کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1960369 1057957 2006 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Viewing Dynamic Assembly of Molecular Complexes by Multi-Wavelength Single-Molecule Fluorescence
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Viewing Dynamic Assembly of Molecular Complexes by Multi-Wavelength Single-Molecule Fluorescence
چکیده انگلیسی

Complexes of macromolecules that transiently self-assemble, perform a particular function, and then dissociate are a recurring theme in biology. Such systems often have a large number of possible assembly/disassembly intermediates and complex, highly branched reaction pathways. Measuring the single-step kinetic parameters in these reactions would help to identify the functionally significant pathways. We have therefore constructed a novel single-molecule fluorescence microscope capable of efficiently detecting the colocalization of multiple components in a macromolecular complex when each component is labeled using a different color fluorescent dye. In this through-objective excitation, total internal reflection instrument, the dichroic mirror conventionally used to spectrally segregate the excitation and emission pathways was replaced with small broadband mirrors. This design spatially segregates the excitation and emission pathways and thereby permits efficient collection of the spectral range of emitted fluorescence when three or more dyes are used. In a test experiment with surface-immobilized single-stranded DNA molecules, we directly monitored the time course of a hybridization reaction with three different oligonucleotides, each labeled with a different color dye. The experiment reveals which of the possible reaction intermediates were traversed by each immobilized molecule, measures the hybridization rate constants for each oligonucleotide, and characterizes kinetic interdependences of the reaction steps.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: - Volume 91, Issue 3, 1 August 2006, Pages 1023–1031
نویسندگان
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