Sequence-specific Recognition Of Double-helical DNA Research Articles

Drug interaction with triple-helical nucleic acids

Publisher Summary Triple-helical DNA was first discovered more than 40 years ago. Renewed interest in this structure developed at the end of the 1980s because of two discoveries. First, sequence-specific recognition of double-helical DNA can be achieved by short oligonucleotides forming a short intermolecular triple helix. Moreover, it has been shown that DNA can adopt an intramolecular triple-helical structure, also called H-DNA, under certain conditions. After a brief description of the characteristics of triple-helical DNA, this chapter describes the physicochemical aspects of drug binding to triplex DNA, with special emphasis on the different methods used to study the binding of low molecular weight compounds to triplex DNA, through a review of examples published in the literature. Some methodological and general considerations about targeting triplehelical nucleic acids with drugs are also discussed, and the biological applications of such drugs are presented. Ligands that are selective for triplex DNA can be used to promote triple helix formation, but their interest is much broader as they represent a unique class of structure-specific ligands that could be used as tools to study the biological relevance of triple-stranded structures, or potentially as drugs acting on structure-related gene regulation. Both high-resolution structural information and thermodynamic as well as kinetic studies still need to be pursued to yield a better understanding of how drugs bind specifically to triplex DNA.

Recognition of DNA by designed ligands at subnanomolar concentrations.

Small molecules that specifically bind with high affinity to any predetermined DNA sequence in the human genome would be useful tools in molecular biology and potentially in human medicine. Simple rules have been developed to control rationally the sequence specificity of minor-groove-binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Two eight-ring pyrrole-imidazole polyamides differing in sequence by a single amino acid bind specifically to respective six-base-pair target sites which differ in sequence by a single base pair. Binding is observed at subnanomolar concentrations of ligand. The replacement of a single nitrogen atom with a C-H regulates affinity and specificity by two orders of magnitude. The broad range of sequences that can be specifically targeted with pyrrole-imidazole polyamides, coupled with an efficient solid-phase synthesis methodology, identify a powerful class of small molecules for sequence-specific recognition of double-helical DNA.

Specific recognition of CG base pairs by 2-deoxynebularine within the purine.purine.pyrimidine triple-helix motif.

The sequence-specific recognition of double-helical DNA by oligodeoxyribonucleotide-directed triple-helix formation is limited mostly to purine tracts. Within the geometric constraints of the phosphate-deoxyribose position of a purine-purine-pyrimidine triple-helical structure, model building studies suggested that the deoxyribonucleoside 2'-deoxynebularine (dN) might form one specific hydrogen bond with cytosine (C) or adenine (A) of Watson-Crick cytosine-guanine (CG) or adenine-thymine (AT) base pairs. 2-Deoxynebularine (dN) was incorporated by automated methods into purine-rich oligodeoxyribonucleotides. From affinity cleavage analysis, the stabilities of base triplets within a purine.purine.pyrimidine (Pu.Pu.Py) triple helix were found to decrease in the order N.CG approximately N.AT >> N.GC approximately N.TA (pH 7.4, 37 degrees C). Oligodeoxyribonucleotides containing two N residues were shown to bind specifically within plasmid DNA a single 15 base pair site of the human immunodeficiency virus genome containing two CG base pairs within a purine tract. This binding event occurs under physiologically relevant pH and temperature (pH 7.4, 37 degrees C) and demonstrates the utility of the new base. Quantitative affinity cleavage titration reveals that, in the particular sequence studied, an N.CG base triplet interaction results in a stabilization of the local triple-helical structure by 1 kcal.mol-1 (10 mM NaCl, 1 mM spermine tetrahydrochloride, 50 mM Tris-acetate, pH 7.4, 4 degrees C) compared to an A.CG base triplet mismatch.

Recognition of all four base pairs of double-helical DNA by triple-helix formation: design of nonnatural deoxyribonucleosides for pyrimidine.cntdot.purine base pair binding

The sequence-specific recognition of double-helical DNA by oligonucleotide-directed triple-helix formation is limited mostly to purine tracts. Design leads that could expand the recognition code to all four Watson-Crick base pairs would provide one step toward a general solution targeting single sites in megabase size DNA. The nonnatural deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamidophenyl)imidazole (D3) was synthesized in four steps and incorporated by automated methods into pyrimidine oligodeoxyribonucleotides. Within a pyrimidine oligonucleotide, D3 binds pyrimidine.purine base pairs with higher affinity than it binds purine.pyrimidine base pairs. From affinity-cleaving analysis, the stabilities of base triplets decrease in the order D3.TA is similar to D3.CG > D3.AT > D3.GC. Such specificity allows binding by triple-helix formation at an 18 base pair site in SV40 DNA containing all four base pairs at physiologically relevant pH and temperature. The stabilities of these novel triplets may be an example of shape-selective recognition of CG and TA Watson-Crick base pairs in the major groove.

Sequence-specific alkylation of double-helical DNA by oligonucleotide-directed triple-helix formation

Affinity cleaving, a method that relies on the attachment of a nonspecific cleaving moiety, such as EDTA•Fe(ll), to a DNA binding molecule, facilitates the elucidation of the structural principles for DNA recognition. The determination of the sequence specificities, groove locations, and binding orientations of peptide analogues, protein-DNA binding motifs, and oligonucleotide-triple-helix motifs has provided reliable models for the sequence-specific recognition of double-helical DNA. It now becomes possible to combine these binding molecules with domains capable of base-specific and quantitative modification of DNA (Figure 1). We report the design and synthesis of an ligodeoxyribonucleotide equipped with an electrophile at the 5'-end that binds to double-helical DNA by triple-helix formation and alkylates predominantly at a single guanine base adjacent to the target DNA sequence in high yield.