Structural Elucidation of Bisulfite Adducts to Pseudouridine That Result in Deletion Signatures during Reverse Transcription of RNA.

The recent report of RBS-Seq to map simultaneously the epitranscriptomic modifications N1-methyladenosine, 5-methylcytosine, and pseudouridine (Ψ) via bisulfite treatment of RNA provides a key advance to locate these important modifications. The locations of Ψ were found by a deletion signature generated during cDNA synthesis after bisulfite treatment for which the chemical details of the reaction are poorly understood. In the present work, the bisulfite reaction with Ψ was explored to identify six isomers of bisulfite adducted to Ψ. We found four of these adducts involved the heterocyclic ring, similar to the reaction with other pyrimidines. The remaining two adducts were bonded to the 1' carbon, which resulted in opening of the ribose ring. The utilization of complementary 1D- and 2D-NMR, Raman, and electronic circular dichroism spectroscopies led to the assignment of the two ribose adducts being the constitutional isomers of an S- and an O-adduct of bisulfite to the ribose, and these are the final products after heating. A mechanistic proposal is provided to rationalize chemically the formation and stereochemistries of all six isomeric bisulfite adducts to Ψ; conversion of intermediate adducts to the two final products is proposed to involve E2, SN2', and [2,3]-sigmatropic shift reactions. Lastly, a synthetic RNA template with Ψ at a known location was treated with bisulfite, leading to a deletion signature after reverse transcription, supporting the RBS-Seq report. This classical bisulfite reaction used for epigenomic and epitranscriptomic sequencing diverges from the C nucleoside Ψ to form stable bisulfite end products that yield signatures for next-generation sequencing.

Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers.

Reactive oxygen species, both endogenous and exogenous, can damage nucleobases of RNA and DNA. Among the nucleobases, guanine has the lowest redox potential, making it a major target of oxidation. Although RNA is more prone to oxidation than DNA is, oxidation of guanine in RNA has been studied to a significantly lesser extent. One of the reasons for this is that many tools that were previously developed to study oxidation of DNA cannot be used on RNA. In the study presented here, the lack of a method for seeking sites of modification in RNA where oxidation occurs is addressed. For this purpose, reverse transcription of RNA containing major products of guanine oxidation was used. Extension of a DNA primer annealed to an RNA template containing 8-oxo-7,8-dihydroguanine (OG), 5-guanidinohydantoin (Gh), or the R and S diastereomers of spiroiminodihydantoin (Sp) was studied under standing start conditions. SuperScript III reverse transcriptase is capable of bypassing these lesions in RNA inserting predominantly A opposite OG, predominantly G opposite Gh, and almost an equal mixture of A and G opposite the Sp diastereomers. These data should allow RNA sequencing of guanine oxidation products by following characteristic mutation signatures formed by the reverse transcriptase during primer elongation past G oxidation sites in the template RNA strand.

Zika Virus Genomic RNA Possesses Conserved G-Quadruplexes Characteristic of the Flaviviridae Family.

Zika virus has emerged as a global concern because neither a vaccine nor antiviral compounds targeting it exist. A structure for the positive-sense RNA genome has not been established, leading us to look for potential G-quadruplex sequences (PQS) in the genome. The analysis identified >60 PQSs in the Zika genome. To minimize the PQS population, conserved sequences in the Flaviviridae family were found by sequence alignment, identifying seven PQSs in the prM, E, NS1, NS3, and NS5 genes. Next, alignment of 78 Zika strain genomes identified a unique PQS near the end of the 3'-UTR. Structural studies on the G-quadruplex sequences found four of the conserved Zika virus sequences to adopt stable, parallel-stranded folds that bind a G-quadruplex-specific compound, and one that was studied caused polymerase stalling when folded to a G-quadruplex. Targeting these PQSs with G-quadruplex binding molecules validated in previous clinical trials may represent a new approach for inhibiting viral replication.