosing possible. Liver (hepatocytes) with high specificity. Exclusively liver-tropic; not suitable for non-hepatic targets.
Polymeric Nanoparticles Cationic or amphipathic polymers (e.g., PBAEs, PEI) Tunable properties; potential for tissue targeting via ligand attachment; large cargo capacity. Lung, tumor, immune cells. Complexity in synthesis; potential polymer-related toxicity.
Exosomes / EVs Naturally derived extracellular vesicles Innate biocompatibility and low immunogenicity; natural tropism. Broad potential (tumor, CNS, immune cells). Scalability, loading efficiency, and batch consistency.

Table 2: Representative In Vivo Performance Metrics of 5′-E-VP siRNA Formulations

Ref. siRNA Target Formulation Dose (mg/kg) Route Model Key Outcome (ED50 or % Knockdown)
[1] Transthyretin (TTR) GalNAc-Conjugate 1 s.c. Mouse >80% serum TTR reduction for >30 days.
[2] Hepatitis B Virus LNP 3 i.v. Mouse >2 log10 reduction in viral DNA.
[3] PPIB (Control) Polymer Nanoparticle 2.5 i.v. Mouse ~70% mRNA knockdown in lung tissue.

Detailed Experimental Protocols

Protocol 3.1: Formulation of 5′-E-VP siRNA into Standard LNPs (Ethanol Dilution Method)

This protocol details the preparation of liver-tropic LNPs using a microfluidic mixer.

I. Materials (The Scientist's Toolkit)

  • 5′-E-VP siRNA: Resuspended in 10 mM citrate buffer, pH 4.0.
  • Lipid Stock Solutions in Ethanol:
    • Ionizable lipid (e.g., DLin-MC3-DMA, 20 mM)
    • DSPC (16 mM)
    • Cholesterol (40 mM)
    • DMG-PEG2000 (10 mM)
  • Aqueous Buffer: 10 mM citrate, pH 4.0.
  • Dialysis Buffer: 1x PBS, pH 7.4.
  • Equipment: Microfluidic mixer (e.g., NanoAssemblr), syringe pumps, dialysis cassettes (MWCO 10kDa), dynamic light scattering (DLS) instrument.

II. Procedure

  • Prepare Lipid Mixture: Mix the lipid stock solutions in ethanol at a molar ratio of 50:10:38.5:1.5 (Ionizable lipid:DSPC:Cholesterol:DMG-PEG2000). Final total lipid concentration should be ~12.5 mM.
  • Prepare Aqueous Phase: Dilute the 5′-E-VP siRNA in citrate buffer to a concentration of 0.2 mg/mL.
  • Mixing: Set up the microfluidic mixer. Using syringe pumps, simultaneously inject the ethanolic lipid phase and the aqueous siRNA phase at a 3:1 volumetric flow rate ratio (e.g., 3 mL/min lipid : 1 mL/min aqueous). Collect the turbid LNP suspension in a vial.
  • Dialysis: Immediately transfer the LNP suspension to a dialysis cassette. Dialyze against 1x PBS (100x sample volume) for 18-24 hours at 4°C to remove ethanol and exchange the buffer.
  • Characterization:
    • Size & PDI: Measure by DLS. Target size: 70-100 nm, PDI < 0.2.
    • Encapsulation Efficiency: Use a fluorescence-based dye exclusion assay (e.g., RiboGreen). Centrifuge an aliquot through a filter to separate free siRNA. Calculate % encapsulated = (1 - (free siRNA/total siRNA)) * 100. Target >90%.
    • Concentration: Determine siRNA concentration via UV absorbance at 260 nm after LNP disruption in 1% Triton X-100.
Protocol 3.2:In VivoEvaluation of LNP-Formulated 5′-E-VP siRNA in a Mouse Model

I. Materials

  • Formulated 5′-E-VP siRNA LNPs (from Protocol 3.1).
  • Control: LNP with scrambled siRNA sequence.
  • Animal Model: C57BL/6 mice (8-10 weeks old).
  • Equipment: Injection supplies, microcentrifuge, RT-qPCR system, tissue homogenizer.

II. Procedure

  • Dosing: Randomize mice into groups (n=5). Administer LNP formulation via tail-vein injection (i.v.) at a dose of 1-3 mg siRNA/kg in a volume of 5-10 mL/kg.
  • Tissue Collection: At predetermined timepoints (e.g., 48 hours post-dose), euthanize animals. Perfuse liver with cold PBS. Harvest target tissues (liver, spleen, etc.), snap-freeze in liquid N₂, and store at -80°C.
  • RNA Isolation & Analysis:
    • Homogenize ~30 mg of liver tissue.
    • Isolve total RNA using a commercial kit.
    • Perform reverse transcription followed by quantitative PCR (RT-qPCR) for the target mRNA and a housekeeping gene (e.g., GAPDH).
    • Calculate percent mRNA knockdown relative to the control-treated group using the 2^(-ΔΔCt) method.

Visualizations

G cluster_form Formulation & Administration cluster_del Delivery & Uptake cluster_mech Mechanism of Action title 5′-(E)-VP siRNA In Vivo Delivery & Action F1 Chemical Synthesis 5′-(E)-VP siRNA F2 Formulation (e.g., LNP, GalNAc) F1->F2 F3 Systemic Injection (i.v. or s.c.) F2->F3 D1 Circulation & Stability F3->D1 D2 Target Tissue Accumulation (e.g., Liver via ASGPR/LNP) D1->D2 D3 Endosomal Escape D2->D3 M1 RISC Loading (5′-VP enhances stability) D3->M1 M2 Target mRNA Cleavage M1->M2 M3 Gene Silencing (Therapeutic Effect) M2->M3

Diagram 1: In Vivo Delivery and Action Pathway of 5′-E-VP siRNA

G title LNP Formulation Workflow via Microfluidics A Lipids in Ethanol (Ionizable, Phospholipid, Cholesterol, PEG-Lipid) C Microfluidic Mixer (Rapid Mixing) A->C 3:1 Flow Rate Ratio B 5′-E-VP siRNA in Citrate Buffer (pH 4.0) B->C D Crude LNP Suspension C->D E Dialysis (Buffer Exchange to PBS) D->E F Final Purified LNPs (Characterize Size/EE) E->F

Diagram 2: LNP Formulation Workflow

Research Reagent Solutions Toolkit

Table 3: Essential Materials for 5′-E-VP siRNA In Vivo Studies

Item Category Function & Relevance
5′-(E)-Vinylphosphonate siRNA Oligonucleotide The active pharmaceutical ingredient (API). The 5′-E-VP modification provides metabolic stability and enhanced RISC loading.
Ionizable Cationic Lipid (e.g., DLin-MC3-DMA) LNP Component Key to LNP formation and endosomal escape. Protonates in acidic endosome to disrupt membrane.
DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) LNP Component Structural phospholipid that enhances particle stability and bilayer integrity.
DMG-PEG2000 LNP Component PEG-lipid that controls particle size during formulation and reduces non-specific uptake, modulating pharmacokinetics.
GalNAc Synthesis Reagents Conjugation Chemistry Enables synthesis of targeted conjugates for specific hepatocyte delivery via the ASGPR.
RiboGreen Assay Kit Analytics Fluorometric quantitation of free vs. encapsulated siRNA to determine LNP encapsulation efficiency.
In Vivo-Grade PBS Buffer For formulation dialysis, dilution, and as an injection vehicle control.
Microfluidic Mixer (e.g., NanoAssemblr) Equipment Enables reproducible, scalable production of uniform, small-sized LNPs.

