Volume 7 Issue 6
Dec.  2021
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Ghimire Chiran, Guo Peixuan. Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting[J]. Biophysics Reports, 2021, 7(6): 449-474. doi: 10.52601/bpr.2021.210003
Citation: Ghimire Chiran, Guo Peixuan. Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting[J]. Biophysics Reports, 2021, 7(6): 449-474. doi: 10.52601/bpr.2021.210003

Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting

doi: 10.52601/bpr.2021.210003
Funds:  The research in P. Guo’s lab was supported by NCI/NIH grant (U01CA207946) and NIBIB/NIH grant (R01EB019036). P. Guo's Sylvan G. Frank Endowed Chair position in Pharmaceutics and Drug Delivery is funded by the CM Chen Foundation. We thank Hui Zhang for initiation of the instrumentation of smTIRF; Mario Vieweger and Hanbin Mao for their collaboration in instrumentation of the optical tweezers; Yinmei Li for communication of optical tweezer 20 years ago; Eckhard Jankowsky, David Rueda, Nils Walter, Noji Hiroyuki, Taekjip Ha, Toshio Yanagida, Kazuhiko Kinosita Jr., Wulf-Dieter Moll, Chris Meiners, Meredith Lambert, Peter Stockley, Faqing Huang and Masasuke Yoshida for their technical assistance and valuable comments on smTIRF. We thank Lixia Zhou, Nic Burns and Archie Bhullar for proofreading of the manuscript.
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  • Corresponding author: guo.1091@osu.edu (P. Guo)
  • Received Date: 07 February 2021
  • Accepted Date: 23 March 2021
  • Available Online: 07 July 2021
  • Publish Date: 31 December 2021
  • Life science is often focused on the microscopic level. Single-molecule technology has been used to observe components at the micro- or nanoscale. Single-molecule imaging provides unprecedented information about the behavior of individual molecules in contrast to the information from ensemble methods that average the information of many molecules in various states. A typical feature of living systems is motion. The lack of synchronicity of motion biomachines in living systems makes it challenging to image the motion process with high resolution. Thus, single-molecule technology is especially useful for real-time study on motion mechanism of biomachines, such as viral DNA packaging motor, or other ATPases. The most common optical instrumentations in single-molecule studies are optical tweezers and single molecule total internal refection fluorescence microscopy (smTIRF). Optical tweezers are the force-based technique. The analysis of RNA using optical tweezer has led to the discovery of the rubbery or amoeba property of RNA nanoparticles for compelling vessel extravasation to enhance tumor targeting and fast renal excretion. The rubbery property of RNA lends mechanistic evidence for RNAs use as an ideal reagent in cancer treatment with undetectable toxicity. Single molecule photobleaching allows for the direct counting of biomolecules. This technique was invented for single molecule counting of RNA in the phi29 DNA packaging motor to resolve the debate between five and six copies of RNA in the motor. The technology has subsequently extended to counting components in biological machines composed of protein, DNA, and other macromolecules. In combination with statistical analysis, it reveals biomolecular mechanisms in detail and leads to the development of ultra-sensitive sensors in diagnosis and forensics. This review focuses on the applications of optical tweezers and fluorescence-based techniques as single-molecule technologies to resolve mechanistic questions related to RNA and DNA nanostructures.
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