Covalent ligation studies on the human telomere quadruplex.
Qi J, Shafer RH
Nucleic Acids Research (2005)
Category: DNA structure, telomere ¤ Added: Jun 06, 2005 ¤ Rating: ◊◊
Recent X-ray crystallographic studies on the human telomere sequence d[AGGG(TTAGGG)3] revealed a unimolecular, parallel quadruplex structure in the presence of potassium ions, while earlier NMR results in the presence of sodium ions indicated a unimolecular, antiparallel quadruplex. In an effort to identify and isolate the parallel form in solution, we have successfully ligated into circular products the single-stranded human telomere and several modified human telomere sequences in potassium-containing solutions. Using these sequences with one or two terminal phosphates, we have made chemically ligated products via creation of an additional loop. Circular products have been identified by polyacrylamide gel electrophoresis, enzymatic digestion with exonuclease VII and electrospray mass spectrometry in negative ion mode. Optimum pH for the ligation reaction of the human telomere sequence ranges from 4.5 to 6.0. Several buffers were also examined, with MES yielding the greatest ligation efficiency. Human telomere sequences with two phosphate groups, one each at the 3' and 5' ends, were more efficient at ligation, via pyrophosphate bond formation, than the corresponding sequences with only one phosphate group, at the 5' end. Circular dichroism spectra showed that the ligation product was derived from an antiparallel, single-stranded guanine quadruplex rather than a parallel single-stranded guanine quadruplex structure.