The properties of duplex CTG⅐CAG and CGG⅐CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 ؋ 10 ؊19 erg⅐cm for CTG and 1.27 ؋ 10 ؊19 erg⅐cm for CGG, ϳ40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were ϳ60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2. Eleven human genetic disorders (including fragile X syndrome, myotonic dystrophy, Kennedy's disease, Huntington's disease, spinocerebellar ataxia type 1, dentatorubral-pallidoluysian atrophy, and Friedreich's ataxia) are characterized at the molecular level by the expansion of DNA triplet repeats (CTG, CGG, or AAG) 1 from Ͻ15 copies in normal individuals to scores of copies in affected cases (1-6). In some cases, the CTG and CGG tracts are transcribed into mature mRNAs, whereas the AAG tracts in Friedreich's ataxia are in the first intron of the frataxin gene. The mechanism for expansion is not known, but it may involve slippage of the complementary strands during DNA synthesis (7-10). Expanded alleles undergo further expansions upon passage to offspring and, in some diseases, are associated with the clinical observation called anticipation, whereby the symptoms become more severe in each successive generation and with an earlier age of onset (1-5). This is a novel type of mutation and shows non-mendelian genetic transmission (11,12).Prior investigations suggested that triplet repeat sequences (TRS) 2 do not have the properties of random B-DNA. First, CTG tracts greatly facilitate nucleosome assembly (13-15), which, in turn, may repress transcription. Second, DNA synthesis in vitro pauses at specific loci in fragments containing CTG and CGG (16). Third, long tracts of AAG and AGG form intramolecular triplexes that arrest DNA synthesis (17). Fourth, CTG and CGG migrate up to 30% more rapidly than expected on polyacrylamide gel electrophoresis, whereas their migration is normal on agarose gels (18). Fifth, CTG is preferentially expanded in Escherichia coli compared with the other nine TRS (8). Sixth, the frequency of expansions and deletions in E. coli (7,9,10) is influenced by the direction of replication, suggesting the formation of stable hairpin loops in the lagging strand template or the newly synthesized nascent strand.Conformational investigations were conducted on plasmids and restriction fragments containing CTG and CGG to evaluate their role in the biological behaviors described above. Several methods were applied, including circularization kinetics, apparent helical repeat determinations, the rate of migration through acrylamide and agarose gel electrophoresis, chemical and enzymatic probe analyses, two-dimensional gel electrophoresis, and the induction of an immune response. The analyses indicate that both CTG and CGG exist as fully paired, r...