The Investigation Of Long G-quadruplex DNA By AFM Imaging And Single Molecule Force Spectroscopy

Telomeres are non-coding DNAs located at the termini of linear chromosomes,whose structures are essential for genome stability. Human telomeric DNA consists ofa duplex region composed of TTAGGG repeats, ending in a100-200nucleotide (nt)G-rich single-stranded overhang. G-rich stretches of DNA have a high propensity toself-associate into planar guanine quartets (G-quartets) to give unusual structurescalled G-quadruplex. The folding and stabilization of a variety of G-quadruplex inG-rich sequences at the end of telomeres will obviously have an impact on the lengthof the telomere and hence the normal regulation of telomeres in the cell cycle. Severalresearch groups have mentioned the possibility that long-telomeric-overhang DNAmay form a higher-order G-quadruplex structure. However, higher-order telomericDNA structures with relatively large molecular sizes are not amenable to study bytraditional methods such as NMR spectroscopy and crystallography. Atomic forcemicroscopy (AFM) is a powerful and widely used imaging technique for visualizingmolecular structure at the single-molecule level. Single molecule force spectroscopy(SMFS) based AFM allows investigation of intramolecular or intermolecularinteractions of macromolecules, thus it makes the investigation of long G-quadruplexDNA possible.In our research, the rolling circle amplification (RCA) technology was firstlyemployed to prepare two types of long single-strand DNAs (ssDNA) containingguanine-rich telomeric motifs (G-4motifs), one with long spacer sequence (sample1)and the other without spacer sequence (sample2) in between the repeating G-4motifs.The structures of G-4motifs were characterized by using circular dichroism spectra (CD) and UV measurements. Then, we used AFM imaging to investigate thestructural characterisitics of those two long-telomeric DNAs. It was found that sample2can form higher-order DNA structure called consecutive G-quadruplex in thepresence of K~+. However, sample1exists mainly in the form of random aggregates,which may be ascribed to the effect of the spacer sequence. Finally, we systematicallystudied the mechanical properties of sample1and sample2in different buffersolutions using AFM-based single molecule force spectroscopy, and obtained a seriesof force fingerprints of random coil ssDNA and the unfolding of consecutiveG-quadruplexes.