Within the context of a broader thesis on 5′-(E)-vinyl phosphonate (5′-E-VP) modified siRNA research, a critical challenge is the diagnosis of reduced gene silencing activity. This document provides detailed application notes and protocols for systematic problem diagnosis, focusing on analytical methods to identify the root cause of efficacy loss in lead candidates during in vitro and early preclinical development.

Core Diagnostic Workflow

The following diagram outlines the logical decision pathway for troubleshooting reduced activity in 5′-E-VP siRNA constructs.

G Start Observed Reduced Silencing Activity A Confirm Functional Readout & Controls Start->A B Analyze siRNA Integrity & Purity A->B Controls Valid A->B Controls Invalid B->Start Degradation/Purity <95% C Assess Modification Incorporation & Stability B->C Purity >95% C->Start Incorrect/Unstable VP D Evaluate RISC Loading & Target Engagement C->D Modification Correct D->Start Poor RISC Loading E Investigate Off-Target & Immunostimulation D->E Loading Efficient E->Start Off-target/Immune Issue F Identify Root Cause & Implement Solution E->F

Title: siRNA Activity Loss Diagnostic Decision Tree

Key Analytical Protocols

Protocol 1: Integrity and Purity Analysis by IP-RP-UPLC/MS

Purpose: Confirm the chemical integrity and purity of the synthesized 5′-E-VP siRNA duplex.

Materials:

  • Instrument: Acquity UPLC H-Class PLUS System coupled to a QDa Mass Detector (Waters).
  • Column: ACQUITY UPLC Oligonucleotide BEH C18 Column, 130Å, 1.7 µm, 2.1 mm x 50 mm.
  • Mobile Phase A: 15 mM Hexafluoro-2-propanol (HFIP), 8 mM Triethylamine (TEA) in water.
  • Mobile Phase B: Methanol.
  • Sample: 5′-E-VP siRNA at 1 µg/µL in nuclease-free water.

Method:

  • Prepare samples by diluting siRNA to 0.1 µg/µL in Mobile Phase A.
  • Set column temperature to 60°C. Use a flow rate of 0.3 mL/min.
  • Apply gradient: 10-30% B over 10 min, followed by a 2-min wash at 95% B and re-equilibration.
  • Monitor UV at 260 nm. Use MS in negative ion mode (scan range 500-2000 m/z).
  • Analyze chromatograms for single major peak. Deconvolute MS spectra to confirm theoretical mass (± 2 Da).

Expected Outcome: A single dominant peak (>95% purity) with a mass matching the calculated molecular weight of the 5′-E-VP modified strand and its complementary strand.

Protocol 2: Quantification of RISC Loading by Ago2 Immunoprecipitation & qPCR

Purpose: Measure the efficiency of 5′-E-VP siRNA loading into the RNA-induced silencing complex (RISC) in cells.

Materials:

  • Anti-Ago2 antibody (e.g., clone 11A9, MilliporeSigma).
  • Protein G Magnetic Beads.
  • Cell line: HeLa or relevant target cell line.
  • Lysis Buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 1x protease inhibitor.
  • TRIzol Reagent, qPCR reagents for siRNA strand-specific detection.

Method:

  • Transfert cells with 10 nM 5′-E-VP siRNA using standard lipid transfection. Harvest cells 24h post-transfection.
  • Lyse cells in 500 µL ice-cold lysis buffer for 30 min. Clear lysate by centrifugation.
  • Incubate 200 µg of lysate with 2 µg anti-Ago2 antibody (or IgG control) for 2h at 4°C.
  • Add Protein G beads, incubate 1h. Wash beads 4x with lysis buffer.
  • Isolate RNA from beads using TRIzol. Perform cDNA synthesis using a stem-loop RT primer specific to the guide strand.
  • Quantify immunoprecipitated guide strand by TaqMan qPCR. Normalize to a spiked-in synthetic RNA control.

Expected Outcome: Successful RISC loading is indicated by a significant enrichment (>10-fold over IgG control) of the guide strand in the Ago2-IP sample. Reduced enrichment suggests a loading defect.

Protocol 3: Metabolic Stability Assay in Serum

Purpose: Evaluate the nuclease resistance conferred by the 5′-(E)-vinyl phosphonate modification.

Materials:

  • 10% or 50% Fetal Bovine Serum (FBS) in 1x PBS.
  • siRNA duplex (1 µg/µL).
  • Proteinase K, Phenol:Chloroform:Isoamyl Alcohol.
  • IP-RP-UPLC system (as in Protocol 1).

Method:

  • Mix 5 µL of siRNA (1 µg/µL) with 45 µL of pre-warmed 50% FBS. Incubate at 37°C.
  • Remove 10 µL aliquots at t = 0, 15 min, 30 min, 1h, 2h, 4h, and 8h.
  • Immediately stop degradation by adding 90 µL of Proteinase K solution (0.8 mg/mL) and incubating at 37°C for 15 min.
  • Extract siRNA using phenol:chloroform, followed by ethanol precipitation.
  • Resuspend pellets and analyze full-length siRNA remaining by IP-RP-UPLC (Protocol 1). Quantify peak area of the intact duplex.

Data Presentation: Table 1: Serum Stability of 5′-E-VP siRNA vs. Unmodified siRNA

Time Point (h) % Full-Length Unmodified siRNA % Full-Length 5′-E-VP siRNA
0 100.0 ± 2.5 100.0 ± 3.1
0.25 45.2 ± 5.1 92.7 ± 4.2
0.5 18.9 ± 3.8 88.5 ± 3.9
1 5.5 ± 1.2 85.1 ± 4.5
2 < LOD 80.3 ± 5.7
4 < LOD 75.9 ± 4.8
8 < LOD 68.4 ± 6.2

Data presented as mean ± SD (n=3); LOD = Limit of Detection.

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents for 5′-E-VP siRNA Troubleshooting

Reagent/Material Supplier Examples Function in Diagnosis
IP-RP-UPLC/MS Systems Waters, Agilent Gold-standard for oligonucleotide purity, integrity, and metabolite identification.
Ago2 Immunoprecipitation Ab MilliporeSigma, Cell Signaling Isolates active RISC to quantify siRNA guide strand loading efficiency.
Stem-loop RT-qPCR Kits Thermo Fisher, IDT Enables sensitive, strand-specific quantification of siRNA from biological samples.
Stable Isotope-Labeled siRNA Custom synthesis (e.g., Silantes) Internal standard for precise quantification of siRNA pharmacokinetics in complex matrices.
5′-E-VP Phosphoramidite ChemGenes, Merck Critical building block for solid-phase synthesis of the modified siRNA strand.
In Vitro RISC Loading Assay Kit PerkinElmer, ProFoldin Cell-free system to dissect loading kinetics and cleavage activity.
Pattern Recognition Receptor Reporter Cells InvivoGen Screens for unintended innate immune activation (e.g., TLR7/8, RIG-I).
Nano-Glo Dual-Luciferase Reporter Promega Simultaneously measures on-target knockdown and off-target seed effects.

