Postsynthetic On-Column 2' Functionalization of RNA by Convenient Versatile Method.

Olga A Krasheninina,Alya G Venyaminova,Veniamin S Fishman,Alexander A Lomzov,Alexey V Ustinov

doi:10.3390/ijms21145127

Abstract

We report a universal straightforward strategy for the chemical synthesis of modified oligoribonucleotides containing functional groups of different structures at the 2′ position of ribose. The on-column synthetic concept is based on the incorporation of two types of commercial nucleotide phosphoramidites containing orthogonal 2′-O-protecting groups, namely 2′-O-thiomorpholine-carbothioate (TC, as “permanent”) and 2′-O-tert-butyl(dimethyl)silyl (tBDMS, as “temporary”), to RNA during solid-phase synthesis. Subsequently, the support-bound RNA undergoes selective deprotection and follows postsynthetic 2′ functionalization of the naked hydroxyl group. This convenient method to tailor RNA, utilizing the advantages of solid phase approaches, gives an opportunity to introduce site-specifically a wide range of linkers and functional groups. By this strategy, a series of RNAs containing diverse 2′ functionalities were synthesized and studied with respect to their physicochemical properties.

Highlights

Over the past decades, synthetic oligoribonucleotides have gained significant importance since the advent of a large number of functional noncoding RNAs (siRNAs, miRNAs, ribozymes, riboswiches, anti-replicative RNAs, and sgRNAs of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, etc.) which play key roles in various cellular processes and have potential as therapeutic agents
The support-attached protected oligoribonucleotide containing ribonucleosides bearing two orthogonal 2 -O-protecting groups is assembled by phosphoramidite chemistry using 5 -O-DMTr nucleoside-modified polystyrene support
We have introduced a rather simple and cost-effective universal approach to the 2 functionalization of oligoribonucleotides (1) that utilizes commercially available reagents without any need for pre-synthesis of all four (U, A, C, and G), specially functionalized nucleotide phosphoramidites, (2) that allows to modify any nucleotide of the RNA of interest during the solid phase phosphoramidite synthesis, and (3) that is based on a combination of modern synthetic methods of bioorganic chemistry characterized by high yields

Summary

Introduction

Synthetic oligoribonucleotides have gained significant importance since the advent of a large number of functional noncoding RNAs (siRNAs, miRNAs, ribozymes, riboswiches, anti-replicative RNAs, and sgRNAs of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, etc.) which play key roles in various cellular processes and have potential as therapeutic agents. Beaucage and coauthors have proposed a convenient and efficient method for the preparation of reversible or permanent ribonucleoside 2 conjugates using oxime formation from aldehydes or ketones [30,31] They demonstrated that chimeric oligoribonucleotides modified with the 2 -O-(pyren-1-ylmethanimine-N-oxymethyl)uridine can be successfully converted to native RNAs by exposure to TBAF in DMSO. The siRNAs with the 2 -O-acetalester modifications possess increased stability to nucleases, enhanced permeability into cells, and targeted suppression efficiency Later, these authors reported an elegant postsynthetic solid-phase method for the synthesis of chimeric 2 -O-alkyl-, benzyldithiomethyl-modified (2 -O-RSSM, where R is a lipophilic moiety or polar groups), or doxorubicin-bearing oligoribonucleotides, which are turned into native RNAs under reduction conditions [13,35,36]

Methods

Results

Conclusion