Characterization of a K+-Induced Conformational Switch in a Human Telomeric DNA Oligonucleotide Using 2-Aminopurine Fluorescence

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Abstract
Human telomeric DNA consists of tandem repeats of the DNA sequence d(GGGTTA). Oligodeoxynucleotide telomere models such as d[A(GGGTTA)3GGG] (Tel22) fold in a cation-dependent manner into quadruplex structures consisting of stacked G-quartets linked by d(TTA) loops. NMR has shown that in Na+ solutions Tel22 forms a “basket” topology of four antiparallel strands; in contrast, Tel22 in K+ solutions consists of a mixture of unknown topologies. Our previous studies on the mechanism of folding of Tel22 and similar telomere analogues utilized changes in UV absorption between 270 and 325 nm that report primarily on G-quartet formation and stacking showed that quadruplex formation occurs within milliseconds upon mixing with an appropriate cation. In this study, we assess the dynamics and equilibria of folding of specific loops by using Tel22 derivatives in which the dA residues were serially substituted with the fluorescent reporter base, 2-aminopurine (2-AP). Tel22 folding induced by Na+ or K+ assessed by changes in 2-AP fluorescence consists of at least three kinetic steps with time constants spanning a range from milliseconds to several hundred seconds. Na+-dependent equilibrium titrations of Tel22 folding could be approximated as a cooperative two-state process. In contrast, K+-dependent folding curves were biphasic, revealing that different conformational ensembles are present in 1 and 30 mM K+. This conclusion was confirmed by 1H NMR. Molecular dynamics simulations revealed a K+ binding pocket in Tel22 located near dA1 that is specific for the so-called hybrid-1 conformation in which strand 1 is in a parallel arrangement. The possible presence of this topologically specific binding site suggests that K+ may play an allosteric role in regulating telomere conformation and function by modulating quadruplex tertiary structure.