High-resolution single-molecule optical trapping measurements of transcription with basepair accuracy: instrumentation and methods

William J. Greenleaf; Kirsten L Frieda; Elio A. Abbondanzieri; Michael T. Woodside; Steven M. Block

doi:10.1117/12.739631

5 September 2007 High-resolution single-molecule optical trapping measurements of transcription with basepair accuracy: instrumentation and methods

William J. Greenleaf, Kirsten L Frieda, Elio A. Abbondanzieri, Michael T. Woodside, Steven M. Block

Author Affiliations +

Proceedings Volume 6644, Optical Trapping and Optical Micromanipulation IV; 664406 (2007) https://doi.org/10.1117/12.739631
Event: NanoScience + Engineering, 2007, San Diego, California, United States

Abstract

Optical traps allow the single-molecule investigation of the chemo-mechanical properties of biomolecules¹. We have developed an ultra-stable optical trapping system capable of ångstr&diaero;m-level position resolution and used it to monitor transcriptional elongation by single molecules of E. coli RNAP polymerase (RNAP). This optical trapping system uses the anharmonic region of the trapping potential, where differential stiffness vanishes, to generate a force-clamp that operates without feedback-associated noise². We demonstrate methods of calibrating this anharmonic trapping region and strategies to eliminate common sources of noise associated with air currents. Records of transcriptional elongation obtained with this device showed discrete steps averaging ~3.7 Å, a distance equivalent to the mean rise per base found in B-DNA³. To determine the absolute position of the RNAP on the DNA template, we monitored transcription under conditions in which a single nucleotide species was held rate-limiting and then aligned the resulting transcriptional pauses with the occurrence of this rate-limiting species in the underlying template. The aligned pause patterns recorded from four molecules, each measured with a different ratelimiting nucleotide species, were used to determine the sequence of a short region of unknown DNA, demonstrating that the motion of a single processive nucleic acid enzyme may be used to extract sequence information directly from DNA⁴.

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William J. Greenleaf, Kirsten L Frieda, Elio A. Abbondanzieri, Michael T. Woodside, and Steven M. Block "High-resolution single-molecule optical trapping measurements of transcription with basepair accuracy: instrumentation and methods", Proc. SPIE 6644, Optical Trapping and Optical Micromanipulation IV, 664406 (5 September 2007); https://doi.org/10.1117/12.739631

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KEYWORDS

Optical tweezers

Polymers

Calibration

Molecules

Optical testing

Motion measurement

Helium

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