Abstract
Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki–Miyaura cross-coupling reaction. 5-Iodouridine triphosphate (IUTP) is efficiently incorporated into RNA ONs at one or more sites by T7 RNA polymerase. Further, using a catalytic system made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), we established a modular method to functionalize iodouridine-labeled RNA ONs in the presence of various boronic acid and ester substrates under very mild conditions (37°C and pH 8.5). This method is highly chemoselective, and offers direct access to RNA ONs labeled with commonly used fluorescent and affinity tags and new fluorogenic environment-sensitive nucleoside probes in a ligand-controlled stereoselective fashion. Taken together, this simple approach of generating functional RNA ON probes by Suzuki–Miyaura coupling will be a very important addition to the resources and tools available for analyzing RNA motifs.
Highlights
Understanding of RNA structure and function, and its use in therapeutics are greatly aided by recent developments in the nucleic acid functionalization strategy based on bioorthogonal chemical reactions [1,2,3,4]
Base-functionalized nucleoside analogs containing fluorescent, isotope, heavy atom or spin labels serve as excellent tools for biophysical investigation of nucleic acid structure, dynamics and function [50,51,52,53,54,55,56,57,58,59,60,61]
Necessity to prepare individual substrates, and the challenges associated with their synthesis and incorporation can be circumvented by developing a modular post-synthetic RNA labeling method, which would allow direct installation of the probes by Suzuki–Miyaura reaction between iodo-labeled RNA ONs with various accessible boronic acid/ester substrates (Figure 1)
Summary
Understanding of RNA structure and function, and its use in therapeutics are greatly aided by recent developments in the nucleic acid functionalization strategy based on bioorthogonal chemical reactions [1,2,3,4]. In several instances, elaborate chemical manipulations to synthesize the functionalized monomers (e.g. phosphoramidites and triphosphates) and challenges associated with their incorporation (e.g. stability under reaction conditions, poor coupling and enzymatic incorporation efficiency) limit the applications of these methods [4] In this context, postsynthetic modification of RNA by using bioorthogonal reactions is proving as a valuable tool to generate functional RNA probes. Reactions like azide-alkyne cycloaddition [7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22], Staudinger ligation [21], inverse electron demand Diels– Alder [23,24,25,26,27,28], to name a few, have emerged as valuable tools to label, image and profile RNA in cell-free and cellular environments These methods often use bulky activated building blocks (e.g. cyclooctyne, tetrazine, norbornyl etc.) to promote efficient post-synthetic reaction under mild conditions. Establishment of new post-synthetic RNA modification strategies that allow direct introduction of various functionalities by using accessible tags and reporters remains a high priority
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