Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1.
By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.
Department of Applied Physics, University of Twente, Enschede, The Netherlands.
M L
ML
Bennink
O D
OD
Schärer
R
R
Kanaar
K
K
Sakata-Sogawa
J M
JM
Schins
J S
JS
Kanger
B G
BG
de Grooth
J
J
Greve
D010582:Bacteriophage lambda / Q000235:genetics; D001583:Benzoxazoles; D004279:DNA, Viral / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007700:Kinetics; D008846:Micromanipulation; D008856:Microscopy, Fluorescence / Q000379:methods; D055095:Optics and Photonics; D011806:Quinolinium Compounds; D011938:Rec A Recombinases / Q000378:metabolism*
D001583:Benzoxazoles; D004279:DNA, Viral; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D011938:Rec A Recombinases