Diastereoselectivity of 5-Methyluridine Osmylation Is Inverted inside an RNA Chain

Abstract

In this study, we investigated the reaction of the osmium tetroxide-bipyridine complex with pyrimidines in RNA. This reagent, which reacts with the diastereotopic 5-6 double bond, thus leading to the formation of two diastereomers, was used in the past to label thymidine and 5-methylcytosine in DNA. In light of the growing interest in post-transcriptional RNA modifications, we addressed the question of whether this reagent could be used for labeling of the naturally occurring RNA modifications 5-methylcytosine and 5-methyluridine. On nucleoside level, 5-methylcytosine and 5-methyluridine revealed a 5- and 12-fold preference, respectively, over their nonmethylated equivalents. Performing the reaction on an RNA level, we could show that the steric environment of a pentanucleotide has a major detrimental impact on the reaction rate of osmylation. Interestingly, this drop in reactivity was due to a dramatic change in diastereoselectivity, which in turn resulted from impediment of the preferred attack via the si side. Thus, while on the nucleoside level, the absolute configuration of the major product of osmylation of 5-methyluridine was (5R,6S)-5-methyluridine glycol-dioxoosmium-bipyridine, reaction with an RNA pentanucleotide afforded the corresponding (5S,6R)-diastereomer as the major product. The change in diastereoselectivity lead to an almost complete loss of selectivity toward 5-methylcytosine in a pentanucleotide context, while 5-methyluridine remained about 8 times more reactive than the canonical pyrimidines. On the basis of these findings, we evaluate the usefulness of osmium tetroxide-bipyridine as a potential label for the 5-methyluridine modification in transcriptome-wide studies.

Thioguanosine Conversion Enables mRNA‐Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC‐seq DUAL)

“…Furthermore, we note that there is no indication for oxidation of the 5,6‐double bond of pyrimidines under the conditions used. This could however become a potential side reaction if higher concentrations of OsO 4 and additives, such as pyridines or bipyridines, were applied that stabilize the pyrimidine OsO 4 adducts …”

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

“…OsBp addition to the pyrimidine C5-C6 double bond occurs from either the top or from the bottom of the pyrimidine ring yielding two topoisomers. 40,41,52 Due to the strand directionality these two isomers are detectable by either HPLC or CE (see profiles of osmylated 31nt oligos in Supplementary Information, part C). Two peaks were observed for every singly osmylated oligo tested here with a product ratio not far from unity, so that both are detectable.…”

Osmium Tag for Post‐transcriptionally Modified RNA

“…Unlike in the case of short RNA strands, HPLC traces that originated from osmium complex formation were negligible if m 5 C was incorporated into the inside of a 10‐nt single‐stranded (ss) RNA sequence AA(m 5 C)AAGAUCU. Previous studies performed by Tserovski and Helm showed that the reaction rate of m 5 C towards osmium oxidation was low and decreased significantly with an increase in the size of the RNA chain . Therefore, an indirect approach, specifically, enzymatic digestion of the target RNA prior to osmium oxidation, was considered.…”