Login / Register Order My Items
mob-logo
Contact Us
800.227.0627
Contact Us Quote Order Login / Register

siRNA Modification Strategies for Potency, Stability & Delivery

Custom RNAi oligos built around guide/passenger design, 2′ modification patterning, terminal protection, delivery conjugates, purification, LC-MS QC and scalable production.

45+ Years of Expertise ISO 9001:2015 / ISO 13485:2016 Bench to Kilo Scale Confidential & IP-Protected U.S.A Facilities - Texas
View Modification Toolkit Request RNAi Quote
Talk to a chemist Request a quote
Overview Cell Journey RNAi Formats Modification Toolkit Design Guide Scale-Up Related Services FAQ Contact
Overview

Why siRNA Modifications Matter

siRNA performance depends on more than sequence selection. The final duplex must support guide-strand loading, preserve RISC activity, avoid unwanted passenger-strand activity, resist nucleases, reduce immune stimulation and reach the right intracellular compartment.

Bio-Synthesis supports RNAi programs with strand-specific modification placement, 2′-OMe and 2′-F patterning, terminal phosphorothioate protection, 5′ phosphate strategies, GalNAc, lipid, peptide and spacer conjugates, plus purification, LC-MS identity confirmation and custom release documentation.

siRNA • miRNA Mimic • anti-miR 2′-OMe • 2′-F • PS • LNA/BNA GalNAc • Lipid • CPP HPLC/UPLC • LC-MS • CE/PAGE Bench to Kilo Scale
siRNA Modification Map

Protect the duplex without losing RISC activity. Modification placement should consider guide strand, passenger strand, seed region, 3′ overhangs, 5′ phosphate and delivery-conjugate position.

Passenger / Sense Strand
PS2′FGal3′
Guide / Antisense Strand
5′PSeed2′OMePS
Guide Loading

Preserve 5′ guide requirements and Ago2 compatibility.

Seed Control

Use careful 2′-OMe placement to manage off-target activity.

Stability

Use 2′-F, 2′-OMe and terminal PS to resist nucleases.

Delivery

Place GalNAc, lipids or CPPs to support uptake without blocking activity.

Mechanism-Driven Workflow

How Modified siRNA Becomes an Active Gene-Silencing Agent

This workflow is focused on the biology of RNAi rather than a generic manufacturing timeline.

Outside Cell

🧬

Design

Guide/passenger optimization, target selection and strand-bias engineering.

🛡️

Stabilize

2′-OMe, 2′-F, PS and selected affinity chemistries improve resistance.

🚚

Deliver

GalNAc, lipids, CPPs and targeting ligands support cellular uptake.

📦

Endosomal Escape

siRNA must escape vesicles before entering the RNAi pathway.

⚙️

RISC Loading

The guide strand is incorporated into Ago2 and the RISC complex.

🎯

Gene Silencing

Target mRNA is recognized and cleaved, reducing protein expression.

Inside Cell

RNAi Formats

RNAi Formats and Advanced Architectures

Bio-Synthesis can support standard and advanced RNAi formats, from duplex siRNA to anti-miR and dual-guide triplex concepts.

RISC

Duplex siRNA

Guide/passenger duplexes for knockdown, screening, assay development and development research.

  • 19–21 bp duplexes
  • 2-nt 3′ overhangs
  • 5′ guide strategy
Dicer

DsiRNA

Dicer-substrate style siRNA formats for RNAi processing and potency exploration.

  • Longer duplex formats
  • Dicer processing
  • Strand bias review
Dual Guide

Tri-siRNA

Dual-guide triplex siRNA with one passenger strand and two guide strands for multi-target or combinatorial silencing research.

  • 1 passenger + 2 guide strands
  • multi-target designs
  • custom QC required
miRNA

miRNA Mimics / anti-miR

Mimics restore miRNA function; anti-miR and antagomir designs suppress endogenous miRNAs.

  • LNA/PS anti-miR
  • miRNA mimic duplex
  • antagomir formats
Delivery

Conjugated siRNA

GalNAc, lipid, peptide and spacer-modified siRNA formats for uptake and biodistribution tuning.

  • GalNAc liver targeting
  • cholesterol / fatty acids
  • CPP and peptide options
Modification Toolkit

RNAi Modification Toolkit: What Bio-Synthesis Offers

The page is centered on modified siRNA synthesis, stereopure siRNA chemistry, GalNAc siRNA, lipid-modified RNAi conjugates, phosphorothioate siRNA, therapeutic RNAi, DsiRNA, anti-miR and RNAi delivery strategies.

