The bacterium Escherichia coli possesses 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Nanopore direct RNA sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to unmodified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knockout E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modification's signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. The double modification m 4 C m 1402 resides in 16S rRNA, and it decreased with both stressors. Using the helicase dwell time, it was determined that the N 4 methyl group is lost during both stressors, and the 2′-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons (rrn), and the earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to all seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for >16 different RNA modifications in a strand is achievable.

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We report the synthesis of N 2 -aryl (benzyl, naphthyl, anthracenyl, and pyrenyl)-deoxyguanosine (dG) modified phosphoramidite building blocks and the corresponding damaged DNAs. Primer extension studies using E. coli Pol IV, a translesion polymerase, demonstrate that translesion synthesis (TLS) across these N 2 -dG adducts is error free. However, the efficiency of TLS activity decreases with increase in the steric bulkiness of the adducts. Molecular dynamics simulations of damaged DNA-Pol IV complexes reveal the van der Waals interactions between key amino acid residues (Phe13, Ile31, Gly32, Gly33, Ser42, Pro73, Gly74, Phe76, and Tyr79) of the enzyme and adduct that help to accommodate the bulky damages in a hydrophobic pocket to facilitate TLS. Overall, the results presented here provide insights into the TLS across N 2 -aryl-dG damaged DNAs by Pol IV.

The stabilization of G-quadruplex DNA structures by using small molecule ligands having simple structural scaffolds has the potential to be harnessed for developing next generation anticancer agents. Because of the structural diversity of G-quadruplexes, it is challenging to design stabilizing ligands, which can specifically bind to a particular quadruplex topology. To address this, herein, we report the design and synthesis of three benzothiazole hydrazones of furylbenzamides having different side chains (ligands 1, 2 and 3), which show preferential stabilization of promoter quadruplex DNAs (c-MYC and c-KIT1) having parallel topologies over telomeric and duplex DNAs. The CD melting study revealed that all the ligands, in particular ligand 2, exhibit higher stabilization toward parallel promoter quadruplexes (ΔTm = 10-15 °C) as compared to antiparallel promoter quadruplex (h-RAS1), telomeric quadruplex and duplex DNAs (ΔTm = 0-3 °C). FID assay and fluorimetric titration results also reveal the preferential binding of ligands toward c-MYC and c-KIT1 promoter quadruplex DNAs over telomeric and duplex DNAs. Validating these results further, Taq DNA polymerase stop assay showed IC50∼ 6.4 μM for ligand 2 with the c-MYC DNA template, whereas the same for the telomeric DNA template was found to be >200 μM. Molecular modeling and dynamics studies demonstrated a 1 : 1 binding stoichiometry in which stacking and electrostatic interactions play important roles in stabilizing the c-MYC G-quadruplex structure. Taken together, the results presented here provide new insights into the design of structurally simple scaffolds for the preferential stabilization of a particular G-quadruplex topology.

The transfer RNA (tRNA) modification 4-thiouridine (s 4 U) acts as a near-ultraviolet (UVA) radiation sensor in Escherichia coli (E. coli), where it induces a growth delay upon exposure to the UVA radiation (∼310−400 nm). Herein, we report sequencing methodology for site-specific profiling of s 4 U modification in E. coli tRNAs. Upon the addition of iodoacetamide (IA) or iodoacetyl-PEG2-biotin (BIA), the nucleophilic sulfur of s 4 U forms a reaction product that is extensively characterized by liquid chromatography−mass spectrometry (LC−MS/MS) analysis. This method is readily applied to the alkylation of natively occurring s 4 U on E. coli tRNA. Next-generation sequencing of BIAtreated tRNA from E. coli revealed misincorporations at position 8 in 19 of the 20 amino acid tRNA species. Alternatively, tRNA from the ΔthiI strain, which cannot introduce the s 4 U modification, does not exhibit any misincorporation at the corresponding positions, directly linking the base transitions and the tRNA modification. Independently, the s 4 U modification on E. coli tRNA was further validated by LC−MS/MS sequencing. Nuclease digestion of wild-type and deletion strains E. coli tRNA with RNase T1 generated smaller s 4 U/U containing fragments that could be analyzed by MS/MS analysis for modification assignment. Furthermore, RNase T1 digestion of tRNAs treated either with IA or BIA showed the specificity of iodoacetamide reagents toward s 4 U in the context of complex tRNA modifications. Overall, these results demonstrate the utility of the alkylation of s 4 U in the site-specific profiling of the modified base in native cellular tRNA.