Covalent ligation studies on the human telomere quadruplex

Abstract

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.

Extreme conformational diversity in human telomeric DNA

“…It is now well established that G-rich DNA can form a wide range of structures, depending on the sequence and conditions (10,19,33,34). Our single-molecule investigation of G-quadruplexes derived from human telomeric DNA detected six distinct conformational states, which differ in static spectroscopic signals and͞or in their dynamic characteristics.…”

Polymorphism of human telomeric quadruplex structures

“…Structure polymorphism of the human telomeric G-quadruplexes, particularly for the 22-nt wtTel22 sequence, has been reported by various methods. The different CD spectra have been observed for human telomeric sequences in Na + and K + solution [32,37,39,40]. Multiple structures formed by wtTel22 in Na + and K + solution have also been reported in a 125 I-radioprobing study [36], a FRET study [33], a platinum cross-linking study [34], a chemical ligation study [40], and a photon correlation spectroscopic study [39].…”

“…A large number of biophysical and crosslinking studies have been reported on the K + -structure of human telomeric G-quadruplexes [32][33][34][36][37][38][39][40]. Structure polymorphism of the human telomeric G-quadruplexes, particularly for the 22-nt wtTel22 sequence, has been reported by various methods.…”

“…The different CD spectra have been observed for human telomeric sequences in Na + and K + solution [32,37,39,40]. Multiple structures formed by wtTel22 in Na + and K + solution have also been reported in a 125 I-radioprobing study [36], a FRET study [33], a platinum cross-linking study [34], a chemical ligation study [40], and a photon correlation spectroscopic study [39]. The hybrid-type G-quadruplex structure can explain the results and predictions made in previous biophysical studies where physiologically relevant conditions were used with the wild-type human telomeric sequence.…”

“…Because the K + structure is considered to be biologically more relevant due to the higher intracellular concentration of K + , it has been the subject of intense investigation [32][33][34][35][36][37][38][39][40][41][42][43][44][45], although the Na + structure was reported more than a decade ago [46]. In 2002, a crystal structure of the parallel-stranded G-quadruplex was reported using a 22-nt human telomeric sequence in the presence of K + [47].…”