Single-Molecule Manipulation of Double-Stranded DNA Using Optical Tweezers: Interaction Studies of DNA with RecA and YOYO-1

Martin L. Bennink, Orlando D. Scharer, Ronald Kanaar, Kumiko Sakata-Sogawa, J.M. Schins, Johannes S. Kanger, B.G. de Grooth, Jan Greve

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

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° ± 3.
Original languageUndefined
Pages (from-to)200-208
Number of pages9
JournalCytometry
Volume36
Issue number3
DOIs
Publication statusPublished - 1999

Keywords

  • fluorescent imaging
  • binding kinetics
  • Optical tweezers
  • single-molecule manipulation
  • METIS-128407
  • DNA
  • YOYO-1
  • RecA
  • IR-60733

Cite this

Bennink, Martin L. ; Scharer, Orlando D. ; Kanaar, Ronald ; Sakata-Sogawa, Kumiko ; Schins, J.M. ; Kanger, Johannes S. ; de Grooth, B.G. ; Greve, Jan. / Single-Molecule Manipulation of Double-Stranded DNA Using Optical Tweezers: Interaction Studies of DNA with RecA and YOYO-1. In: Cytometry. 1999 ; Vol. 36, No. 3. pp. 200-208.
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abstract = "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° ± 3.",
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author = "Bennink, {Martin L.} and Scharer, {Orlando D.} and Ronald Kanaar and Kumiko Sakata-Sogawa and J.M. Schins and Kanger, {Johannes S.} and {de Grooth}, B.G. and Jan Greve",
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Single-Molecule Manipulation of Double-Stranded DNA Using Optical Tweezers: Interaction Studies of DNA with RecA and YOYO-1. / Bennink, Martin L.; Scharer, Orlando D.; Kanaar, Ronald; Sakata-Sogawa, Kumiko; Schins, J.M.; Kanger, Johannes S.; de Grooth, B.G.; Greve, Jan.

In: Cytometry, Vol. 36, No. 3, 1999, p. 200-208.

Research output: Contribution to journalArticleAcademicpeer-review

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AU - Bennink, Martin L.

AU - Scharer, Orlando D.

AU - Kanaar, Ronald

AU - Sakata-Sogawa, Kumiko

AU - Schins, J.M.

AU - Kanger, Johannes S.

AU - de Grooth, B.G.

AU - Greve, Jan

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AB - 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° ± 3.

KW - fluorescent imaging

KW - binding kinetics

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KW - METIS-128407

KW - DNA

KW - YOYO-1

KW - RecA

KW - IR-60733

U2 - 10.1002/(SICI)1097-0320(19990701)36:3<200::AID-CYTO9>3.0.CO;2-T

DO - 10.1002/(SICI)1097-0320(19990701)36:3<200::AID-CYTO9>3.0.CO;2-T

M3 - Article

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JO - Cytometry

JF - Cytometry

SN - 0196-4763

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