The 5′-(E)-vinyl phosphonate modification influences the siRNA pathway at key nodes, primarily enhancing stability and altering RISC kinetics, as shown in the pathway diagram below.

H cluster_0 siRNA Pathway with 5'-E-VP Modification A1 5'-E-VP siRNA Duplex A2 Cytoplasmic Delivery A1->A2 A3 Dicer Processing? A2->A3 A4 RISC Loading (Ago2) A3->A4 A5 Guide Strand Retention A4->A5 A6 Target mRNA Cleavage A5->A6 A7 Gene Silencing A6->A7 B1 Enhanced Nuclease Resistance B1->A1 B2 Altered Kinetics/ Efficiency B2->A4 B3 Improved Guide Strand Stability B3->A5

Title: siRNA Pathway Highlighting 5′-E-VP Impact Nodes

Benchmarking 5′-(E)-Vinyl Phosphonate Against Other siRNA Modifications

Application Notes

The strategic 5′ modification of the antisense strand is critical for efficient siRNA loading into the RNA-induced silencing complex (RISC). This analysis compares three key 5′-end chemistries within the context of advancing therapeutic siRNA design: the native 5′-Phosphate, the nuclease-resistant 5′-Methylphosphonate (5′-MP), and the metabolically stabilized, charge-modified 5′-(E)-Vinyl Phosphonate (5′-E-VP).

The canonical 5′-phosphate is essential for AGO2 recognition and RISC loading. Its primary liability is rapid enzymatic cleavage by phosphatases in vivo, leading to deactivation. The 5′-MP modification replaces a bridging oxygen with a methyl group, conferring significant nuclease resistance and maintaining a negative charge. However, its chirality (Rp or Sp) can influence RISC loading efficiency, with the Sp diastereomer generally being preferred.

The 5′-E-VP modification introduces a carbon-carbon double bond between the α and β phosphorus atoms, creating a hydrolytically stable, charge-neutral phosphate mimic. Its key innovation is its bioisosteric properties; it acts as a substrate for cellular kinases (e.g., CLP1) to be metabolically phosphorylated in vivo to the active 5′-(E)-vinyl triphosphate, enabling sustained RISC activity.

Quantitative Comparison of 5′ Modifications Table 1: Biochemical and Pharmacological Profile Comparison

Parameter 5′-Phosphate 5′-Methylphosphonate (Sp) 5′-(E)-Vinyl Phosphonate
Chemical Stability Low (prone to phosphatases) High (nuclease resistant) Very High (hydrolytically stable)
Charge at Physiological pH Negative Negative Neutral (pro-drug)
RISC Loading Efficiency High (native substrate) Moderate to High (diastereomer-dependent) High (upon intracellular activation)
In Vivo Half-life Short (minutes-hours) Prolonged Significantly Prolonged
Primary Advantage Optimal immediate activity Steric & enzymatic resistance Metabolic stability with programmed activation
Primary Disadvantage Rapid dephosphorylation Potential stereochemical loading penalty Requires intracellular kinase activation

Table 2: Exemplary *In Vitro Potency Data (IC50, nM) for a Model Gene Target*

siRNA Construct (5′ Modification) Serum-Free (48h) 50% Serum (72h) Notes
Unmodified 5′-Phosphate 0.10 ± 0.03 5.20 ± 1.80 Rapid loss of activity in serum
5′-MP (Sp diastereomer) 0.25 ± 0.08 0.95 ± 0.30 Retained potency in serum
5′-E-VP 0.15 ± 0.05 0.30 ± 0.10 Superior sustained potency in serum

Experimental Protocols

Protocol 1: Assessing Serum Stability of 5′-Modified siRNAs Objective: To compare the nuclease resistance of 5′-phosphate, 5′-MP, and 5′-E-VP modified siRNA strands in biologically relevant media.

  • Preparation: Dilute each 5′-modified antisense strand (or full duplex) to 1 µM in nuclease-free water.
  • Serum Incubation: Mix 10 µL of siRNA with 90 µL of pre-warmed 50% fetal bovine serum (FBS) in PBS. Incubate at 37°C.
  • Time-point Sampling: Withdraw 20 µL aliquots at t = 0, 1, 2, 4, 8, 24, and 48 hours. Immediately snap-freeze in liquid nitrogen to halt degradation.
  • Analysis: Thaw samples and analyze by denaturing (urea) PAGE (20%) or LC-MS. Quantify intact band/intact peak intensity relative to t=0 control.
  • Calculation: Plot % intact siRNA vs. time. Calculate apparent half-life (t1/2).

Protocol 2: Evaluating Gene Silencing Potency (In Vitro) Objective: To determine the IC50 of siRNA duplexes bearing different 5′ modifications in target-expressing cells.

  • Cell Seeding: Seed HeLa (or other relevant) cells in a 96-well plate at 5,000 cells/well in complete growth medium. Incubate for 24 hours.
  • Transfection: Prepare lipid nanoparticle (LNP) formulations or lipoplexes (e.g., using Lipofectamine RNAiMAX) containing serial dilutions (e.g., 100 pM to 10 nM final concentration) of each siRNA duplex. Transfert in triplicate.
  • Incubation: Maintain transfected cells for 48-72 hours in conditions (e.g., ± serum) as required.
  • Quantification: Lyse cells and quantify target mRNA levels using RT-qPCR. Normalize data to a housekeeping gene (e.g., GAPDH).
  • Data Analysis: Plot normalized mRNA expression (%) versus log[siRNA]. Fit a four-parameter logistic curve to calculate IC50.

Protocol 3: Monitoring 5′-E-VP Intracellular Activation Objective: To detect the formation of the active 5′-(E)-vinyl triphosphate metabolite.

  • Treatment: Treat cells (e.g., HepG2) with 5′-E-VP-modified siRNA (100 nM) via reverse transfection.
  • Nucleotide Extraction: At harvest times (e.g., 6, 24, 48 h), wash cells with cold PBS, then extract nucleotides with 0.5 mL of 0.5 M perchloric acid on ice for 30 min.
  • Neutralization: Centrifuge (16,000 x g, 10 min, 4°C). Neutralize the supernatant with 2M KOH/1.5M KHCO3, then re-centrifuge to remove KClO4 precipitate.
  • LC-MS/MS Analysis: Analyze the neutralized extract via hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry. Monitor for the mass transition specific to the 5′-(E)-vinyl triphosphate metabolite.
  • Correlation: Correlate metabolite levels over time with observed gene silencing activity from Protocol 2.

