Determination of the fraction and stoichiometry of femtomolar levels of biomolecular complexes in an excess of monomer using single-molecule, two-color coincidence detection
We have extended the method of single-molecule fluorescence, two-color coincidence detection (TCCD) to detect coincident events due to a low fraction of a complex against a background of chance coincident events, due to monomers. We developed two complementary methods to determine the number of chance coincident events using the experimental data and without the need for additional experiments. We show that the subtraction of the chance coincidence level is essential for accurate quantification of the relative number of complexes and their stoichiometry. By performing experiments on model samples made from fluorophore-labeled duplex DNA and free dye, a linear dependence on the fraction of duplex DNA was found, independent of the level or ratio of free dye, with quantification down to a level of 0.5% and 500 fM duplex DNA. The method was then used to measure the equilibrium dissociation constant and offrate of a 9-mer duplex DNA, demonstrating the application of this method to systems with nanomolar dissociation constants. These improvements to the method of TCCD analysis significantly extend the application of TCCD to weakly bound complexes and large multicomponent biomolecular systems.
机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Deniz, AA
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Dahan, M
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Dahan, M
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Grunwell, JR
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Grunwell, JR
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Ha, TJ
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Ha, TJ
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Faulhaber, AE
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Faulhaber, AE
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Chemla, DS
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Chemla, DS
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Weiss, S
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Weiss, S
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Schultz, PG
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Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USAUniv Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Deniz, AA
;
Dahan, M
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h-index: 0
机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Dahan, M
;
Grunwell, JR
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Grunwell, JR
;
Ha, TJ
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Ha, TJ
;
Faulhaber, AE
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Faulhaber, AE
;
Chemla, DS
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机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Chemla, DS
;
Weiss, S
论文数: 0引用数: 0
h-index: 0
机构:Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA
Weiss, S
;
Schultz, PG
论文数: 0引用数: 0
h-index: 0
机构:
Univ Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USAUniv Calif Berkeley, Lawrence Berkeley Lab, Div Sci Mat, Berkeley, CA 94720 USA