Probing DNA triple helix structure by chemical ligation

Abstract

A series of oligomeric double and triple helical DNAs with irregular sequences of homo?urine and homopyrimidine strands were prepared. DNA triplexes were identified by CD spectroscopy and thermal denaturation profiles (biphaslc helix-coil transition). Condensation of ohgonucleotides on single and double-stranded DNA templates was performed using water-soluble carbodiimide, phosphodiester and pyrophosphate internucleotide bonds being newly formed. Such chemical ligation proved to be a sensitive monitor of changes in the sugar-phosphate backbone resulting from conversion of double to triple helix and of third-strand binding.DNA triplex; Chemical ligation of nucleic acids 1, INTRODUCTIONRenewed interest in DNA triple helices has recently intensified because of their potential role in the regulation of the eukaryotic genome [ 1,2]. There is also interest in generating nucleic acid ligands that recognize double-stranded targets. Such probes could serve as artificial gene repressors [3] and as DNA cleaving agents in sequencing procedures or in chromosome mapping [4]. Triple helices are believed to adopt an A-form structure, the purine strand and one pyrimidine strand being Watson-Crick paired and the other pyrimidine strand being Hoogsteen base-paired parallel to the purine strand along the major grooves [5-g]. In addition to physical methods [5-71, chemical approaches such as chemical and enzymatic cleavage-protection assays [8] and sequence-specific cIeavage of double helical DNA [9] have been used for studying the molecular structure of triplexes. Recently, it was also reported that double-stranded DNA can serve as a template to promote BrCN-induced condensation of homopyrimidine Hoogsteen paired third strands [lo], Using a different condensing agent, the present study demonstrates the potentialities of chemical ligation for monitoring changes in the sugar-phosphate backbone on converting a DNA duplex to triplex and for following third-strand binding. Because the two homopyrimidine strands of a triplex are antiparallel, they cannot replace each other in the triplex structure when irregular sequences are used. This makes it possible to indepen- MATERIALS AND METHODS Oligodeoxyribonucleotideswere synthesized by the phosphoramidite method on a Cyclon (Biosearch) DNA synthesizer. TTCCCTC and TCCTCTCTA were phosphorylated at the 5' end with polynucleotide kinase and [r-"P]ATP.ACTCCCTTCp was obtained from ACTCCCTTCrU as described in Ill]. CD1 and MES were from Merck. 2.1, Optical tneasurenientsEquimolar solutions of oligonucleotidcs l-7 (Scheme 1) were mixed to give comptexcs I-VI (Scheme 2). e260 VdUeS for oligomers 3-5 were assumed to be 8200, 13300 and 9000 (per monomer), respectively. iMelting curves of oligomer mixtures in 0.05 M MES, pH 6.0,0.02 M MgClz (buffer A) were determined on a Cary 219 spectrophotometer. The data were processed on a'tiewlett Packard 9825A computer. Circular dichroism measurements were made using a Jasco J-500C spectrometer. Cliet~lical ligation reactiorrOligomer mixtures (0.5 x IO-' M (...