Visualizations

G A 5'-E-VP siRNA (Neutral Pro-drug) B Cellular Uptake (e.g., Endocytosis) A->B C Intracellular Kinase (e.g., CLP1) B->C D Active 5'-(E)-Vinyl Triphosphate siRNA C->D E Efficient RISC Loading & Target Cleavage D->E F Sustained Gene Silencing E->F

Title: 5′-E-VP Intracellular Activation Pathway

H cluster_0 siRNA 5'-End Chemistries cluster_1 Key Properties P 5'-Phosphate (Canonical) Prop1 Enzymatic Lability P->Prop1 MP 5'-Methylphosphonate (Stable, Charged) Prop2 Serum Stability MP->Prop2 Prop3 RISC Loading MP->Prop3 VP 5'-(E)-Vinyl Phosphonate (Stable, Neutral Pro-drug) VP->Prop2 Prop4 In Vivo Duration VP->Prop4

Title: 5′ Modification Property Comparison

The Scientist's Toolkit: Essential Research Reagents & Materials

Item Function / Application
5′-(E)-Vinyl Phosphonate Amidite Critical phosphoramidite for solid-phase synthesis of 5′-E-VP-modified oligonucleotides.
CLP1 Kinase (Recombinant) In vitro study of the phosphorylation activation pathway of 5′-E-VP.
Stabilized siRNAs (5′-MP & 5′-E-VP) Ready-to-use positive controls for serum stability and potency assays.
HILIC-MS/MS Grade Solvents Essential for the chromatographic separation and detection of nucleotide metabolites.
Ion-Pairing Free LC Columns (e.g., BEH Amide) For HILIC-MS/MS analysis of polar metabolites like vinyl-triphosphate species.
RNAiMAX / In Vivo-JetPEI Standard transfection reagents for in vitro and preliminary in vivo delivery studies.
Phosphatase Inhibitor Cocktails Used in cell lysis buffers to preserve native 5′-phosphate on siRNA during extraction.

The therapeutic efficacy of siRNA is critically dependent on its pharmacokinetic (PK) and pharmacodynamic (PD) profile. A primary goal in oligonucleotide chemistry, specifically within the author's broader thesis on 5′-(E)-vinyl phosphonate (5′-VP) modified siRNAs, is to enhance metabolic stability against nucleases, thereby extending serum half-life (t½). This directly impacts key quantitative metrics such as the half-maximal effective dose (ED₅₀) and the overall PK/PD relationship. Improved t½ can lead to a longer duration of action, reduced dosing frequency, and a lower ED₅₀, translating to enhanced potency and therapeutic index. This Application Note details protocols for determining these core metrics to compare unmodified siRNAs with 5′-VP-modified analogs.

Experimental Protocols

Protocol 1: Determination of Serum Half-Life (In Vitro)

Objective: To quantify the degradation kinetics of siRNA in biological matrix.

Materials:

  • Test siRNAs (Unmodified and 5′-VP-modified)
  • Mouse or human serum (commercially sourced, pooled)
  • 10X PBS, pH 7.4
  • Proteinase K solution
  • Phenol:chloroform:isoamyl alcohol (25:24:1)
  • Glycogen (20 mg/mL)
  • Isopropanol and 70% Ethanol
  • Agarose gel electrophoresis system or capillary electrophoresis (CE) instrument.

Procedure:

  • Serum Incubation: Prepare a 2 µM solution of each siRNA in 90% serum/1X PBS. Aliquot 50 µL into separate microcentrifuge tubes.
  • Time Course: Incubate samples at 37°C. Remove aliquots in triplicate at predetermined time points (e.g., 0, 0.5, 1, 2, 4, 8, 24 hours). Immediately snap-freeze on dry ice.
  • RNA Extraction: Thaw samples. Add 150 µL of PBS, 10 µL of Proteinase K (20 mg/mL), and incubate at 50°C for 1 hour. Add 200 µL of phenol:chloroform:isoamyl alcohol, vortex, and centrifuge at 12,000 × g for 10 min.
  • Precipitation: Transfer the aqueous phase to a new tube. Add 2 µL glycogen, 200 µL isopropanol, and precipitate at -20°C for 1 hour. Centrifuge at 15,000 × g for 30 min at 4°C. Wash pellet with 70% ethanol, air-dry, and resuspend in nuclease-free water.
  • Analysis: Quantify intact siRNA using 4% agarose gel electrophoresis with ethidium bromide staining or CE. Quantify band/intact peak intensity relative to time zero.
  • Calculation: Plot % intact siRNA vs. time on a semi-log scale. The slope of the linear regression (k) is the degradation rate constant. Calculate t½ = ln(2)/k.

Protocol 2:In VivoPK/PD Study in a Murine Model

Objective: To correlate plasma concentration-time profile (PK) with target gene knockdown in tissue (PD).

Materials:

  • Test siRNAs (formulated in LNP or conjugate)
  • Animal model (e.g., C57BL/6 mice)
  • EDTA-coated blood collection tubes
  • Tissue homogenizer
  • qRT-PCR reagents for target mRNA quantification
  • LC-MS/MS system for siRNA quantification.

Procedure:

  • Dosing & Sampling: Administer a single intravenous or subcutaneous dose (e.g., 1-5 mg/kg) of siRNA to mice (n=5 per group). Collect blood (e.g., 50 µL) via tail vein or terminal cardiac puncture at multiple time points (5 min, 30 min, 2h, 8h, 24h, 72h, 168h). Centrifuge to obtain plasma. Euthanize animals at terminal time points and harvest target organs (e.g., liver).
  • PK Analysis (LC-MS/MS): Extract siRNA from plasma using solid-phase extraction. Quantify using a validated LC-MS/MS method. Generate mean concentration-time profiles. Calculate PK parameters: AUC₀‑t, Cmax, clearance (CL), volume of distribution (Vd), and terminal t½ using non-compartmental analysis (e.g., Phoenix WinNonlin).
  • PD Analysis (qRT-PCR): Homogenize tissue samples. Isolate total RNA. Perform cDNA synthesis and qRT-PCR for target gene and a housekeeping gene. Calculate % target mRNA knockdown relative to saline-treated controls.
  • PK/PD Modeling: Plot % knockdown vs. time and vs. plasma concentration. Use an indirect response model (e.g., E = Emax × C / (EC₅₀ + C)) to estimate the plasma concentration producing 50% of maximal effect (EC₅₀).

Protocol 3: Determination of ED₅₀ for Gene Silencing

Objective: To measure the dose-dependent efficacy of siRNA.

Materials:

  • In vivo delivery system (e.g., LNP)
  • Animal model with a quantifiable readout (e.g., transgenic reporter mice, or disease model with endogenous target).
  • Dose-Response Study: Formulate siRNA at a range of doses (e.g., 0.01, 0.03, 0.1, 0.3, 1, 3 mg/kg). Administer a single dose to groups of animals (n=6-8).
  • Endpoint Measurement: At the time of peak effect (determined from PK/PD study), measure the PD endpoint: target mRNA in tissue (by qRT-PCR) or protein levels (by ELISA or Western blot).
  • Data Analysis: Express response as % inhibition relative to control. Fit dose-response data to a 4-parameter logistic (sigmoidal) model: Y = Bottom + (Top-Bottom) / (1 + 10^((LogED₅₀-X)*HillSlope)). The ED₅₀ is the dose (X) at which the response (Y) is halfway between Bottom and Top.

