A novel method to the kinetics of secondary structure formation of individual single-stranded DNA study (ssDNA) and RNA molecules is described. The method exploits the discovery that hairpin DNA molecules threaded through the approximately 1 nanometer pore made by the alpha-hemolysin membrane-channel can be unzipped in a controllable way by a voltage gradient. Information about the unzipping and re-zipping processes is obtained by the concurrent monitoring of the ion current flowing through the pore, and the Forster Resonance Energy Transfer (FRET) signals arising from labeled polynucleotides, during their transit through the pore. The unique combination of these two single-molecule methods in a robust instrument will enable the study of unfolding and refolding dynamics of DNA and RNA, ranging from the relatively simple case of a single hairpin structure to the study of the kinetics of ribozyme folding that contain multiple hairpins and pseudo knots. The folding kinetics of RNA, in general, and ribozymes in particular is difficult to measure because these molecules fold as they're transcribed. In the case of the HDV ribozyme an early bifurcation point is predicted to be responsible for the main misfolded path, which leads to the non-catalytic form. Biopolymer threading through the nanopore in conjunction with SM optical detection makes it possible to study sequential folding kinetics in general. Using this novel technique, we will study the kinetics of co-transcriptional folding of the HDV genomic ribozyme via the formation of the native and nonnative hairpins. The remarkably powerful capabilities of this novel method can directly be applied to a wide range of applications in molecular, cellular and genomic studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory/Developmental Grants (R21)
Project #
7R21GM072893-03
Application #
7273429
Study Section
Special Emphasis Panel (ZRG1-BPC-A (50))
Program Officer
Lewis, Catherine D
Project Start
2005-02-01
Project End
2008-01-31
Budget Start
2006-07-01
Budget End
2007-01-31
Support Year
3
Fiscal Year
2006
Total Cost
$137,680
Indirect Cost
Name
Boston University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
049435266
City
Boston
State
MA
Country
United States
Zip Code
02215
Dudko, Olga K; Mathe, Jerome; Szabo, Attila et al. (2007) Extracting kinetics from single-molecule force spectroscopy: nanopore unzipping of DNA hairpins. Biophys J 92:4188-95
Edel, Joshua B; Eid, John S; Meller, Amit (2007) Accurate single molecule FRET efficiency determination for surface immobilized DNA using maximum likelihood calculated lifetimes. J Phys Chem B 111:2986-90
Viasnoff, Virgile; Meller, Amit; Isambert, Herve (2006) DNA nanomechanical switches under folding kinetics control. Nano Lett 6:101-4