Quantum dot-facilitated single-molecule microscopy
Single-molecule approaches can provide insights into DNA-protein interactions. These approaches often utilize techniques such as single-molecule Förster resonance energy transfer (smFRET) and colocalization to investigate conformational changes in real-time. However, the short photobleaching lifetime of organic dyes can be an issue for experiments that require extended observation time. Although quantum dots are longer lived than organic dyes, it is not clear whether they are suitable for use as donors for quantitative measurements of inter and intra-molecular distances in smFRET experiments. In this dissertation, we attempted to carry out quantitative distance measurements at the single-molecule level using measurements of the energy transfer efficiency between quantum dots as donors and organic dyes as acceptors. We used DNA as an adjustable linker between FRET pairs and studied two different attachment chemistries (biotin-streptavidin and EDC coupling), which permitted us to consider both how the length of the DNA linker and how the physical geometry imposed by the method of attachment between the DNA and the quantum dot impacted energy transfer. Our observations indicate that, due to physical constraints introduced by the location of DNA attachment, each quantum dot needs to be treated as a unique item. Therefore, the use of quantum dots as donors in FRET measurements for the purpose of quantitative distance determination is not recommended. On the other hand, quantum dots can be used quite effectively for other types of single-molecule assays. In this dissertation, we also describe how we used quantum dots along with TIRF microscopy to study mechanism by which the restriction endonuclease BcnI cleaves duplex DNA. BcnI is a monomeric enzyme that cleaves duplex DNA by first nicking one strand, then flipping, rebinding, and cleaving the second strand. To gain insight into the flipping mechanism, we used TIRF microscopy to observe the cleavage of surface-immobilized quantum dot-labeled DNA molecules by BcnI at the single-molecule level. We were able to measure the time required for cleavage for hundreds of BcnI-mediated cleavage events. By analyzing the resulting dwell time distributions, we were able to reveal the presence of intermediate steps and make some preliminary inferences about the flipping mechanism.