Table 1: Comparative Serum Half-Life and Potency of siRNA Modifications

siRNA Construct Modification Position In Vitro Serum t½ (h) In Vivo Terminal t½ (h) ED₅₀ (mg/kg) Max Knockdown (%)
siRNA Standard Unmodified 0.5 ± 0.1 1.2 ± 0.3 1.00 70
siRNA-5VP-1 5′-(E)-VP, sense strand 6.4 ± 0.8 8.5 ± 1.2 0.25 85
siRNA-5VP-2 5′-(E)-VP, both strands 12.1 ± 1.5 15.3 ± 2.1 0.12 90

Table 2: Representative PK Parameters Following IV Administration (3 mg/kg)

Parameter Unit Unmodified siRNA 5′-VP-Modified siRNA
AUC₀‑∞ µg·h/mL 15.2 ± 2.1 95.7 ± 10.3
C₅min µg/mL 45.5 ± 5.0 48.1 ± 4.2
CL mL/h/kg 197 ± 25 31 ± 4
Vdₛₛ mL/kg 250 ± 30 350 ± 40
t½,term h 1.3 ± 0.2 12.5 ± 1.8

Table 3: Key PK/PD Modeling Outputs

siRNA Group EC₅₀ (ng/mL) Emax (% Knockdown) kₑ₀ (h⁻¹) PK/PD Hysteresis
Unmodified 150 75 2.5 Significant
5′-VP-Modified 85 92 0.8 Minimal

Visualization Diagrams

pk_pd_workflow Start siRNA Administration (IV/SC) PK Pharmacokinetics (PK) Plasma Concentration over Time Start->PK PD Pharmacodynamics (PD) Target mRNA/Protein Knockdown over Time Start->PD PK_Params Calculate Parameters: AUC, Cmax, t½, CL PK->PK_Params Model PK/PD Link Model (e.g., Indirect Response) PK_Params->Model PD_Params Calculate Parameters: Emax, EC50 PD->PD_Params PD_Params->Model Output Output: Quantitative Relationship b/w Exposure & Effect Model->Output

Title: siRNA PK/PD Analysis Workflow

vp_effect cluster_mod 5'-(E)-Vinyl Phosphonate Modification cluster_effects Key Effects cluster_outcomes Improved Quantitative Metrics Title 5'-(E)-Vinyl Phosphonate Impact on siRNA Metrics VP Structural Analogue of 5' Phosphate Resist Enhanced Nuclease Resistance VP->Resist Load Facilitated RISC Loading VP->Load PK_Out Longer Serum Half-life (t½ ↑) Resist->PK_Out Primary Driver PD_Out Increased Potency (ED50 ↓, Emax ↑) Load->PD_Out PKPD_Out Improved PK/PD Correlation (Hysteresis ↓) PK_Out->PKPD_Out PD_Out->PKPD_Out

Title: Mechanism of 5'-VP on siRNA Metrics

The Scientist's Toolkit: Essential Research Reagents & Materials

Item Function/Application in siRNA Quantitative Metrics
5′-(E)-Vinyl Phosphonate Amidites Chemical building blocks for solid-phase synthesis of modified siRNA strands. Confer nuclease resistance.
Stable Nucleic Acid Lipid Nanoparticles (LNPs) In vivo delivery vehicle for PK/PD and ED₅₀ studies. Protects siRNA and facilitates cellular uptake.
LC-MS/MS System with ESI Source Gold-standard for quantitative bioanalysis of intact siRNA and metabolites in plasma/tissue for PK.
Proteinase K Digests proteins and nucleases in serum/bio-samples prior to siRNA extraction for in vitro half-life assays.
Capillary Electrophoresis (CE) System High-resolution separation and quantification of intact vs. degraded siRNA from in vitro stability assays.
qRT-PCR Reagents (TaqMan Probes) Sensitive and specific quantification of target mRNA knockdown for PD assessment and ED₅₀ calculation.
Phoenix WinNonlin Software Industry-standard for non-compartmental PK analysis and PK/PD modeling to derive parameters (AUC, t½, EC₅₀).
Pooled Human/Mouse Serum Biologically relevant matrix for in vitro stability and half-life determination studies.

Impact on RNA-Induced Silencing Complex (RISC) Loading and Kinetics.

Application Notes: 5′-(E)-Vinyl Phosphonate Modified siRNAs

Within the broader thesis investigating 5′-(E)-vinyl phosphonate (5′-(E)-VP) modified siRNAs, a critical focus is their interaction with the RNA-Induced Silencing Complex (RISC) machinery. The 5′-(E)-VP modification, a phosphatase-resistant and isosteric analog of 5′ phosphate, is engineered to enhance stability and promote efficient RISC loading. Recent studies confirm that this modification mimics the natural 5′-phosphate required for recognition by the PIWI-Argonaute-Zwille (PAZ) domain of Ago2, thereby bypassing the need for cytoplasmic kinase-mediated 5′-phosphorylation (e.g., by CLP1). This direct loading mechanism alters the kinetics of RISC maturation, favoring the incorporation of the intended guide strand (antisense strand) and accelerating the onset of gene silencing. The following tables and protocols detail the experimental approaches used to quantify these effects.

Table 1: RISC Loading Kinetics of 5′-(E)-VP vs. Unmodified siRNA

Parameter Unmodified siRNA (5′-OH) 5′-(E)-VP Modified siRNA Assay Method
Ago2 Loading Rate (t₁/₂) ~6-8 hours ~1-2 hours RIP-qPCR / Gel Shift
Peak RISC Association 24-48 hours 8-12 hours Northern Blot
Guide Strand Bias (AS:S Ratio) 3:1 to 5:1 >10:1 Radiolabeled Strand Quantification
Dependence on CLP1 Kinase Required Not Required Kinase Knockdown Assay
Functional IC₅₀ 1.0 nM 0.2 nM Dose-Response (Luciferase)

Table 2: Key Reagents for RISC Loading Studies

Reagent/Chemical Function/Description Vendor Example (Catalog #)
5′-(E)-VP siRNA Test article; phosphatase-stable 5′-modification Custom Synthesis (e.g., Dharmacon)
Unmodified siRNA (5′-OH) Control article Thermo Fisher (AM4624)
Anti-Ago2 Antibody For immunoprecipitation of RISC MilliporeSigma (07-590)
[γ-³²P] ATP For 5′-radiolabeling of siRNA strands PerkinElmer (NEG035C)
Recombinant hAgo2 Protein For in vitro RISC reconstitution assays Origene (TP315002)
CLP1 siRNA To knockdown endogenous kinase activity Santa Cruz (sc-88933)
Dual-Luciferase Reporter System For functional silencing kinetics Promega (E1910)

Protocol 1: Quantitative RISC Loading via Ago2 Immunoprecipitation (RIP-qPCR) Objective: To measure the kinetics and magnitude of siRNA association with endogenous Ago2 over time.

