Chemoproteomic discovery of a human RNA ligase.

RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5'-PO and 3'-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as a human enzyme promoting RNA ligation between 5'-PO and 3'-OH termini.

Concerted dynamics of metallo-base pairs in an A/B-form helical transition.

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-Hg-T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium.

HgII binds to C-T mismatches with high affinity.

Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C-T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C-T mismatches (kon ≈ 105 M-1 s-1, koff ≈ 10-3 s-1) as compared to T-T mismatches (kon ≈ 104 M-1 s-1, koff ≈ 10-4 s-1), resulting in very similar equilibrium binding affinities for both types of 'all natural' metallo base pairs (Kd ≈ 10-150 nM).

DNA Polymerase Inhibition by High Kinetic Stability of T-Hg-T Base Pairs.

A fluorescent surrogate of thymidine called DMAT was used for the first fluorescence-based study of HgII binding to discrete T-T sites in duplex DNA. The fluorescent properties of DMAT-A base pairs were highly sensitive to wild-type T-HgII-T base pair formation at an adjacent site, allowing for a determination of the precise thermodynamic and kinetic parameters of these metal binding reactions. T-HgII-T complexes exhibited equilibrium dissociation constants of Kd ≈ 8-50 nM.

DNA-Targeted Inhibition of MGMT.

The cationic porphyrin 5,10,15,20-tetrakis (diisopropyl-guanidine)-21H,23H-porphine (DIGPor) selectively binds to DNA containing O -methylguanine (O -MeG) and inhibits the DNA repair enzyme O -methylguanine-DNA methyltransferase (MGMT). The O -MeG selectivity and MGMT inhibitory activity of DIGPor were improved by incorporating Zn into the porphyrin. The resulting metal complex (Zn-DIGPor) potentiated the activity of the DNA-alkylating drug temozolomide in an MGMT-expressing cell line.

Fluorescent Base Analogue Reveals T-Hg-T Base Pairs Have High Kinetic Stabilities That Perturb DNA Metabolism.

The thymidine analogue T was used for the first fluorescence-based study of direct, site-specific metal binding reactions involving unmodified nucleobases in duplex DNA. The fluorescence properties of T-A base pairs were highly sensitive to mercury binding reactions at T-T mismatches located at an adjacent site or one base pair away. This allowed for precise determination of the local kinetic and thermodynamic parameters of T-Hg-T binding reactions.

A fluorescent surrogate of thymidine in duplex DNA.

is a new fluorescent thymidine mimic composed of 2'-deoxyuridine fused to dimethylaniline. exhibits the same pKa and base pairing characteristics as native thymidine residues, and its fluorescence properties are highly sensitive to nucleobase ionization, base pairing and metal binding.