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DNA/2'-O-Methyl RNA Chimera Modification Service

Introduction

DNA/2'-O-methyl RNA chimeric oligonucleotides blend DNA and 2'-O-methyl RNA to balance target specificity and nuclease resistance, leveraging DNA's stability and modified RNA's enzymatic resilience, ideal for gene silencing and therapies. They overcome unmodified oligos' degradation and off-target issues, enhancing delivery and durability. We offer tailored design, synthesis, stability profiling, and functional validation. With 20+ years' expertise, we accelerate gene-modulation programs.

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DNA/2'-O-Methyl RNA Chimera Modification

2'-O-alkyl-modified structure: 2'-O-methoxy RNA (2'-O-ME RNA). (OA Literature)Fig.1 Structure of 2'-O-Methyl RNA.1

Structural characteristics

In DNA/2'-O-methyl RNA chimeras, the 2'-O-methyl RNA segment has a typical C3'-endo conformation, with its 2'-O-methyl group pointing to the double strand's minor groove. In the central RNA/DNA hybrid, DNA residues (except the middle thymidine) also adopt C3'-endo, giving an overall structure close to a typical A-type double strand.

Chemical properties

2'-O-methyl modification boosts RNA stability by reducing susceptibility to alkaline hydrolysis, nuclease cleavage, and oxidation, via altering 2'-hydroxyl hydrogen bonding and inhibiting nucleophilicity. It also increases Tm and stability of hybrid duplexes with complementary RNA versus DNA sequences.

Biological functions

DNA/2'-O-methyl RNA chimeras act via the RNAi pathway: For example, asymmetric 18-base-pair med-siRNAs (alternating DNA/2'-O-methyl RNA) bind Ago 2, cleave target mRNA efficiently, and reduce target protein expression.

The synergistic combination of 2'-O-Methyl RNA and phosphorothioate linkages creates highly stable and potent chimeric oligonucleotides. This dual modification strategy is particularly advantageous for:

  • Antisense Therapy: Enhancing the stability and efficacy of antisense oligonucleotides designed to modulate gene expression by binding to target mRNA.
  • miRNA Modulation: Improving the stability and activity of oligonucleotides used to inhibit or mimic microRNAs.
  • Gene Silencing: Providing a durable and effective tool for silencing specific genes implicated in disease.
  • Genetic Disorder Therapies: As exemplified by research into Duchenne muscular dystrophy, where such chimeras are explored for inducing exon skipping to restore functional protein production.

Workflow

  1. Sequence Design & Modification Mapping

    The DNA/2'-O-methyl RNA chimeric sequence was designed using algorithms to avoid off-target risks, optimize the position of modifications in the gene sequence, enhance the recruitment of RNase H, eliminate CpG sequences or TLR activation sequences that may cause side effects, and reduce immunogenicity while ensure specificity.

  2. Solid-Phase Synthesis

    We achieved a coupling efficiency of over 99.5% by using amide phosphorus chemistry and an auto-synthesizer. This high precision reduces the formation of incomplete sequences and truncated impurities. Our unique synthetic method enables the smooth integration of DNA/2'-O-methyl RNA chimeras, supporting complex chimeric designs with precise modification patterns.

  3. Purification & QC

    Employ UHPLC for size-exclusion purification, followed by MS to confirm mass accuracy. PAGE analysis ensures diastereomer impurities <5%, critical for maintaining consistent biological activity. Endotoxin levels are controlled (<0.1 EU/mg) for in vivo applications.

  4. Functional Validation

    Nuclease Stability: Incubate with serum nucleases to quantify half-life improvements over unmodified oligonucleotides.

    Thermodynamic Profiling: Differential scanning calorimetry measures Tm shifts, ensuring optimal duplex stability for target engagement.

  5. Timeline & Deliverables

    Research-Grade: 4-8 weeks, including design iterations and basic validation.

    GMP-Grade: 10-14 weeks, with full compliance documentation.

    Outputs: Lyophilized products with >95% purity, stability profiles, HPLC/MS reports, and application-specific optimization guides.

What We Can Offer

Customized Design & Synthesis

Tailored oligonucleotide design/synthesis to meet unique sequence/modification needs, ensuring optimal performance for specific applications.

Enhanced Stability & Nuclease Resistance

Produces chimeras with improved enzymatic degradation resistance, extending half-life and efficacy in biological systems.

Superior Target Affinity

Modifications boost binding affinity to target mRNA, enabling more potent and specific therapeutic effects.

Advanced Polymerase Engineering

Uses cutting-edge polymerase engineering for efficient enzymatic synthesis of long 2'-O-Methyl RNA and MOE-RNA oligomers, overcoming traditional limitations.

Support for Diverse Applications

Solutions for antisense therapy, miRNA modulation, gene silencing, and development of novel nucleic acid catalysts (2'OMezymes) and aptamers.

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Customer Reviews

"The enhanced stability using Creative Biolabs' DNA/2'-O-Methyl RNA Chimera in our antisense research has significantly improved the longevity of our constructs in cell culture, allowing for more robust experimental results."

2 months ago, Jame

"Using Creative Biolabs' 2'-O-Methyl RNA modification, we observed a remarkable increase in target mRNA binding affinity, which was crucial for the efficacy of our gene silencing studies. The precision and quality were outstanding."

4 weeks ago, Mari

"Creative Biolabs' expertise in DNA/2'-O-Methyl RNA Chimera synthesis greatly accelerated our project timeline. The high-quality product and detailed reports made our downstream applications seamless and highly successful."

1 month ago, Davi

FAQs

Q: How does the design of DNA/2'-O-Methyl RNA chimeras influence siRNA activity, and what structural factors balance efficiency and off-target effects?

A: Placing 2'-O-Methyl RNA in the guide strand's seed region (positions 2-8) preserves Ago2 loading for efficient silencing while reducing off-target mRNA binding. Limiting modifications in the passenger strand avoids disrupting strand separation, balancing potency and specificity.

Q: What are the optimal storage conditions for DNA/2'-O-Methyl RNA chimeras, and why are they critical?

A: Store at -80°C in RNase-free Tris-EDTA (pH 8.0); avoid >3 freeze-thaw cycles. Low temperature and neutral pH prevent 2'-O-Methyl group hydrolysis, while minimizing cycles preserves structural integrity and functional activity.

Q: What strategies mitigate immune activation in DNA/2'-O-Methyl RNA chimera design, and how do they interact with innate immune receptors?

A: Designs exclude CpG motifs (TLR9 triggers) and use UNA spacers in TLR7/8-binding regions. 2'-O-Methyl modifications further reduce TLR recognition by altering backbone flexibility, lowering IFN-α secretion by ~60% without losing therapeutic function.

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Reference

  1. Liczner, Christopher, et al. "Beyond ribose and phosphate: selected nucleic acid modifications for structure–function investigations and therapeutic applications." Beilstein Journal of Organic Chemistry 17.1 (2021): 908-931. DOI: 10.3762/bjoc.17.76. Distributed under Open Access license CC BY 4.0, the picture was cropped.

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