EXPERIMENTAL SETUP FOR STUDY OF IMMOBILIZED SINGLE NUCLEOSOMES USING TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY
Abstract
An experimental setup for study of imm obilized molecules and their complexes by fluorescence microscopy with sensitivity at the single fluorophore level was developed. The installation records fluorescence images of immobilized molecules in two spectral ranges simultaneously, allowing analysis based on the Förster resonance energy transfer effect. The fluorescence excitation is caused by evanescent light wave formed by the total internal reflection technique, and registration of signal with a highly sensitive detection system allows conducting measurements with a temporal resolution of about 100 ms. The glass surface modification protocol was developed for immobilization of nucleosomes via the high-affinity streptavidin-biotin interactions. To ensure immobilization, one of the DNA ends of fluorescently labelled nucleosomal DNA was biotinylated. The algorithm of image processing for analysis of structural rearrangements in single nucleosomes was developed. Fluorescence microscopy of single immobilized molecules and their complexes allows the analysis of nucleosome structural dynamics during transcription and its interaction with various nuclear proteins.
About the Authors
K. S. Kudryashova
Bioengineering Department, School of Biology, Lomonosov Moscow State University, Leninskiye gory 1-12, Moscow, 119234, Russia
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya ul. 16/10, GSP-7, 117997, Moscow, Russia
Russian Federation
Russian Federation
O. V. Chertkov
Bioengineering Department, School of Biology, Lomonosov Moscow State University, Leninskiye gory 1-12, Moscow, 119234, Russia
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya ul. 16/10, GSP-7, 117997, Moscow, Russia
Russian Federation
Russian Federation
Y. O. Ivanov
Bioengineering Department, School of Biology, Lomonosov Moscow State University, Leninskiye gory 1-12, Moscow, 119234, Russia
Russian Federation
Russian Federation
V. M. Studitsky
Bioengineering Department, School of Biology, Lomonosov Moscow State University, Leninskiye gory 1-12, Moscow, 119234, Russia
Cancer Epigenetics Program Team, Fox Chase Cancer Center; Cottman Avenue 333, Philadelphia, PA 19111, USA
Russian Federation
Russian Federation
A. V. Feofanov
Bioengineering Department, School of Biology, Lomonosov Moscow State University, Leninskiye gory 1-12, Moscow, 119234, Russia
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya ul. 16/10, GSP-7, 117997, Moscow, Russia
Russian Federation
Russian Federation
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Review
For citations:
Kudryashova K.S., Chertkov O.V., Ivanov Y.O., Studitsky V.M., Feofanov A.V. EXPERIMENTAL SETUP FOR STUDY OF IMMOBILIZED SINGLE NUCLEOSOMES USING TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY. Vestnik Moskovskogo universiteta. Seriya 16. Biologiya. 2016;(2):37-42. (In Russ.)
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