Tetraplex formation of surface-immobilized human telomere sequence probed by surface plasmon resonance using single-stranded DNA binding protein

Abstract

Many sequences in genomic DNA are able to form unique tetraplex structures. Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases. Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces. Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions. In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe. We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method. Our results also show that immobilization destabilized the tetraplex structure.

Fast detection of quadruplex structure in DNA by the intrinsic fluorescence of a single‐stranded DNA binding protein

“…We also used the SSB to detect the pH-induced structural transition in (C 3 TA 2 ) 3 C 3 , the core sequence of the C-rich strand of human telomeric DNA. From neutral to pH 5.0, the fluorescence quenching of the SSB clearly indicated a pH-dependent structural transition of the DNA (Figure 6) similar to those observed by other techniques (Manzini et al, 1994;Zeng et al, 2005). Within this range, pH change did not significant affect the fluorescence and binding activity of the SSB.…”

“…The G24 loaded with SSB in the Li þ solution resulted in retarded migration compared to the free G24 indicating that it was bound by the SSB. When the quadruplex formed in K þ solution was UV-crosslinked to prevent it from unfolding (Williamson et al, 1989;Zhao et al, 2004;Zeng et al, 2005), the DNA was not bound by the SSB since the inclusion of SSB did not affect its migration.…”

“…UV crosslinking and gel mobility shift assay UV crosslinking of G24 G-quadruplex was carried out as described in Williamson et al (1989), Zhao et al (2004) and Zeng et al (2005). 32 P-5 0 -end-labeled G24, crosslinked or uncrosslinked, were mixed with SSB or C24 at equimolar ratio in buffer C (25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM DTT, 150 mM LiCl).…”

Real-time Investigation of SV40 Large T-antigen Helicase Activity Using Surface Plasmon Resonance

“…into the foldover quadruplex. We used E. coli single-strand binding protein (SSB) to verify in situ folding of the quadruplex on the sensor chip [30] and the sequence was demonstrated to be greater than 95% folded (data not shown). It is very unlikely that any bimolecular or four-stranded quadruplex structures would form on the sensor chip since the DNA was folded after immobilization and the surface coverage of immobilized DNA ligand was kept low.…”

“…buffer. The extent of folding was verified using E. coli single strand binding protein (SSB) [30]. The maximum response, R max , that could be observed from SSB interacting with immobilized G4'DNA if it were 100% single-stranded was calculated using Eq.…”