Identification of the New Type of G-Quadruplex with Multiple Vacant Sites in Human Telomeric DNA

Abstract

To date, few studies have reported on the folding mechanism of tandem G-quadruplexes in human telomeric DNA. Hence, the control of the biofunctions of G-quadruplex, which requires a thorough understanding of its dynamic behavior, is limited. Here, we investigated the folding/unfolding behavior of human telomeric sequences with lengths over 10 kilonucleotide (knt) by circular dichroism (CD) spectroscopy, UV melting assay, and atomic force microscopy (AFM)-based single-molecule force spectroscopy. A novel G-quadruplex with multiple vacant sites was captured in the long human telomeric DNA and denoted as pre-G-quadruplex (pre-GQ). According to the number of vacant sites, pre-GQ is divided into two types (four vacant sites in type 1 and two vacant sites in type 2), among which type 1 is the dominant one. The unfolding force of a tandem pre-GQ was 10 pN lower than that of a complete G-quadruplex, suggesting the destabilized structure of the tandem pre-GQ due to its incompletely folded state. Our results revealed that the folding of long-telomere G-strand could be achieved by a two-step process involving fast transition (in seconds) from unstructured ssDNA to tandem G-hairpin states and slow folding of free guanines into the vacant sites in tetraplex to generate type 1 pre-GQ, type 2 pre-GQ, or complete GQ.