Measuring Unzipping and Rezipping of Single Long DNA Molecules with Optical Tweezers.

The unwinding of double-stranded DNA is a frequently occurring event during the cellular processes of DNA replication, repair, and transcription. To help further investigate properties of this fundamental process as well as to study proteins acting on unzipped DNA at the single molecule level, we describe a novel method for efficient preparation of long DNA constructs (arbitrary sequences of many kilobasepairs (kbp) in length) that can be forcibly unzipped and manipulated with optical tweezers or other single-molecule manipulation techniques. This method utilizes PCR, a nicking endonuclease, and strand displacement synthesis by the Klenow fragment of DNA polymerase I to introduce labeled nucleotides at appropriate positions to facilitate unzipping of the DNA by application of force. We also describe various optical tweezers measurement modes for measuring DNA unzipping and rezipping. These methods have applications to studying helicases and DNA binding proteins.

Single-Molecule Measurements of Motor-Driven Viral DNA Packaging in Bacteriophages Phi29, Lambda, and T4 with Optical Tweezers.

Viral DNA packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a preformed prohead shell in ~5 min at room temperature. We describe procedures for measuring the packaging of single DNA molecules into single viral proheads with optical tweezers. Three viral packaging systems are described in detail: bacteriophages phi29 (φ29), lambda (λ), and T4. Two different approaches are described: (1) With φ29 and T4, prohead-motor complexes can be preassembled in bulk and packaging can be initiated in the optical tweezers by "feeding" a single DNA molecule to one of the complexes; (2) With φ29 and λ, packaging can be initiated in bulk then stalled, and a single prohead-motor-DNA complex can then be captured with optical tweezers and restarted. In both cases, the prohead is ultimately attached to one trapped microsphere and the end of the DNA being packaged is attached to a second trapped microsphere such that packaging of the DNA pulls the two microspheres together and the rate of packaging and force generated by the motor is directly measured in real time. These protocols allow for the effect of many experimental parameters on packaging dynamics to be studied such as temperature, ATP concentration, ionic conditions, structural changes to the DNA substrate, and mutations in the motor proteins. Procedures for capturing microspheres with the optical traps and different measurement modes are also described.