500+ RNAi Modification Combinations Therapeutic RNAi GalNAc Lipid Conjugates Stereopure Chemistry XNA Options Bench → Kilo Scale
Modification Description siRNA Use Code
Phosphorothioate (PS) Sulfur substitution in the phosphate backbone Terminal or partial PS for nuclease resistance and pharmacokinetic tuning [PS]
Stereodefined PS (Rp / Sp) Controlled phosphorothioate stereochemistry Controls nuclease stability, protein binding, potency and PK behavior [Rp-PS]/[Sp-PS]
Stereopure Oligonucleotides Defined backbone stereochemistry pattern Advanced RNAi and therapeutic-development chemistry optimization [Stereo-PS]
Phosphorodithioate (PS₂) Both non-bridging oxygens replaced by sulfur High nuclease resistance and hydrophobicity tuning [PS2]
Methylphosphonate (PM) Non-ionic methyl substitution Neutral backbone and uptake studies [PM]
Phosphoramidate (PN) P–N linkage Stability and altered enzyme interactions [PN]
Boranophosphate (BPh) Boron substitution Metabolic stability and nuclease resistance [BPh]
PMO Morpholino phosphorodiamidate scaffold Maximum nuclease resistance and steric-blocking RNA-targeting research [PMO]
PNA Peptide nucleic acid backbone High-affinity, enzyme-resistant RNA targeting and clamp-style designs [PNA]
Custom Backbone Systems Stereopure PS, PS₂, PMO, PNA and proprietary backbone chemistries Contact Bio-Synthesis for specialized RNAi modification requirements Contact Us

Backbone note: Duplex siRNA typically uses selective or terminal PS placement. Stereopure and stereodefined backbone chemistries can be evaluated when protein binding, PK, nuclease stability or therapeutic RNAi performance requires tighter control.

Modification Description siRNA Use Code
2′-OMe 2′-O-methyl ribose Nuclease resistance, immune reduction and seed management [2OMe]
2′-F 2′-fluoro ribose Stability and potency support in RNAi duplexes [2F]
LNA / BNA Locked or bridged ribose Affinity and potency; use sparingly in RISC-active designs [LNA]/[BNA]
cEt Constrained ethyl bridge Affinity and safety tuning [cEt]
MOE 2′-O-methoxyethyl Affinity and nuclease resistance; evaluate RISC compatibility [MOE]
2′-Amino Primary amine at 2′ Affinity increase and conjugation handle [2NH2]
2′-O-Propargyl Alkyne at 2′-O Click chemistry handle for RNAi conjugation workflows [2O-PRG]
2′-ONMA 2′-O-(N-methylacetamide) Affinity and nuclease resistance [2ONMA]
UNA Unlocked ribose Flexibility and structure-function tuning [UNA]
Ara-FANA Arabinose 2′-fluoro-arabino Stability and antisense exploration [Ara-FANA]
Additional FANA Variants Specialty fluoro-arabino nucleic acid analogs Project-specific stability and binding studies [FANA-var]
ENA Ethylene-bridged nucleic acid Affinity increase; experimental RNAi design support [ENA]
TNA Threose nucleic acid Minimal backbone and orthogonal pairing research [TNA]
GNA Glycol nucleic acid Simplified backbone and base-pairing studies [GNA]
SNA Serinol nucleic acid Biostability and synthetic RNAi research [SNA]
HNA Hexitol nucleic acid Specialty XNA chemistry for nuclease resistance and structure studies [HNA]
CeNA Cyclohexenyl nucleic acid Backbone rigidity and duplex-behavior tuning [CeNA]
tcDNA Tricyclo-DNA sugar framework High-affinity specialty RNA-targeting research [tcDNA]
Custom Sugar & XNA Chemistries Additional proprietary sugar analogs and specialty XNA systems Contact Bio-Synthesis for project-specific RNAi chemistry requirements Contact Us

Patterning note: 2′-OMe and 2′-F are common modified siRNA synthesis options. Bio-Synthesis also supports custom sugar analogs, XNA technologies and emerging RNAi chemistries when a standard pattern is not enough.

