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RNA dimerization monitored by fluorescence correlation spectroscopy

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Abstract

Fluorescence correlation spectroscopy (FCS) provides a versatile tool to investigate molecular interaction under native conditions, approximating infinite dilution. One precondition for its application is a sufficient difference between the molecular weights of the fluorescence-labelled unbound and bound ligand. In previous studies, an 8-fold difference in molecular weights or correspondingly a 1.6-fold difference in diffusion coefficients was required to accurately distinguish between two diffusion species by FCS. In the presented work, the hybridization of two complementary equally sized RNA single strands was investigated at an excellent signal-to-noise ratio enabled by the highly photostable fluorophore Atto647N. The fractions of ssRNA and dsRNA were quantified by applying multicomponent model analysis of single autocorrelation functions and globally fitting several autocorrelation functions. By introducing a priori knowledge into the fitting procedure, 1.3- to 1.4-fold differences in diffusion coefficients of single- and double-stranded RNA of 26, 41, and 54 nucleotides could be accurately resolved. Global fits of autocorrelation functions of all titration steps enabled a highly accurate quantification of diffusion species fractions and mobilities. At a high signal-to-noise ratio, the median of individually fitted autocorrelation functions allowed a robust representation of heterogeneous data. These findings point out the possibility of studying molecular interaction of equally sized molecules based on their diffusional behavior, which significantly broadens the application spectrum of FCS.

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Fig. 1a–c
Fig. 2a–f
Fig. 3a–d
Fig. 4
Fig. 5a, b

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Abbreviations

AF:

Autocorrelation function

bp:

Base pair

dsRNA:

Double-stranded RNA

FCS:

Fluorescence correlation spectroscopy

FCCS:

Fluorescence crosscorrelation spectroscopy

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

K d :

Dissociation constant

nt:

Nucleotide

ssRNA:

Single-stranded RNA

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Acknowledgments

We thank Johan Strömqvist for his advice in nonlinear regression analysis.

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Authors and Affiliations

  1. Institute for Biochemistry and Molecular Biology, Department of Chemistry, Hamburg University, Hamburg, Germany

    Arne Werner, Cindy Meyer & Ulrich Hahn

  2. Department of Systems Analysis, Faculty of Radio Physics and Electronics, Belarusian State University, Minsk, Belarus

    Victor V. Skakun

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  1. Arne Werner
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  2. Victor V. Skakun
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  3. Cindy Meyer
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  4. Ulrich Hahn
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Correspondence to Arne Werner or Ulrich Hahn.

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Werner, A., Skakun, V.V., Meyer, C. et al. RNA dimerization monitored by fluorescence correlation spectroscopy. Eur Biophys J 40, 907–921 (2011). https://doi.org/10.1007/s00249-011-0701-8

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  • DOI: https://doi.org/10.1007/s00249-011-0701-8

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