Acyclic Linker Cap Analogs for RNA Capping Efficiency
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Solution Overview
Problem
Current cap analogs used in in vitro transcription reactions have limitations such as low efficiency in co-transcriptional capping and high immunostimulation, leading to incomplete or incorrectly oriented cap structures, which affect the expression levels of capped RNAs.
Innovation Solution
Development of new cap analogs with specific structural modifications, including linear unbranched or single-branched acyclonucleosides at the 5' terminal, which enhance binding efficiency to eIF-4E and reduce recognition by IFIT1 and IFIT3 proteins, thereby improving capping efficiency and expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If m7G(5′)ppp is used as cap analog, then co-transcriptional capping can be achieved, but capping efficiency is low because m7G(5′)ppp competes with guanine nucleotide G for transcription initiation
Solution Approach 1:
The cap analog is divided into two functional parts: a dinucleotide cap structure (m7GpppG) that binds to eIF-4E and ensures correct cap orientation, and a separate 3'-OH group that serves as the transcription initiation site. This segmentation allows the cap structure to fulfill its binding function while the 3'-OH group initiates transcription without competition from free guanine nucleotides.
Solution Approach 2:
The patent introduces a dinucleotide intermediate structure (m7GpppG) that acts as a mediator between the cap structure and the transcription initiation process. The 3'-OH group of this dinucleotide serves as the initiating nucleophile, while the 5'-terminal m7G provides the cap structure that binds to eIF-4E, thus mediating both cap formation and transcription initiation functions.
2Productivity
If m7G(5′)ppp(5′)G is used as cap analog, then transcription initiation can be achieved, but reverse orientation of cap structure occurs because the 3'-OH group of m7G or G can serve as initiating nucleophile
Solution Approach 1:
The cap analog is designed with asymmetric connectivity where the 3'-OH group of the dinucleotide (m7GpppG) is positioned to serve as the initiating nucleophile, while the 5'-terminal m7G provides the cap structure. This asymmetric arrangement prevents reverse orientation by ensuring that only the correct 3'-OH group can initiate transcription, while the 5'-terminal m7G maintains proper cap structure orientation through its binding to eIF-4E.
3Ease of manufacture
If conventional cap analogs are used, then in vitro transcription can be performed, but immunostimulation occurs due to recognition by IFIT1 and IFIT3 proteins
Solution Approach 1:
The patent modifies the chemical structure of the cap analog by changing the sugar moiety from ribose to acyclonucleoside with linear unbranched or single-branched structure. This structural parameter change reduces recognition by IFIT1 and IFIT3 proteins, thereby reducing immunostimulation while maintaining the cap analog's ability to function in in vitro transcription reactions.
Data Source
AI summary
The present invention is inter alia concerned with (A) a compound of formula (I) as defined herein or a salt, stereoisomer, tautomer or deuterated version thereof, (B) a cap analog comprising a 5′ terminal acyclonucleoside, wherein the acyclonucleoside comprises a linear unbranched structure or a linear single-branched structure instead of a ribose, wherein the 5′ terminal acyclonucleoside is optionally deuterated (C) an RNA molecule comprising at least three nucleotides and comprising a 5′ end of formula (III) as defined herein, wherein the 5′ end is optionally deuterated (D) an RNA molecule comprising at least three nucleotides and comprising a 5′ terminal acyclonucleoside, wherein the acyclonucleoside comprises a linear unbranched structure or a linear single-branched structure instead of a ribose, wherein the 5′ terminal acyclonucleoside is optionally deuterated (E) an in vitro method for synthesizing an RNA molecule, (F) the RNA molecule obtained thereby, (G) compositions comprising the RNA molecule, (H) kits comprising the compound of formula (I) or the cap analog, (I) uses as well as (J) methods as outlined herein.