Modification Description siRNA Use Code
5′-Phosphate Guide 5′ phosphate or pro-phosphate Ago2 loading and potency support [5P]
3′ End Caps 3′ inverted dT or cap Exonuclease protection [3CAP]
3′ dTdT / UU Two-base 3′ overhangs Dicer-style duplex design and stability [OVR-2NT]
Amino C6 / C12 Primary amine linker handles Labels, surfaces, ligands and downstream coupling [NH2-C6]/[NH2-C12]
Thiol C6 / C12 Reactive thiol handles Maleimide conjugation, surfaces and custom bioconjugation [SH-C6]/[SH-C12]
Azide Bioorthogonal click handle Click chemistry and ligand attachment [N3]
DBCO Strain-promoted click handle Copper-free click conjugation [DBCO]
Alkyne Terminal alkyne handle CuAAC click chemistry [Alkyne]
Biotin Streptavidin-binding tag Pulldown, capture, immobilization and detection [Biotin]
Desthiobiotin Reversible biotin analog Milder elution capture workflows [DesthioBio]
Fluorescent Labels Fluorophore-modified siRNA Uptake, localization, tracking and assay readout [FAM]/[Cy]/[Dye]
PEG / TEG Linkers Flexible hydrophilic spacer systems Conjugate orientation and steric control [PEG]/[TEG]
C3 / C6 / C12 Spacers Alkyl spacer arms Distance control for conjugates, labels and surfaces [C3]/[C6]/[C12]
Cleavable Linkers Triggerable release linkers Payload release and delivery optimization [CL]
Enzyme-Cleavable Linkers Protease or enzyme-sensitive linkers Intracellular release strategies [Enz-CL]
Photocleavable Linkers Light-triggered release spacers Controlled release and photoactivation studies [PC-Linker]
Custom Linkers & Handles Cleavable, photocleavable, enzyme-sensitive and project-specific attachment chemistries Contact Bio-Synthesis for specialized linker requirements Contact Us

Terminal note: Terminal design affects Ago2 loading, strand bias, exonuclease resistance and conjugation behavior. Delivery handles are often placed on passenger strand termini, but final placement should be validated.

Conjugate Description Delivery Use Code
GalNAc / GalNAc3 / GalNAc4 ASGPR ligands Liver-targeted siRNA delivery [GalNAc]
Cholesterol / PC-Chol Sterol conjugates Uptake and membrane association [Chol]
Tocopherol Vitamin E derivative Hydrophobic delivery and formulation research [Toco]
DHA / EPA / Fatty Acids Hydrophobic delivery modifiers Biodistribution and extrahepatic delivery research [DHA]/[EPA]/[FA]
Folate Folate receptor ligand Receptor-mediated targeting [Folate]
Mannose Mannose receptor ligand Immune-cell and macrophage-targeting research [Mannose]
CPPs / Targeting Peptides TAT, R9, Penetratin and related peptides Cellular uptake and tissue delivery [CPP]
RGD / iRGD Integrin-binding peptides Tumor and tissue-targeting research [RGD]
Angiopep-2 LRP1-binding peptide CNS delivery research [Ang2]
RVG29 Rabies virus glycoprotein-derived peptide Neuronal targeting research [RVG29]
Aptamer-siRNA Chimeras Aptamer-guided RNAi construct Cell-specific targeting and RNAi delivery [Apt-siRNA]
Antibody-Oligo Conjugates Antibody or binding protein conjugated to siRNA/oligo Targeted delivery and cell-specific uptake [AOC]
PEG / TEG Spacers Flexible hydrophilic linkers Conjugate orientation and reduced steric hindrance [TEG]/[PEG]
Cleavable Linkers Disulfide, Val-Cit-PAB, hydrazone, photocleavable Cytosolic, enzyme, pH or light-triggered release [SS]/[Val-Cit]
Custom Targeting Ligands Antibodies, aptamers, peptides, receptor ligands and specialized delivery systems Contact Bio-Synthesis for custom RNAi delivery strategies Contact Us

Delivery note: Passenger 3′ placement with a short spacer is often a practical starting point, but final placement should be validated for activity, uptake and release. Additional targeting ligands are available upon request.