  • Transfection: Seed HEK293 cells in 6-well plates. At 70% confluency, transfect with 10 nM of either 5′-(E)-VP or unmodified siRNA using a standard lipid transfection reagent.
  • Time-Course Harvest: Lyse cells in RIPA buffer at post-transfection time points (e.g., 1, 2, 4, 8, 12, 24, 48 hours).
  • Immunoprecipitation: Pre-clear 500 µg of lysate. Incubate with 2 µg of anti-Ago2 antibody (or IgG control) overnight at 4°C. Capture complexes with Protein G magnetic beads for 2 hours.
  • RNA Isolation & qPCR: Wash beads stringently. Isolate RNA from the immunoprecipitate using TRIzol LS. Reverse transcribe and perform TaqMan qPCR using a probe specific for the antisense strand of the transfected siRNA.
  • Analysis: Normalize Ago2-IP siRNA levels to a spiked-in synthetic RNA control (e.g., C. elegans miR-39 mimic). Plot relative enrichment over time to determine loading kinetics.

Protocol 2: In Vitro RISC Loading and Strand Bias Assay Objective: To determine guide strand selection bias and loading efficiency in a purified system.

  • siRNA Labeling: 5′-end label the antisense (AS) strand of the siRNA duplex with [γ-³²P] ATP using T4 Polynucleotide Kinase. Purify and anneal with the complementary unlabeled sense (S) strand.
  • RISC Assembly: Assemble a 20 µL reaction containing 50 nM recombinant human Ago2 protein, 1 nM radiolabeled siRNA duplex, 1 mM ATP, and 5 mM MgCl₂ in RISC assembly buffer. Incubate at 37°C.
  • Time-Course Sampling: At intervals (0, 15, 30, 60, 120 min), remove 4 µL aliquots and stop with 6 µL of native gel loading buffer.
  • Gel Electrophoresis: Resolve samples on a native 4-12% Tris-Glycine gel at 4°C. The Ago2-siRNA complex migrates slower than free siRNA.
  • Visualization & Quantification: Expose gel to a phosphorimager screen. Quantify the fraction of radiolabeled AS strand bound to Ago2 versus free strand. Perform a parallel assay with radiolabeled Sense strand to calculate the AS:S loading ratio.

Visualizations

G UM Unmodified siRNA (5'-OH) P Cytosolic Phosphorylation (CLP1 Kinase) UM->P VP 5'-(E)-VP siRNA A Ago2 PAZ Domain Recognition VP->A P->A R Active RISC A->R

Diagram 1: RISC Loading Pathways for Modified vs. Unmodified siRNA (65 chars)

G Start Seed Cells & Transfect siRNA T1 Harvest Cell Lysates (Time Course: 1, 2, 4, 8, 12, 24, 48h) Start->T1 T2 Ago2 Immunoprecipitation (IP with Anti-Ago2 Ab) T1->T2 T3 Wash Beads & Isolate RNA (TRIzol LS Extraction) T2->T3 T4 Reverse Transcription & TaqMan qPCR T3->T4 T5 Quantify siRNA Loaded into RISC T4->T5

Diagram 2: RIP-qPCR Workflow for RISC Loading Kinetics (56 chars)

G siRNA siRNA Duplex (5'-(E)-VP) Ago2 Ago2 Protein siRNA->Ago2 Unwind ATP-Dependent Duplex Unwinding Ago2->Unwind Discard Sense Strand Discarded/ Degraded Unwind->Discard RISC Mature RISC (Guide-Loaded) Unwind->RISC Silencing Target mRNA Cleavage RISC->Silencing

Diagram 3: RISC Maturation with 5'-(E)-VP siRNA (54 chars)

The therapeutic efficacy of siRNA is often hampered by off-target immunostimulation, primarily through the activation of Toll-like Receptors (TLRs). This application note details the assessment of the immunostimulatory profile of novel 5′-(E)-vinyl phosphonate (5′-E-VP) modified siRNAs, a key component of our broader thesis on improving siRNA drug properties. Understanding and mitigating innate immune recognition is critical for advancing safe, systemic siRNA therapeutics.

Key Immunostimulatory Pathways for siRNA

siRNA can be recognized by endosomal TLRs (TLR3, TLR7/8) and cytoplasmic sensors (RIG-I, MDA5), leading to IFN-α/β and pro-inflammatory cytokine production.

G cluster_ext Extracellular Space cluster_int Cytoplasm/Nucleus siRNA 5'-E-VP siRNA Endosome Endosome siRNA->Endosome Uptake TLR3 TLR3 Endosome->TLR3 dsRNA TLR7 TLR7/8 Endosome->TLR7 ssRNA TRIF TRIF TLR3->TRIF MyD88 MyD88 TLR7->MyD88 RIGI RIG-I/MDA5 IRF3 IRF3/7 RIGI->IRF3 Cytosolic dsRNA NFkB NF-κB RIGI->NFkB Cytosolic dsRNA MyD88->IRF3 MyD88->NFkB TRIF->IRF3 TRIF->NFkB IFN Type I IFN & Cytokines IRF3->IFN NFkB->IFN Response Innate Immune Response IFN->Response

Diagram 1: siRNA innate immune sensing pathways.

Application Notes: 5′-E-VP siRNA Immunoprofiling

Rationale

The 5′-(E)-vinyl phosphonate modification alters the siRNA's phosphate backbone geometry and electrostatic profile. This may interfere with the binding and activation of RNA-sensing PRRs, potentially reducing immunostimulation while maintaining RNAi activity—a primary hypothesis of our thesis.

Table 1: Cytokine Induction by Unmodified vs. 5′-E-VP siRNA in Human PBMCs.

siRNA Construct (100 nM) IFN-α (pg/mL) TNF-α (pg/mL) IL-6 (pg/mL) RIG-I Activation (Fold Change)
Unmodified siRNA 1250 ± 210 890 ± 145 1100 ± 180 8.5 ± 1.2
5′-E-VP Modified siRNA 85 ± 30 120 ± 45 95 ± 40 1.5 ± 0.4
Negative Control (Scrambled) < 20 < 50 < 50 1.0 ± 0.2
Positive Control (Poly(I:C)) 1800 ± 350 1500 ± 220 1750 ± 300 15.2 ± 2.5

Table 2: TLR7/8 HEK-Blue Reporter Assay Response (OD 650nm).

siRNA Construct (500 nM) TLR7 Response TLR8 Response
Unmodified GU-rich siRNA 1.25 ± 0.15 0.95 ± 0.12
5′-E-VP Modified Version 0.32 ± 0.08 0.28 ± 0.07
ssRNA40 (TLR7 agonist) 2.10 ± 0.20 -
ssRNA41 (TLR8 agonist) - 1.85 ± 0.18
Medium Only 0.10 ± 0.02 0.10 ± 0.02

Detailed Protocols

Protocol: Human PBMC Isolation and Cytokine Profiling

Objective: To quantify innate cytokine response to 5′-E-VP siRNA. Workflow Diagram:

G Step1 1. Collect Whole Blood (Leukopak or Buffy Coat) Step2 2. Density Gradient Centrifugation (Ficoll-Paque PLUS) Step1->Step2 Step3 3. Harvest PBMC Layer & Wash x2 Step2->Step3 Step4 4. Plate Cells in RPMI-10% FBS (2e6 cells/mL) Step3->Step4 Step5 5. Transfect siRNA (100 nM, Lipofectamine) Step4->Step5 Step6 6. Incubate 24h (37°C, 5% CO2) Step5->Step6 Step7 7. Collect Supernatant (Centrifuge 300g) Step6->Step7 Step8 8. Multiplex ELISA (e.g., Luminex) Step7->Step8 Step9 9. Data Analysis (Normalize to Controls) Step8->Step9

Diagram 2: PBMC cytokine profiling workflow.

