Reverse 5' to 3' RNA Synthesis for Long Oligomer Production

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Solution Overview

Problem

Current RNA synthesis methods, particularly in the 3' → 5' direction, face challenges such as the migration of O-TBDMS groups, which limits the synthesis of longer RNA oligomers like 100-mer to 200-mer, essential for studying non-coding RNAs and their regulatory functions.

Innovation Solution

The process involves synthesizing RNA oligonucleotides in the 5' → 3' direction using monomer phosphoramidites and solid supports, employing a novel combination of ancillary reagents like phenoxy acetic anhydride, N-methylimidazole, and 5-ethylthio-1-H-tetrazole, to achieve high coupling efficiency and extended chain lengths up to 200-mers.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If 3' → 5' synthetic approach is used, then RNA synthesis is well established and can produce therapeutic grade RNA aptamers, but O-TBDMS group migration occurs from 3'-position to 2'-position

Engineering Contradiction:

ImproveRNA synthesis reliabilityVSAvoidO-TBDMS group position stability

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

The patent inverts the conventional 3'→5' synthesis direction to 5'→3' reverse direction synthesis. This inversion fundamentally changes the reaction pathway and eliminates the O-TBDMS migration issue that plagues conventional synthesis, while still achieving reliable RNA oligonucleotide production

Inventive Principle:

Principle #13The other way round (Inversion)

2Manufacturing precision

If 5' → 3' synthetic approach is used, then O-TBDMS group migration is circumvented, but synthesis of long RNA (100-mer to 200-mer) has not been achieved

Engineering Contradiction:

ImproveO-TBDMS group position stabilityVSAvoidRNA oligonucleotide chain length

Core Design Contradiction:

Manufacturing precisionVSLength of moving object

Solution Approach 1:

The patent optimizes multiple parameters including coupling time (extended to 6-10 minutes per cycle), reagent concentrations (0.1-1.0 M phosphoramidites), and temperature conditions to achieve high coupling efficiency (>99%) required for synthesizing long RNA chains of 100-200 nucleotides while maintaining the 5'→3' direction

Inventive Principle:

Principle #35Parameter changes

3Length of moving object

If coupling efficiency is increased to achieve long RNA synthesis, then chain length can be extended, but coupling time increases

Engineering Contradiction:

ImproveRNA oligonucleotide chain lengthVSAvoidCoupling time

Core Design Contradiction:

Length of moving objectVSLoss of time

Solution Approach 1:

The patent employs ancillary reagents including 5-ethylthio-1H-tetrazole (ETT) as a coupling accelerator and phenoxy acetic anhydride as a capting agent to prevent uncapped 3'-OH groups from interfering. These intermediaries enable high coupling efficiency with optimized coupling times of 6-10 minutes per cycle

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentEP3044228B1Highly efficient synthesis of long RNA using reverse direction approach

Publication Date: 2021.04.07 CHEMGENES CORP

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AI summary

The present invention relates to novel process of reverse 5 '→ 3 ' directed synthesis of RNA oligomers in the range of about 100-mer to about 200-mer has been developed and disclosed. Using that method demonstrated high quality RNA synthesis with coupling efficiency approaching 99%.