Investigating G-quadruplex Stabilizing Compound Alters Unfolding Pathway of Human Telomeric DNA Using Optical Tweezers
Date Issued
2014
Date
2014
Author(s)
Huang, Tao
Abstract
We used an optical tweezers platform to study the folding and unfolding pathway of individual molecules containing single-stranded DNA human telomeric G-quadruplex (G4) sequence, (TTAGGG)4. We home-built an optical tweezers platform with force-clamp capability, which held a DNA tether at a constant force, to determine the DNA length in high spatro-temperal resolution. We included an acousto-optic modulator (AOM) to maintain the laser stability and madulate laser output intensity. By modulating the applied voltage of AOM, feedbacked from the laser power measurement, we achieved to maintain the laser power with standard deviation less than 0.01mW over 20 minutes. For G4 experiments, these G4 containing DNA molecules are found to form the G-quadruplex structure based on Hoogsteen basepairing in 150 mM Na+ solution. When forces were applied to unfold the G4-containing DNA molecules, most of the unfolding traces showed one or two transitions, suggesting the existence of one stable intermediate state. The total unfolding distance was consistent with the expected value of unfolded G4 structure. However, when the DNA molecules were pre-incubated with a G4 stabilizing ligand BMVC, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole, most DNA molecules showed three unfolding transitions, suggesting of two stable unfolding intermediate states. Using the force-clamp assay, we found that three extension states exist in the traces of G4 DNA. When G4 was pre-incubated with BMVC, the number of extension states also increases to four. Both force-extension and force-clamp results suggest that BMVC-bound G4 structures are able to withstand higher force than the ligand-free G4 structure, thus revealing more intermediate states.
Subjects
鳥嘌呤-四股結構
解構機制
人類端粒序列
BMVC
單分子光學鉗
Type
thesis
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