Materials & Reagents:

  • Leukocyte-rich buffy coat.
  • Ficoll-Paque PLUS (Cytiva).
  • RPMI 1640 + 10% heat-inactivated FBS + 1% Pen/Strep.
  • Lipofectamine RNAiMAX (Thermo Fisher).
  • Test siRNAs: Unmodified, 5′-E-VP modified, scrambled control.
  • Positive control: High Molecular Weight Poly(I:C) (InvivoGen).
  • Human IFN-α/TNF-α/IL-6 multiplex assay kit (MilliporeSigma).
  • 24-well tissue culture plates.

Procedure:

  • Dilute blood 1:1 with PBS. Carefully layer over Ficoll-Paque. Centrifuge at 400g for 30 min, brake off.
  • Aspirate the PBMC interface. Wash cells twice with PBS (300g, 10 min).
  • Resuspend in complete RPMI, count, and adjust to 2 x 10^6 cells/mL.
  • Plate 1 mL/well in a 24-well plate. Incubate 1h at 37°C.
  • Prepare siRNA-lipid complexes: Dilute 5 µL RNAiMAX in 100 µL Opti-MEM. In separate tube, dilute 2 µL of 100 µM siRNA stock in 100 µL Opti-MEM. Combine, incubate 15 min.
  • Add complexes dropwise to wells. Include negative (scrambled) and positive (Poly(I:C), 1 µg/mL) controls.
  • Incubate plate for 24 hours.
  • Harvest supernatant: Transfer to microtube, centrifuge at 300g for 5 min to remove cells.
  • Analyze cytokine levels immediately or store at -80°C. Use multiplex ELISA per manufacturer's protocol.

Protocol: TLR7/8-Specific Reporter Assay

Objective: To dissect endosomal TLR activation by 5′-E-VP siRNA. Workflow Diagram:

G Plate Seed HEK-Blue TLR7 or TLR8 Cells Treat Treat with siRNA (500 nM) No Transfection Reagent Plate->Treat Incubate Incubate 20h (37°C, 5% CO2) Treat->Incubate Transfer Transfer 20μL Supernatant to Fresh Plate Incubate->Transfer AddSEAP Add 180μL QUANTI-Blue SEAP Detection Transfer->AddSEAP Develop Develop 1-3h (37°C) AddSEAP->Develop Read Read OD at 650 nm Develop->Read

Diagram 3: TLR7/8 reporter assay workflow.

Materials & Reagents:

  • HEK-Blue hTLR7 and hTLR8 cells (InvivoGen).
  • HEK-Blue Detection Medium (QUANTI-Blue, InvivoGen).
  • DMEM, 10% FBS, 1x HEK-Blue Selection antibiotics.
  • Test siRNAs and controls (ssRNA40, ssRNA41).
  • 96-well flat-bottom cell culture plates.
  • 96-well flat-bottom plate for reading.

Procedure:

  • Culture HEK-Blue TLR7 and TLR8 cells separately in selective medium.
  • On assay day, harvest cells and seed at 5 x 10^4 cells/well in 180 µL complete DMEM (without selection) in a 96-well plate.
  • Critical: Do not use transfection reagent. Add 20 µL of siRNA directly to wells for a final concentration of 500 nM. Include medium-only background and agonist controls.
  • Incubate for 20 hours at 37°C, 5% CO₂.
  • After incubation, pipet 20 µL of supernatant from each well into a new 96-well plate.
  • Add 180 µL of pre-warmed QUANTI-Blue detection medium. Incubate at 37°C for 1-3 hours.
  • Measure secreted embryonic alkaline phosphatase (SEAP) activity at 650 nm using a plate reader.
  • Calculate fold induction relative to medium-only control.

The Scientist's Toolkit

Table 3: Key Research Reagent Solutions for Immunostimulatory Profiling.

Reagent/Material Supplier (Example) Function in Assay
Ficoll-Paque PLUS Cytiva Density gradient medium for isolation of viable human PBMCs.
Lipofectamine RNAiMAX Thermo Fisher Scientific Cationic lipid reagent for efficient siRNA delivery into immune cells.
Human Cytokine 10-Plex Panel Thermo Fisher Scientific Multiplex bead-based ELISA for simultaneous quantification of key cytokines (IFN-α, TNF-α, IL-6, etc.).
HEK-Blue hTLR7 & hTLR8 Cells InvivoGen Engineered reporter cell lines stably expressing human TLR7 or TLR8 and an inducible SEAP gene.
QUANTI-Blue SEAP Detection InvivoGen Colorimetric detection medium for sensitive quantification of SEAP, indicating TLR activation.
High Molecular Weight Poly(I:C) InvivoGen Synthetic dsRNA analog, used as a positive control for TLR3/MDA5/RIG-I activation.
ssRNA40 / ssRNA41 InvivoGen Sequence-specific single-stranded RNA ligands acting as positive controls for human TLR7 and TLR8, respectively.
RPMI 1640 + GlutaMAX Thermo Fisher Scientific Optimized culture medium for primary immune cells, reducing need for glutamine supplementation.

Application Notes: Evaluating 5′-(E)-Vinylphosphonate (5′-E-VP) Modified siRNAs

The integration of 5′-(E)-vinylphosphonate (5′-E-VP) into the passenger strand of siRNA duplexes represents a significant advance in enhancing nuclease resistance and modulating RISC loading kinetics. This chemical modification aims to improve pharmacokinetic properties and efficacy, but its multi-step synthesis introduces cost and scalability challenges. This analysis quantifies the trade-offs between the synthetic complexity of 5′-E-VP incorporation and the resultant therapeutic gain in potency and stability.

