RNA Phosphorothioate Modification in Prokaryotes and Eukaryotes

We report the first RNA phosphorothioate modification in both prokaryotes and eukaryotes. The GpsG modification exists in the Rp configuration and was quantified with liquid chromatography coupled with tandem mass spectrometry. Furthermore, we show the Dnd clusters that regulate DNA PT modification also play roles in RNA PT modification

Nature’s Selection of Geranyl Group as a tRNA Modification: The Effects of Chain Length on Base-Pairing Specificity

The recently discovered geranyl modification on the 2-thio position of wobble U34 residues in tRNA^Glu, tRNA^Lys, and tRNA^Gln in several bacteria has been found to enhance the U:G pairing specificity and reduce the frameshifting error during translation. It is a fundamentally interesting question why nature chose a C10 terpene group in tRNA systems. In this study, we explore the significance of the terpene length on base-paring stability and specificity using a series of 2-thiouridine analogues containing different lengths of carbon chains, namely, methyl- (C1), dimethylallyl- (C5), and farnesyl-modified (C15) 2-thiothymidines in a DNA duplex. Our thermal denaturation studies indicate that the relatively long chain length of ≥ C10 is required to maintain the base-pairing discrimination of thymidine between G and A. The results from our molecular dynamics simulations show that in the T:G-pair-containing duplex, the geranyl and farnesyl groups fit into the minor groove and stabilize the overall duplex stability. This effect cannot be achieved by the shorter carbon chains such as methyl and dimethylallyl groups. For a duplex containing a T:A pair, the terpene groups disrupt both hydrogen bonding and stacking interactions by pushing the opposite A out of the helical structure. Overall, as the terpene chain length increases, the xT:G pair stabilizes the duplex, whereas the xT:A pair causes destabilization, indicating the evolutionary significance of the long terpene group on base-pairing specificity and codon recognition

General recognition of U‑G, U‑A, and C‑G pairs by double-stranded RNA-binding PNAs incorporated with an artificial nucleobase

Chemically modified peptide nucleic acids (PNAs) show great promise in the recognition of RNA duplexes by major-groove PNA·RNA–RNA triplex formation. Triplex formation is favored for RNA duplexes with a purine tract within one of the RNA duplex strands, and is severely destabilized if the purine tract is interrupted by pyrimidine residues. Here, we report the synthesis of a PNA monomer incorporated with an artificial nucleobase S, followed by the binding studies of a series of S-modified PNAs. Our data suggest that an S residue incorporated into short 8-mer dsRNA-binding PNAs (dbPNAs) can recognize internal Watson–Crick C-G and U-A, and wobble U-G base pairs (but not G-C, A-U, and G-U pairs) in RNA duplexes. The short S-modified PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. Interestingly, replacement of the C residue in an S·C-G triple with a 5-methyl C results in the disruption of the triplex, probably due to a steric clash between S and 5-methyl C. Previously reported PNA E base shows recognition of U-A and A-U pairs, but not a U-G pair. Thus, S-modified dbPNAs may be uniquely useful for the general recognition of RNA U-G, U-A, and C-G pairs. Shortening the succinyl linker of our PNA S monomer by one carbon atom to have a malonyl linker causes a severe destabilization of triplex formation. Our experimental and modeling data indicate that part of the succinyl moiety in a PNA S monomer may serve to expand the S base forming stacking interactions with adjacent PNA bases.Ministry of Education (MOE)Nanyang Technological UniversityWe thank Prof. Tom Brown for providing us a detailed protocol for the synthesis of the S base. This work was supported by NTU start-up grant, Singapore Ministry of Education (MOE) Tier 1 grants (RGT3/13, RG42/15, and RG152/17), and MOE Tier 2 grants (MOE2013-T2-2-024 and MOE2015-T2-1-028) to G.C. The work was also supported by MOE Tier 1 (RG126/16 and RG31/18) and MOE Tier 2 (MOE2018-T2-1-033) to K.X

Construction and structure studies of DNA-bipyridine complexes as versatile scaffolds for site-specific incorporation of metal ions into DNA

<p>The facile construction of metal–DNA complexes using ‘Click’ reactions is reported here. A series of 2′-propargyl-modified DNA oligonucleotides were initially synthesized as structure scaffolds and were then modified through ‘Click’ reaction to incorporate a bipyridine ligand equipped with an azido group. These metal chelating ligands can be placed in the DNA context in site-specific fashion to provide versatile templates for binding various metal ions, which are exchangeable using a simple EDTA washing-and-filtration step. The constructed metal–DNA complexes were found to be thermally stable. Their structures were explored by solving a crystal structure of a propargyl-modified DNA duplex and installing the bipyridine ligands by molecular modeling and simulation. These metal–DNA complexes could have wide applications as novel organometallic catalysts, artificial ribonucleases, and potential metal delivery systems.</p

RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells

Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation