Stable end-sealed DNA as robust nano-rulers for in vivo single-molecule fluorescence.

Chemical Science
Anne PlochowietzAchillefs N Kapanidis

Abstract

Single-molecule fluorescence and Förster resonance energy transfer (smFRET) are important tools for studying molecular heterogeneity, cellular organization, and protein structure in living cells. However, in vivo smFRET studies are still very challenging, and a standardized approach for robust in vivo smFRET measurements is still missing. Here, we synthesized protected DNAs with chemically linked ends as robust in vivo nano-rulers. We efficiently internalized doubly-labeled end-sealed DNA standards into live bacteria using electroporation and obtained stable and long-lasting smFRET signatures. Single-molecule fluorescence signals could be extended to ∼1 min by studying multi-fluorophore DNA standards. The high stability of protected DNA standards offers a general approach to evaluate single-molecule fluorescence and FRET signals, autofluorescence background, and fluorophore density, and hence, quality check the workflow for studying single-molecule trajectories and conformational dynamics of biomolecules in vivo.

References

Aug 1, 1967·Proceedings of the National Academy of Sciences of the United States of America·L Stryer, R P Haugland
Jun 25, 1996·Proceedings of the National Academy of Sciences of the United States of America·T HaS Weiss
Jan 1, 1996·Annual Review of Biophysics and Biomolecular Structure·E T Kool
Sep 18, 2001·Methods : a Companion to Methods in Enzymology·T Ha
Dec 7, 2002·Nature Biotechnology·Antje KepplerKai Johnsson
Jun 4, 2004·Proceedings of the National Academy of Sciences of the United States of America·Achillefs N KapanidisShimon Weiss
Nov 22, 2005·Nature Methods·Nathan C ShanerRoger Y Tsien
Nov 18, 2006·Science·Achillefs N KapanidisRichard H Ebright
Jan 8, 2008·Nature Methods·Makio TokunagaKumiko Sakata-Sogawa
Jun 25, 2008·Annual Review of Biophysics·X Sunney XieGiuseppe Lia
May 5, 2009·Methods : a Companion to Methods in Enzymology·Rui Zhao, David Rueda
Dec 4, 2009·Proceedings of the National Academy of Sciences of the United States of America·Thomas D ChristianDavid Rueda
Jan 19, 2010·Proceedings of the National Academy of Sciences of the United States of America·Yusdi SantosoAchillefs N Kapanidis
Feb 2, 2010·Nature Methods·John J Sakon, Keith R Weninger
Feb 19, 2010·Analytical Chemistry·Samuel J LordW E Moerner
Mar 24, 2010·Annual Review of Biochemistry·Chang C Liu, Peter G Schultz
Aug 10, 2010·Nature Methods·Richard WombacherMarkus Sauer
Nov 26, 2010·Nature Structural & Molecular Biology·Shixin LiuXiaowei Zhuang
Apr 1, 2011·Nature Methods·Seamus J HoldenAchillefs N Kapanidis
May 10, 2011·Nature Methods·Sara A JonesXiaowei Zhuang
Mar 13, 2012·Annual Review of Physical Chemistry·Taekjip Ha, Philip Tinnefeld
Sep 3, 2013·Journal of Controlled Release : Official Journal of the Controlled Release Society·Sander A A KooijmansPieter Vader
Dec 10, 2013·Biophysical Journal·Robert CrawfordAchillefs N Kapanidis
Apr 4, 2014·Histochemistry and Cell Biology·Marko SustarsicAchillefs Kapanidis
May 20, 2014·Physical Chemistry Chemical Physics : PCCP·A PlochowietzA N Kapanidis
Aug 1, 2015·Nature Methods·Anne Plochowietz, Achillefs N Kapanidis
Aug 22, 2015·Current Opinion in Structural Biology·Marko Sustarsic, Achillefs N Kapanidis

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Methods Mentioned

BETA
FRET
fluorescence correlation spectroscopy
FCS
confocal microscopy
chip

Software Mentioned

smFRET
LabVIEW
MATLAB

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