Study of single-molecule dynamics and reactions with classic light microscopy.
Single-molecule studies in the life sciences often deal with observation or spectroscopy. Studies of reactions are rare, and the light microscope has been used for such experiments only occasionally. In an experimental environment, for example, as is required for most nearfield scanning or electron microscopies, it is difficult to study single-molecule reactions of biological relevance. Therefore, we have developed techniques to study single-molecule reactions with classic (nonscanning) farfield light microscopy. The conversion of nicotinamide adenine dinucleotide (NAD+) and lactate to NADH (a reduced form of NAD+), pyruvate, and H+ catalyzed by a few LDH-1 enzyme molecules has been studied in substrate solutions with different viscosity using the NADH autofluorescence. It is even possible to monitor the progress of the reaction by phase-contrast microscopy via scattering or absorption by product molecules. As an example for a single-molecule reaction with a macromolecule as substrate, the handling and enzymatic cutting of fluorescently stained lambda-DNA is studied. In solutions containing 10 mM magnesium and 66 mM potassium ions at pH 7.9, an individual DNA molecule tends to collapse into a globular structure. When moved through an aqueous solution, it becomes stretched by viscosity drag. After stopping the motion, the molecule collapses and the dynamics of this process can be quantified. When a restriction enzyme is present, sequence-specific cutting can be directly observed in the light microscope. The theoretical restriction pattern, as predicted from the sequence of the molecule, can be generated directly under visual inspection.
Schäfer
B
B
Institut für Molekulare Biotechnologie, Jena, Germany.
Nasanshargal
B
B
Monajembashi
S
S
Gemeinhardt
H
H
Greulich
K O
KO
Uhl
V
V
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism*; D008845:Microinjections; D008856:Microscopy, Fluorescence / Q000379:methods; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism
D004279:DNA, Viral; D009243:NAD; D019344:Lactic Acid; D009249:NADP; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D015252:Deoxyribonucleases, Type II Site-Specific; C038485:GGGCCC-specific type II deoxyribonucleases