This Outlook calls attention to two seemingly disparate and emerging fields regarding viral genomics that may be correlated in a way previously overlooked. First, we describe identification of conserved potential G-quadruplex-forming sequences (PQSs) in viral genomes relevant to human health. Studies have demonstrated that PQSs are highly conserved and can fold to G-quadruplexes (G4s) to regulate viral processes. Key examples include G4s as a countermeasure to the host’s immune system or G4-guided regulation of replication or transcription. Second, emerging data are discussed concerning the epitranscriptomic modification N 6 -methyladenosine (m 6 A) in viral RNA installed by host proteins in a consensus sequence favoring 5′-GG(m 6 A)C-3′. The proposed pathways by which m 6 A is written, read, and erased in viral RNA genomes and the impact this has on viral replication are described. The structural reason why certain sites are selected for modification while others are not is still mysterious. Finally, we discuss our new observations regarding these previous sequencing data that identify m 6 A installation within the loops of two-tetrad PQSs in the RNA genomes of the Zika, HIV, hepatitis B, and SV40 viruses. We hypothesize that conserved viral PQSs can provide a framework (sequence and/or structural) for m 6 A installation. We also discuss literature sources suggesting that PQSs as sites of RNA modification could be a general phenomenon. We anticipate our observations will provide ample opportunities for exciting discoveries regarding the interplay between G4 structures and epitranscriptomic modifications of RNA.

Nucleic acids are chemically modified to fine-tune their properties for biological function. Chemical tools for selective tagging of base modifications enables new approaches; the photosensitizers riboflavin and anthraquinone were previously proposed to oxidize N 6 -methyladenine (m 6 A) or 5-methylcytosine (5mdC) selectively. Herein, riboflavin, anthraquinone, or Rose Bengal were allowed to react with the canonical nucleosides dA, dC, dG, and dT, and the modified bases 5mdC, m 6 A, 8-oxoguanine (dOG), and 8-oxoadenine (dOA) to rank their reactivities. The nucleoside studies reveal that dOG is the most reactive and that the native nucleoside dG is higher or similar in reactivity to 5mdC or m 6 A; competition in both single-and double-stranded DNA of dG vs. 5mdC or 6mdA for oxidant confirmed that dG is favorably oxidized. Thus, photosensitizers are promiscuous nucleic acid oxidants with poor chemoselectivity that will negatively impact attempts at targeted oxidation of modified nucleotides in cells.

Nucleic acids are chemically modified to fine-tune their properties for biological function. Chemical tools for selective tagging of base modifications enables new approaches; the photosensitizers riboflavin and anthraquinone were previously proposed to oxidize N 6 -methyladenine (m 6 A) or 5-methylcytosine (5mdC) selectively. Herein, riboflavin, anthraquinone, or Rose Bengal were allowed to react with the canonical nucleosides dA, dC, dG, and dT, and the modified bases 5mdC, m 6 A, 8-oxoguanine (dOG), and 8-oxoadenine (dOA) to rank their reactivities. The nucleoside studies reveal that dOG is the most reactive and that the native nucleoside dG is higher or similar in reactivity to 5mdC or m 6 A; competition in both single-and double-stranded DNA of dG vs. 5mdC or 6mdA for oxidant confirmed that dG is favorably oxidized. Thus, photosensitizers are promiscuous nucleic acid oxidants with poor chemoselectivity that will negatively impact attempts at targeted oxidation of modified nucleotides in cells.

Positive control (or standard) is an indispensable ingredient in molecular biology assays widely used for the quantification of nucleic acid. The commonly used standards are plasmid DNA, cDNA, or naked RNA, which are unstable and easily degraded by nucleases in the surrounding environment; this might affect the accuracy of quantitative results. In this study, the authors designed and created a positive control for the hepatitis C virus (HCV) quantification based on armored RNA technology. The 5’UTR non-encoding sequence of HCV was cloned into the BH20 plasmid. Armored RNA HCV (AR-HCV) was induced for expression in the E. coli BL21 (DE3) by the addition of an IPTG inducer. AR-HCV was collected by sucrose density gradient ultracentrifugation followed by gel filtration chromatography using Superdex 75 column. Created AR-HCV was determined the concentration and examined the formation of pseudo viral particles by transmission electron microscopy (TEM). Stability assessment of AR-HCV to DNase and RNase treatment simultaneously has demonstrated its ability to resist these nucleases. Moreover, AR-HCV is stable over time and storage conditions. Strikingly, AR-HCV can be directly added to the specimen, allowing better and more accurate control of the whole quantitative procedure of HCV.