Architecture Description Use Design Note
Duplex siRNA Standard guide/passenger RNAi duplex Gene knockdown, screening and validation 19–21 bp duplexes with guide-strand control
DsiRNA Dicer-substrate siRNA Potency and processing exploration Requires Dicer-processing review
Tri-siRNA One passenger strand with two guide strands Dual-guide or multi-target RNAi research Also described as dual-guide triplex siRNA
Multi-Guide RNAi Multiple active guide sequences in one RNAi strategy Simultaneous target silencing Requires off-target and strand-bias review
Multivalent siRNA RNAi construct with multiple targeting or delivery elements Enhanced targeting or avidity strategies Custom synthesis and QC recommended
miRNA Mimic Duplex mimicking endogenous miRNA function Gain-of-function studies Manage passenger strand activity
anti-miR Modified oligo inhibitor of endogenous miRNA miRNA knockdown and pathway studies Often LNA/PS or stabilized single-strand format
LNA anti-miR LNA/PS miRNA inhibitor High-affinity miRNA inhibition Often single-stranded format
Antagomir 2′-OMe + PS + hydrophobic conjugate In vivo miRNA inhibition research Delivery and end protection are central
Aptamer-siRNA Chimeras Aptamer-guided RNAi construct Cell-selective RNAi delivery research Requires aptamer folding and linker review
Self-Delivering RNAi Conjugate-enabled uptake without conventional transfection Delivery and formulation research Optimize conjugate, linker and release behavior
Custom RNAi Architectures Project-specific RNAi designs beyond standard duplexes Advanced therapeutic RNAi research Contact Bio-Synthesis for feasibility review

Architecture note: Advanced RNAi formats may require custom synthesis, strand-specific purification, duplex/triplex annealing strategy and expanded QC.

Need a modification not listed?

Bio-Synthesis supports stereopure phosphorothioates, stereodefined backbone chemistries, phosphorodithioates, PMO, PNA, custom sugar analogs, XNA technologies, targeting ligands, antibody and aptamer conjugates, cleavable linkers and advanced RNAi architectures beyond those shown here. Contact our scientific team to discuss specialized RNAi modification requirements.

Design Guide

Choose by Function, Delivery Route and Risk

Use this compact guide before final sequence review and analytical release planning.

RNAi Design Decision Guide

Modification choices should protect siRNA without preventing RISC loading or target silencing.

Potency

Guide Loading

Preserve guide 5′ requirements and avoid modification patterns that block Ago2 activity.

Watch: seed effects, passenger loading, 19-mer vs 21-mer

Stability

Nuclease Resistance

Use terminal PS, selected 2′-OMe/2′-F and passenger-side stabilization.

Watch: over-modification of antisense seed

Immune Tolerance

Reduce Innate Signals

Place 2′-OMe at immune-sensitive motifs and validate final activity.

Watch: PBMC or cytokine readouts

Liver

GalNAc Route

Use passenger 3′ GalNAc with PEG/TEG geometry when hepatocyte targeting is the goal.

Watch: linker length and conjugate purity

Extrahepatic

Lipid / CPP Route

Consider cholesterol, LMO, fatty acid, CPP or peptide strategies.

Watch: uptake versus release dynamics

miRNA

Mimic or Inhibit

Use mimics for gain-of-function and anti-miR/antagomir designs for inhibition.

Watch: strand bias and functional readout

Controls

Validation Set

Include scrambled, mismatch, passenger-only or unconjugated controls when needed.

Watch: assay background and vehicle controls

Scale

Manufacturable Pattern

Lock chemistry, purification and QC before moving from screening to gram/kilo lots.

Watch: method consistency and documentation

Scale-Up Support

From Modified siRNA Design to Scalable Supply

Manufacturing stays important, but this page keeps the focus on modification-driven RNAi performance.

RNAi Scale-Up Pathway

Move from early duplex screening to manufacturable RNAi supply with scale-matched purification, analytics, formulation and project files.

01

Discovery Screens

µmol lots, sequence panels, guide/passenger pattern testing and basic identity QC.

02

Lead Confirmation

Final duplex length, 5′-P strategy, modification pattern, delivery handle and controls.

03

Pilot Supply

mmol to gram supply, scalable purification route and method consistency review.

04

Gram / Kilo-Class

Large-scale RNAi oligo synthesis with optimized purification and release testing.

05

Documentation

CoA, analytical package, formulation, labeling, packaging and documentation support.

Related Services

Related RNAi & Oligonucleotide Services

Use these links to connect RNAi customers to specialized workflows.