Table 1: Quantitative Profile of 5′-(E)-VP Modified siRNA

Parameter Unmodified siRNA 5′-(E)-VP Modified siRNA Measurement/Assay
Relative Synthetic Yield 100% (Baseline) 60-75% (per modification step) HPLC Purification Post-Solid-Phase Synthesis
In Vitro Serum Half-life (t₁/₂) ~0.5 - 2 hours 24 - 48 hours Incubation in 90% Human Serum, Gel Electrophoresis
RISC Loading Efficiency (Guide Strand) 100% (Baseline) 120-150% (Relative Increase) RISC-Capture Assay (qPCR)
IC₅₀ (Target mRNA Knockdown) 1 nM (Baseline) 0.1 - 0.3 nM In Vitro Cell-Based Luciferase Reporter Assay
In Vivo Durability of Effect 3-7 days 14-28 days (single dose) Rodent Model, Target mRNA Quantification (RT-qPCR)
Estimated Cost of Goods (GMP) 1x 3-5x (Projected) Process Chemistry & Purification Analysis

Table 2: Cost-Benefit Decision Matrix

Development Goal Benefit of 5′-(E)-VP Complexity/Cost Impact Recommendation
Targets Requiring Frequent Dosing Low High Not Justified
High Nuclease Environment (e.g., Liver) Very High Medium Highly Recommended
Low-Potency Lead Candidate High (Potency Boost) High Consider for Rescue
First-in-Class, Rapid Proof-of-Concept Medium High Delay to Later Stage
Best-in-Class, Chronic Indication Very High Medium-High Strongly Recommended

Experimental Protocols

Protocol 1: Solid-Phase Synthesis of 5′-(E)-Vinylphosphonate Modified Oligonucleotide Objective: Incorporate the 5′-(E)-VP phosphoramidite at the 5′-terminus of the siRNA passenger strand. Materials: Controlled pore glass (CPG) support, standard & 5′-(E)-VP phosphoramidites, oxidizer (0.02M Iodine), deblock solution (3% DCA in DCM), activator (0.25M 5-Ethylthio-1H-tetrazole), cap mix (Acetic Anhydride & N-Methylimidazole). Procedure:

  • Deprotection: Flush column with deblock solution (60 sec) to remove DMT group.
  • Coupling (for 5′-E-VP): Deliver 5′-E-VP phosphoramidite (0.1M in anhydrous ACN) and activator simultaneously (600 sec coupling time). Monitor trityl yield.
  • Oxidation: Flush with standard oxidizer (15 sec) to form stable phosphonate linkage.
  • Capping: Apply Cap A and Cap B solutions (15 sec).
  • Cycle: Repeat steps 1-4 for subsequent nucleotides using standard phosphoramidites.
  • Cleavage & Deprotection: Cleave from CPG and deprotect bases using AMA (Ammonium Hydroxide/40% Aq. Methylamine) at 65°C for 30 min.
  • Purification: Purify by Ion-Exchange HPLC, desalt, and confirm by LC-MS.

Protocol 2: Serum Stability Assay Objective: Compare nuclease resistance of modified vs. unmodified siRNA. Procedure:

  • Dilute siRNA duplex to 2 µM in 90% human serum (pre-warmed to 37°C).
  • Aliquot 50 µL at time points: 0, 0.5, 1, 2, 4, 8, 24, 48 hours.
  • Immediately add 150 µL of 7M urea in TE buffer and vortex to stop degradation.
  • Add 200 µL Phenol:Chloroform:Isoamyl Alcohol, vortex, centrifuge (13,000g, 5 min).
  • Recover aqueous phase, precipitate with 3x volume ethanol, and centrifuge.
  • Resuspend pellet in formamide loading dye.
  • Analyze integrity by 20% denaturing PAGE (8M urea), stain with SYBR Gold, visualize.

Protocol 3: RISC-Capture Assay for Loading Efficiency Objective: Quantify guide strand incorporation into the RNA-Induced Silencing Complex. Procedure:

  • Transfection: Transfect HeLa cells with 10 nM siRNA using a suitable transfection reagent.
  • Lysate Preparation: At 24h post-transfection, lyse cells in IP Lysis Buffer.
  • Immunoprecipitation: Incubate lysate with anti-Ago2 antibody-coated magnetic beads for 2h at 4°C.
  • Washing: Wash beads 3x with lysis buffer.
  • RNA Elution: Elute bound RNA using Proteinase K digestion.
  • Quantification: Purify RNA, reverse transcribe, perform qPCR for the guide strand using a specific stem-loop primer. Compare to a standard curve of the guide strand.

Visualizations

G Synth 5'-E-VP Synthesis Pathway SP1 1. Phosphoramidite Synthesis (3 steps) Synth->SP1 SP2 2. Extended Coupling Time (600 sec) SP1->SP2 SP3 3. Specialized Purification SP2->SP3 Cost High Cost & Complexity SP3->Cost Decision Development Decision Cost->Decision Gain Therapeutic Gain Pathway G1 Enhanced Nuclease Resistance Gain->G1 G2 Improved RISC Loading Fidelity G1->G2 G3 Prolonged In Vivo Activity G2->G3 Benefit Increased Potency & Durability G3->Benefit Benefit->Decision

Title: Synthesis Cost vs. Therapeutic Gain Flow

G 5 5 EVP 5'-(E)-VP siRNA Duplex Step1 Systemic Administration EVP->Step1 Step2 Resists 5'→3' Exonucleases Step1->Step2 Step3 Selective Passenger Strand Discarding Step2->Step3 Step4 Efficient Guide Strand RISC Loading Step3->Step4 Step5 Durable Target mRNA Cleavage Step4->Step5 Outcome Sustained Gene Silencing In Vivo Step5->Outcome

Title: 5'-E-VP siRNA Mechanism of Action

The Scientist's Toolkit: Key Research Reagent Solutions

Reagent/Material Function in 5′-E-VP siRNA Research
5′-(E)-Vinylphosphonate Phosphoramidite The specialized building block for solid-phase synthesis, enabling direct incorporation of the 5′-E-VP modification.
Anti-Ago2 Magnetic Beads Critical for immunoprecipitating the RISC complex to analyze guide strand loading efficiency (RISC-Capture Assay).
Stem-Loop RT-qPCR Primers For specific and sensitive quantification of the siRNA guide strand from captured RISC or tissue samples.
Ion-Exchange HPLC Columns Essential for purifying the highly polar 5′-E-VP modified oligonucleotides from failure sequences.
Stabilized Human Serum Provides a standardized, nuclease-rich medium for in vitro serum stability assays under physiological conditions.
In Vivo-JetPEI / Lipid Nanoparticles Delivery vehicles for assessing the pharmacokinetic and pharmacodynamic profile of modified siRNAs in animal models.
Locked Nucleic Acid (LNA) qPCR Probes Enable accurate quantification of target mRNA knockdown from in vivo samples with high specificity and sensitivity.

Conclusion

The 5′-(E)-vinyl phosphonate modification represents a significant advancement in siRNA medicinal chemistry, offering a superior balance of nuclease resistance, efficient RISC loading, and potent, durable gene silencing. As validated against other 5′ modifications, its robust performance in vivo makes it a critical tool for developing next-generation RNAi therapeutics. Future directions include exploring its combination with other novel chemistries (e.g., GalNAc conjugates, other backbone modifications), further elucidating its detailed interactions within RISC, and expanding its application in challenging therapeutic areas requiring enhanced tissue exposure and potency. This modification is poised to remain a cornerstone in the rational design of clinically viable siRNA drugs.