DNA sequence recognition by under-methylated analogues of triostin A

Abstract

Two new analogues of TANDEM (des-N-tetramethyl triostin A) have been synthesised in an effort to elucidate the molecular basis of DNA nucleotide sequence recognition in this series of compounds. Their binding preferences have been investigated by DNAase I footprinting and differential inhibition of restriction nuclease attack. The presence of a single N-methyl group on only one valine residue (in [N-MeVal4] TANDEM) abolishes the ability to recognise DNA, presumably because this antibiotic analogue has suffered an unfavourable conformational change in the depsipeptide ring. A bis-methylated analogue, [N-MeCys3, N-MeCys7]TANDEM, was found to interact quite strongly with DNA and afforded binding sites, rich in AT residues, identical to those of TANDEM. Footprinting with various DNA fragments of known sequence showed that this analogue recognises sequences containing the dinucleotide TpA, although we cannot exclude the possibility that it binds to ApT as well. [N-MeCys3, N-MeCys7]TANDEM inhibits cutting by RsaI, a restriction enzyme that recognises GTAC but not by Sau3AI which recognises GATC. This provides further supportive evidence that the ligand (and, by extension, TANDEM itself) prefers binding to sequences containing the dinucleotide step TpA.

Preferred Binding Sites for [N-MeCys3,N-MeCys7]TANDEM Determined Using a Universal Footprinting Substrate

“…We have shown that the best tetranucleotide binding site for TANDEM contains the sequence ATAT, consistent with the previous observation that it binds best to TpA steps which are flanked by A and T residues (8,9,17). However, the binding is strongly influenced by the identity of the surrounding bases and all TpA steps do not constitute good binding sites.…”

“…Subsequent footprinting studies showed that TANDEM and its derivative [N-MeCys 3 ,N-MeCys 7 ]TANDEM bind to the dinucleotide TpA (8 -11), and this has been confirmed by NMR studies which have suggested mechanisms by which the ligand targets this sequence (12)(13)(14)(15)(16). However, several studies have suggested that TpA alone does not constitute the best binding sequence, and that the preferred sites are flanked by other AT residues (8,9,17,18), consistent with the observation that the ligand binds cooperatively to poly(dA-dT) (7). We have therefore analyzed the binding of this ligand to TpA when flanked by different base pairs, by quantitative DNase I footprinting using a novel DNA substrate which contains all possible 136 tetranucleotide sequences.…”

DNA recognition by quinoxaline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of triostin A and TANDEM

“…Footprinting studies eventually revealed that triostin A binds specifically to sequences centred around a CpG step, whereas TANDEM selectively recognizes TpA sites [14][15][16][17]. Most binding studies have been performed with [N-MeCys$, NMeCys(]TANDEM rather than with TANDEM itself, but the two synthetic drugs appear equivalent in terms of binding strength and specificity.…”

Strand orientation of [α]‐oligodeoxynucleotides in triple helix structures: Dependence on nucleotide sequence

“…However, it must be emphasized that the binding mode of these new lexitropsins is far from fully understood and that it is not yet possible to design reagents specifically targeted to desired sequences by this approach. Another direction of research involves the design of ligands with mixed binding modes, such as intercalator-peptide conjugates like TANDEM (Low et al 1986), and intercalatorminor-groove binder conjugates (Bailly et al, 1990). However, to date our knowledge of the sequencedependent variation in DNA structure and of the perturbation induced by simultaneous intercalation and groove binding is incomplete and does not allow a full * Author to whom correspondence should be addressed.…”