THE FRAGILE-X SYNDROME SINGLE-STRAND D(CGG)(N) NUCLEOTIDE REPEATS READILY FOLD BACK TO FORM UNIMOLECULAR HAIRPIN STRUCTURES

Expansion of a d(CGG)(n) run within the 5'-untranslated region of the X-linked human gene FMR1 blocks FMR1 transcription, delays its replica tion, and precipitates the fragile X syndrome, We showed previously th at d(CGG)(n) tracts aggregate into interstrand tetrahelical complexes (Fry, M,, and Loeb, L, A, (1994) Proc. Natl. Acad. Sci, U. S. A. 91, 4 950-4954), Here we show that these sequences also form under physiolog ical conditions in in vitro unimolecular hairpin structures, Folding i s demonstrated by temperature-dependent mobility of d(CGG)(n) oligomer s in a nondenaturing polyacrylamide gel, by UV-hyperchromicity of ther mally denaturing oligomers, and by UV cross-linking of compact forms o f d(CGG)(n) runs interspersed by thymidine clusters, That the compact d(CGG)(n) structures are unimolecular is suggested by their zero-order kinetics of formation, Diethyl pyrocarbonate modification reveals a s ingle, 4-5 residue-long central or epicentral unpaired loop in folded d(CGG)(n) oligomers. The position of this loop remains unchanged by in sertion of thymidine clusters into 15- or 33-mer d(CGG) tracts as indi cated by KMnO4 probing of unpaired thymidines. The presence of a singl e loop in folded d(CGG)(n) oligomers and the accessibility of every gu anine to dimethyl sulfate modification suggest that they are hairpin a nd not tetraplex structures, Modeling indicates that different d(CGG)( n) hairpins are stabilized by guanine-guanine Hoogsteen hydrogen bonds or by Hoogsteen and Watson-Crick bonds. If formed in vivo, d(CGG)(n) hairpins could entail slippage and trinucleotide expansion during repl ication and could obstruct FMR1 gene transcription and replication.