• Bhisma K Patel

      Articles written in Journal of Chemical Sciences

    • Synthetic nucleases crafted fromL-lysine

      Darshan Ranganathan Rakesh K Mishra Bhisma K Patel Narendra K Vaish

      More Details Abstract Fulltext PDF

      A central strategy for the design of chemical nucleases is presented. This involves the utilization of the α-amino carboxylate unit ofL-lysine to form Cu(II) templates to function as the cleaving centre on the one hand and ω-amino group for the attachment of DNA recognition elements on the other, thus giving rise to a duplex recognition termini, harbouring a centrally placed Cu(II) for potentiation of oxidative scission. The recognition element studied encompasses a spectrum of structures ranging from quinazolines and purine residues to specifically crafted peptide segments that have potential to form secondary structures. These could be represented as R-K-Cu-K-R wherein R is the recognition system and K-Cu-K, a composite crafted from lysine, consisting of the cleaving centre from metal complexation of a-amino acid unit and the spacer consisting of the four methylene groups of the side chain. The binding and DNA scission profile of the sixteen chemical nucleases thus prepared and fully characterized have been probed by UV, fluorescence quenching and electrophoretic studies. Their binding to calf thymus DNA is associated with a decrease in ε and an ∼ 10–15 nm red shift. The involvement of GC sequence in binding is indicated from studies with poly[d(G-C)d(G-C)] and poly[d(A-T).d(A-T)], wherein the hypochromicity and red shift were found to be quite pronounced in the former.

      Fluorescence quenching studies with Bz-Trp-Trp-K-Cu-K-Trp-Trp-Bz demonstrated the binding of one ligand at approximately every stretch of 112 bp and approximately a stretch of 80 bp in the presence of salt. The DNA cleaving properties of all the nucleases were demonstrated with pBR 322 and p blue script 11KS using standard protocols. In all cases, covalently closed supercoiled (form I DNA) is converted largely into open circular (form II) suggesting nicking of the single strand at binding sites. Sequence specificity experiments with the nuclease. Bz-Ala-Gly-K-Cu-K-Gly-Ala-Bz in a32P 3’-end labeled 117 bp restriction fragment (Eco RI/Hind III) of pUC-18 showed almost exclusive attacks at thymidylate residues in particular, thymines corresponding to 5’T of the CTAT(3’-5’) box. Whilst the most preferred site of attack is found at T of 3’-ATC-5’ at the trinucleotide level, cleavage studies at low concentration have shown that at pentanucleotide level, the lone sequence 3’-GATCT-5’ (a part of the inverted repeat -GAGATCTC-) is favoured (fragment 92) over the more frequently occurring 3’-TATCT-5’ segment.

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