RNAi

RNAi / siRNA

Custom siRNA design, modifications and scale-up.

Explore →

RNAi

Advanced RNAi Architectures

DsiRNA, Tri-siRNA, dual-guide architectures, miRNA mimics, anti-miR and advanced RNAi formats.

Explore →

Gal

GalNAc Oligo Conjugation

GalNAc conjugates for liver-targeted RNAi.

Explore →

Lip

Lipid-Modified Oligos

Cholesterol, fatty acid and LMO options.

Explore →

Pep

Peptide-Oligo Conjugates

CPP and peptide targeting strategies.

Explore →

BioC

Oligo Bioconjugation

Handles, linkers, labels and payload conjugates.

Explore →

Pur

Oligo Purification

HPLC, UPLC, PAGE and scale workflows.

Explore →

QC

Oligo Release QC

C-MS, purity, endotoxin and documentation.

Explore →

FAQ

What should this page emphasize?
This page should emphasize siRNA modification strategy: potency, stability, immune tolerance, delivery and RISC compatibility.
Which siRNA modifications are best for nuclease resistance?
Common choices include terminal PS, 2′-OMe, 2′-F, selected LNA/BNA/cEt, 3′ caps and delivery conjugates.
What is Tri-siRNA?
Tri-siRNA, or dual-guide triplex siRNA, is an advanced RNAi architecture with one passenger strand and two guide strands.
Where should delivery conjugates be placed?
Many siRNA conjugates are placed on the passenger/sense strand 3′ end with a short PEG/TEG spacer, but final placement depends on activity and delivery route.
What information is needed for a quote?
Provide target gene, sequence or design request, duplex or single-strand format, scale, modifications, conjugates, purification level, QC requirements and delivery application.
Can modified siRNA scale to gram or kilo-class supply?
Yes. Programs can move from discovery micromole lots to pilot, gram and kilo-class supply with scale-matched purification, QC and documentation.
More FAQ'sAll FAQ's
Contact & Quote Request

Build Your Modified siRNA Program

Format siRNA, DsiRNA, anti-miR, Tri-siRNA
Sequence guide/passenger or design request
Chemistry PS, 2′-OMe, 2′-F, LNA/MOE
Delivery GalNAc, lipid, CPP, PEG
Scale µmol, mmol, gram, kilo
QC LC-MS, HPLC, endotoxin, CoA
Modification-First RNAi Support

Need help selecting siRNA modifications?

Share your target, sequence, RNAi format, modification pattern, delivery conjugate, scale, purification and QC requirements. Bio-Synthesis can help design a practical modified siRNA pathway from discovery lots to development supply.
Request a Quote Speak to a Scientist

Need help before quoting?

Email: info@biosyn.com
Phone: 800-227-0627 (US/CAN)
Phone: +001-972-420-8505 (INT)
2′

Modification Focus

2′-OMe, 2′-F, PS, LNA/BNA/cEt, terminal caps and delivery conjugates.

Potency Stability Delivery QC
QC

Production Package

Purification, LC-MS, analytical purity, endotoxin options, packaging and documentation support.

HPLC/UPLC LC-MS OD260 CoA
Scientific Background & Recommended Reading

Recommended Reading

Selected background references for RNA interference, siRNA chemistry and delivery strategy.

  1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998.
  2. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001.
  3. Deleavey GF, Damha MJ. Designing chemically modified oligonucleotides for targeted gene silencing. Chemistry & Biology. 2012.
  4. Nair JK, et al. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. Journal of the American Chemical Society. 2014.
  5. Setten RL, Rossi JJ, Han SP. The current state and future directions of RNAi-based therapeutics. Nature Reviews Drug Discovery. 2019.

Note: References are provided for scientific background. Final RNAi oligo design should be reviewed in the context of target biology, guide strand behavior, delivery route, chemistry placement, purification and release QC.

Why Choose Bio-Synthesis

Trusted by biotech leaders worldwide for over 45+ years of delivering high quality, fast and scalable synthetic biology solutions.

Greetings Researchers,

Please be advised that any information, guidelines, writeups, and oral/video/graphic content on our website are intended solely as general guidelines.
It is the researcher's sole responsibility to conduct thorough research on the designs of the products intended for their experiments before submitting
their designs to Biosynthesis for review of production feasibility.
Thank you for your understanding.

Quick Links