Characterization of inter-genomic and telomeric G-quadruplex structures through atomic force microscopy

G-quadruplex;Atomic Force Microscopy

Kreig, Alex ; Myong ; Su-A

关键词: G-quadruplex;    Atomic Force Microscopy;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46566/Alex_Kreig.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The G-quadruplex DNA structure, a secondary folding motif of DNA, is found throughout the genome. The dynamics of these structures has been seen to affect processes such as telomerase elongation and regulation of gene transcription.Typical investigation techniques require these structures to be present in very high concentrations and in non-biological buffers.Adding to the issues with current methods of investigation, this structure currently relies on bulk measurements, which limit the accuracy with which it can be measured.In this report, we present newly developed atomic force microscopy-based assays for quantifying G-quadruplex folding patterns with sub nanometer accuracy. The assays are based upon a duplex DNA handle ranging from 18 to 525 base pairs in length containing an overhang sequence utilized to attach the DNA to a mica surface.The overhang sequence pairs with a possible G-quadruplex forming sequence and allows for the retention and identification of the folded structure. Using a 12 base DNA probe, we demonstrate that the location of G-quadruplex folding on long strands of DNA are not preferred to occur at the distal end, contrary to DMS foot printing studies. We demonstrate that this method enables detection of the two major folding motifs and is able to distinguish height differences between them. The high resolution and low concentration requirements, combined with the ability to image hundreds of single molecules per study, allow this method to provide quick and accurate characterization of G-quadruplex folding.