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Single-Molecule Optical-Trapping Measurements with DNA Anchored to an Array of Gold Nanoposts

  • D. Hern Paik
  • Thomas T. PerkinsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 875)

Abstract

Gold–thiol chemistry is one of the most successful chemistries for conjugating biomolecules to surfaces, but such chemistry has not been exploited in optical-trapping experiments because of laser-induced ablation of gold. In this work, we describe a method to combine these two separate technologies without undue heating using DNA anchored to gold nanostructures (r = 50–250 nm; h ≈ 20 nm). Moreover, we demonstrate a quantitative and mechanically robust (>100 pN) optical-trapping assay. By using three dithiol phosphoramidites (DTPAs) incorporated into a polymerase chain reaction (PCR) primer, the gold–DNA bond remained stable in the presence of excess thiolated compounds. This chemical robustness allowed us to reduce nonspecific sticking by passivating the unreacted gold with methoxy-(polyethylene glycol)-thiol (mPEG-SH). Overall, this surface conjugation of biomolecules onto an ordered array of gold nanostructures by chemically and mechanically robust bonds provides a unique way to carry out spatially controlled, repeatable measurements of single molecules.

Key words

Single molecule Optical trap Optical tweezers Gold–thiol bond Gold–DNA bond DNA Force spectroscopy 

Notes

Acknowledgments

We thank Gavin King for a careful reading of this manuscript. This work was supported by a National Research Council Research Associateship (D.H.P.), a W.M. Keck Grant in the RNA Sciences, the National Science Foundation (NSF Phys-0404286 to T.T.P.), and National Institute of Standards and Technology (NIST). Mention of commercial products is for information only; it does not imply NIST recommendation or endorsement, nor does it imply that the products mentioned are necessarily the best available for the purpose. T.T.P. is a staff member of NIST’s Quantum Physics Division.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.JILA, National Institute of Standards and Technology and University of ColoradoBoulderUSA
  2. 2.Department of Molecular, Cellular and Developmental BiologyUniversity of ColoradoBoulderUSA

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