Viruthachalam Thiagarajan

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH-). The CPD repair mechanism involves electron transfer from photoexcited FADH- to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH-. The two electron transfer processes occur on time scales of 10-10 and 10-9 s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T∘-) and electron return from T∘- to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride

BACKGROUND We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA. METHODS The combination of experimental and theoretical methods was used. RESULTS The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by (31)P NMR experiments. The thermodynamics of the ligand-nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory. CONCLUSIONS Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling. GENERAL SIGNIFICANCE The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein-DNA interactions. However, these are not clear for the DNA-small molecule interactions and we believe that our studies will bring a new insight into such phenomena.

NBD-Based Green Fluorescent Ligands for Typing of Thymine-Related SNPs by Using an Abasic Site-Containing Probe DNA

The binding behavior of green fluorescent ligands, derivatives of 7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N1‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)propane‐1,3‐diamine (NBD‐NH2) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52×106 M−1. From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD‐NH2 is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2‐amino‐6,7‐dimethyl‐4‐hydroxypteridine (diMe‐pteridine).

Excited state behaviour of acridinedione dyes in PMMA matrix: inhomogeneous broadening and enhancement of triplet

Photophysics and photochemistry of acridinedione (ADD) dyes doped in PMMA matrix have been studied using fluorescence and flash photolysis technique. The absorption and emission spectra of acridinedione dyes in PMMA matrix are broad. A pronounced influence of λexc on the red shift of ADD fluorescence spectra was found which is independent of the various substituents in the ADD dyes. This observed shift was explained by the inhomogeneity in the PMMA matrix site results in the migration of the electronic excitation energy from the blue centres to red ones. The inhomogeneous distribution of ADD dyes in PMMA matrix causes the non-exponential fluorescence decay. The triplet lifetimes of these dyes in polymer matrix is found to be in millisecond time domain, whereas in solution the triplet lifetime is in microseconds. Laser flash photolysis shows that the photoionisation of ADD dyes in PMMA matrix results in the formation of an electron and a cation radical. Steady state photolysis of ADD dyes in PMMA matrix was carried out. ADD dyes have higher photostability in PMMA matrix than in solution.

Simultaneous recognition of nucleobase and sites of DNA damage: Effect of tethered cation on the binding affinity

BACKGROUND The 3,5-diamino-N-(3-aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH(2)) has been synthesized and characterized by Mass and (1)H NMR. The selective binding of the ligand to thymine (T) target base is investigated by the melting temperature (T(m)) and fluorescence measurements. METHODS Thermal denaturation study of DNA duplex containing T target base revealed the DeltaT(m) of 5.1 degrees C, while least influence was observed for other target bases. The fluorescence of the ligand DCPC-NH(2) is quenched only upon adding the DNA containing T target base. RESULTS The binding constant for the interaction of the ligand to T target base containing DNA duplex was determined to be 4.7 (+/-0.3)x10(6) M(-1). The tethered cation in the ligand is found to enhance the binding constant. The ligand binds to both a target nucleotide and an AP site on the complimentary strand for the target strand in a DNA duplex. GENERAL SIGNIFICANCE Interestingly, the electronic behavior of the ligand depends on the bases flanking the AP site. Its fluorescence is quenched with guanine flanking bases, while it is enhanced with DNA duplex containing T bases flanking an AP site. Finally, the binding modes were visualized by molecular modeling.