A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA–protein interactions, such as the DNA digestion reaction by λ exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual λ exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of λ exonuclease, a stained λ DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by λ exonuclease revealed that the DNA digestion rate was ∼1000 bases/s and also demonstrated high processivity.

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Understanding the mechanisms of DNA compaction is becoming increasingly important for gene therapy and nanotechnology DNA applications. The kinetics of the compaction velocity of single DNA molecules was studied using two non-protein condensation systems, poly(ethylene glycol) (PEG) with Mg²⁺ for the polymer-salt-induced condensation system and spermine for the polyamine condensation system. The compaction velocities of single tandem λ-DNA molecules were measured at various PEG and spermine concentrations by video fluorescent microscopy. Single DNA molecules were observed using a molecular stretching technique in the microfluidic flow. The results show that the compaction velocity of a single DNA molecule was proportional to the PEG or spermine concentration to the power of a half. Theoretical considerations indicate that the compaction velocity is related to differences in the free energy of a single DNA molecule between the random coil and compacted states. In the compaction kinetics with PEG, acceleration of the compaction velocity occurred above the overlap concentration while considerable deceleration occurred during the coexistence state of the random coil and the compacted conformation. This study demonstrates the control factors of DNA compaction kinetics and contributes toward the understanding of the compaction mechanisms of non-protein DNA interactions as well as DNA–protein interactions in vivo.