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Research into the DNA cleavage properties of metallobleomycins conjugated to three solid supports is presented. It is suggested that the bithiazole associates with the DNA substrate in the DNA minor groove or by edgewise (partial) intercalation of one or both thiazole rings.

The rapid isolation of accessible DNA is reported.

The conjugation of bleomycins to a controlled pore glass bead to form a congene with a C-terminal substituent approximately 10 to the 5th power times bigger than the remainder of the bleomycin molecule is described. Bleomycin conjugates were found to have the ability to cause plasmid DNA relaxation and sequence-selective DNA cleavage. The occurrence of DNA cleavage in a system that minimizes the chance for DNA strand separation to facilitate intercalation was also observed.

N 4 -Acetylcytidine (ac 4 C) has been found to affect a variety of cellular and biological processes. For a mechanistic understanding of the roles of ac 4 C in biology and disease, we present an antibody-free, fluorine-assisted metabolic sequencing method to detect RNA ac 4 C, called "FAM-seq". We successfully applied FAM-seq to profile ac 4 C landscapes in human 293T, HeLa, and MDA cell lines in parallel with the reported acRIP-seq method. By comparison with the classic ac 4 C antibody sequencing method, we found that FAM-seq is a convenient and reliable method for transcriptome-wide mapping of ac 4 C. Because this method holds promise for detecting nascent RNA ac 4 C modifications, we further investigated the role of ac 4 C in regulating chemotherapy drug resistance in chronic myeloid leukemia. The results indicated that drug development or combination therapy could be enhanced by appreciating the key role of ac 4 C modification in cancer therapy.

Hundreds of modified bases have been identified and enzymatically modified to transfer RNAs (tRNAs) to regulate RNA function in various organisms. 2-Methylthio- N 6 -isopentenyladenosine (ms 2 i 6 A), a hypermodified base found at tRNA position 37, exists in both prokaryotes and eukaryotes. ms 2 i 6 A is traditionally identified by separating and digesting each tRNA from total RNA using RNA mass spectrometry. A transcriptome-wide and single-base resolution method that enables absolute mapping of ms 2 i 6 A along with analysis of its distribution in different RNAs is lacking. Here, through chemoselective methylthio group bioconjugation, we introduce a new approach ( re dox a ctivated c hemical t agging seq uencing, ReACT-seq) to detect ms 2 i 6 A transcriptome-wide at single-base resolution. Using the chemoselectivity between the methylthio group and oxaziridine group, ms 2 i 6 A is bio-orthogonally tagged with an azide group without interference of canonical nucleotides, advancing enrichment of methylthio group modified RNAs prior to sequencing. ReACT-seq was demonstrated on nine known tRNAs and proved to be highly accurate, and the reverse transcription stop (RT-stop) character enables ReACT-seq detection at single-base resolution. In addition, ReACT-seq identified that the modification of ms 2 i 6 A is conservative and may not exist in other RNAs.

We have first demonstrated the distinctive effects of three newly reported epigenetic modifications, including 5hmC, 5fC, and 5caC, on B-Z transition of CpG dinucleotide DNAs. We have performed detailed assays and compared their effects. We further studied the regulation of B-Z transition of CpG dinucleotide dodecamers by alternating oxidation and alternating reduction.

To surmount the challenges of the locus determination and accurate quantification of 5-methyl-2'-deoxycytidine ((5Me)dC) in DNA fragments that contain multiple (5Me)dC residues, we designed and synthesized two N-halogeno-N-sodiobenzenesulfonamide reagents that provide a new chemical method for probing (5Me)dC in DNA sequences. When the strategy we provided was combined with β-glucosyltransferase, (5Me)dC could be distinguished from 5-hydroxymethyl-2'-deoxycytidine ((5hm)dC) and deoxycytidine (dC) through the introduction of a glucose moiety to the hydroxyl group of (5hm)dC.

Translesion DNA synthesis, the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template, represents a common route toward mutagenesis and possibly disease development. To further define the mechanism of this promutagenic process, we synthesized and tested the enzymatic incorporation of two isosteric 5-substituted indolyl-2'deoxyriboside triphosphates opposite an abasic site. The catalytic efficiency for the incorporation of the 5-cyclohexene-indole derivative opposite an abasic site is 75-fold greater than that for the 5-cyclohexyl-indole derivative. The higher efficiency reflects a substantial increase in the k(pol) value (compare 25 versus 0.5 s(-1), respectively) as opposed to an influence on ground-state binding of either non-natural nucleotide. The faster k(pol) value for the 5-cyclohexene-indole derivative indicates that pi-electron density enhances the rate of the enzymatic conformational change step required for insertion opposite the abasic site. However, the kinetic dissociation constants for the non-natural nucleotides are identical and indicate that pi-electron density does not directly influence ground-state binding opposite the DNA lesion. Surprisingly, each non-natural nucleotide can be incorporated opposite natural templating bases, albeit with a greatly reduced catalytic efficiency. In this instance, the lower catalytic efficiency is caused by a substantial decrease in the k(pol) value rather than perturbations in ground-state binding. Collectively, these data indicate that the rate of the conformational change during translesion DNA synthesis depends on pi-electron density, while the enhancement in ground-state binding appears related to the size and shape of the non-natural nucleotide.