Single-molecule measurements of the persistence length of double-stranded RNA.
Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 +/- 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 +/- 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.
Abels
J A
JA
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, 2628 CJ Delft, The Netherlands.
Moreno-Herrero
F
F
van der Heijden
T
T
Dekker
C
C
Dekker
N H
NH
D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D002021:Buffers; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D007700:Kinetics; D008280:Magnetics; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods*; D008954:Models, Biological; D015233:Models, Statistical; D008962:Models, Theoretical; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000648:ultrastructure*; D043244:Ribonuclease III / Q000737:chemistry; D055672:Static Electricity; D013696:Temperature; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
D002021:Buffers; D012330:RNA, Double-Stranded; D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D043244:Ribonuclease III