Structure Conversion Of Human Telomeric Sequence Studied By Single-molecule Tethered Particle Motion

Abstract

Telomeres contain G-rich tandem repeats of single-stranded DNA sequences at 3′ tail. The G-rich sequences can be folded into a secondary structure named G-quadruplexes by Hoogsteen base pairing in the presence of monovalent cations (such as Na+, K+). The folding of telomeric DNA into the G-quadruplexes may inhibit telomerase activity for the proliferation of cancer cells. Moreover, the change of a quadruplex conformation may play an important role in biological effect. Thus, understanding structure conversion between the folded and unfolded G-quadruplex structures, and how the structure conversion is mediated by ions, its anti-sense sequence and its stabilizers are important to telomere biology. Here, we have directly monitored the conversion between the folded and unfolded structures in human telomeric AGGG(TTAGGG)3 sequence by the single-molecule tethered particle motion (TPM) method. TPM method monitors the DNA length change caused by the G-quadruplex formation, and allows us to monitor the conversion mechanism of telomeric sequences in real-time. In the presence of its antisense sequence, the folded G-quadruplex structures (in 150 mM Na+) can be disrupted and converted to the unfolded conformation, and the conversion frequency depends strongly on the antisense concentrations. In the great excess of antisense sequence, the conversion efficiency is about 10 % in our single-molecule essay (N >100). However, in the presence of Li+ ions, the efficiency of antisense interaction increases significantly to 50 %. Since Li+ ions have been proposed to destabilize the G-quadruplex structure, our results suggest the antisence sequence interacts with the unfolded or, at least, partially unfolded state of telomeric sequences. Experiments of structure conversion between 150 mM Na+ and 100 mM K+ ions, and effects of structure conversion in the presence of a G-quadruplex stabilizer (BMVC) will also be discussed.