In vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerases

Triplet repeats are the most abundant simple sequencerepeats in the coding and non-coding sequences of allknown eukaryotic genomes [1]. The frequency of spe-cific types of triplet repeats and their localization ingenes vary significantly between genomes, reflectingtheir important role in genome evolution [1,2]. Expan-sions of DNA triplet repeat sequences are associatedwith 16 inherited neurological disorders known astriplet repeat expansion diseases [3–5], which can leadto total disability and death. The severity of a tripletrepeat expansion disease is increased anticipativelyand the age of onset is reduced with each successivegeneration [6,7]. The high mutation rate of tripletrepeats makes them a rich source of quantitativegenetic variation [8–11]. The tendency for repeatingDNA strands to form hairpin loops or slipped confor-mations, and their inherent conformational properties,for example their high degree of flexibility, writhingand the stability of the hairpin formation, are impor-tant in the investigation of DNA slippage phenomena[3,11,12].Among the non-B-form DNA conformations formedby triplet repeats, simple bulged structures (one ormore unpaired bases) have been postulated as inter-mediates in the synthesis of slipped DNA and areassociated with the unstable expansion of tripletrepeats on the basis of their entropy [13]. Severalgroups have shown an interest in developing smallmolecules that possess specific effects for DNA tripletrepeat strand slippage [14–23]. The most promisingand successful bulge-specific agent discovered to dateoriginated from studies on the enediyne natural prod-uct neocarzinostatin chromophore (NCS-chrom) [24].Its isostructural mimic, NCSi-gb (Scheme 1A) bindsbulge DNA at sub-micromolar concentrations [25],and is also able to induce formation of the bulge-bind-ing pocket by stacking between the base pairs thatflank the bulge site in the oligonucleotide [26,27].