DIRECT MEASUREMENT OF DNA MOLECULAR LENGTH IN SOLUTION USING OPTICAL TWEEZERS - DETECTION OF LOOPING DUE TO BINDING-PROTEIN INTERACTIONS

DNA looping is caused by the interaction between DNA binding proteins located at separate positions on a DNA molecule and may play an import ant role in transcription regulation. We have developed a system to st retch single DNA molecules and to measure changes in molecular length. DNA molecules were prepared and 5' end-labeled by PCR amplification. Two beads and the intervening DNA molecule were trapped and manipulate d independently with dual trap optical tweezers, The trapped DNA molec ule was then stretched and the extension (the distance between the two beads) was measured. The extension at the specific tension force of 3 0 pN was calculated and used as a molecular length. The molecular leng th was found to be proportional to the base pair number. The rise per residue was calculated to be 3.31 +/- 0.05 Angstrom. The length measur ement was applied to DNA fragments containing GC box sequences at two different locations separated by a distance of 2.428 kbp. The addition of GC box binding transcription factor Spl shortened the molecular le ngth, suggesting DNA looping forms as a result of interaction between transcription factors.