Molecular surgery of DNA based on electrostatic micromanipulation

A novel method for the space-resolved dissection (molecular surgery) of deo xyribonucleic acid (DNA) using electrostatic molecular manipulation is prop osed and demonstrated. In conventional biochemistry, DNA-cutting enzymes an d DNA are mixed in water, so the cutting reactions occur only by stochastic chances. In contrast, the present method is based upon a physical manipula tion and enables the reproducible cutting of DNA at any desired position al ong the DNA molecule. In order to realize this space-resolved cutting, the target DNA is stretched straight by electrostatic orientation and anchored on a solid surface by dielectrophoresis, using the high-intensity (1 MV/m) high-frequency (1 MHz) held created in microfabricated electrodes. It is fo und that, for the enzymatic cutting to occur, the DNA strand must be immobi lized in such a way as to allow the enzyme to bind and interact with DNA. F or this purpose, an electrode system is developed, in which DNA is anchored to the substrate only at the ends of the molecule, leaving the middle free . The enzyme, on the other hand, is immobilized on a latex particle having 1-mu m diameter, and optical tweezers are used to hold it and press it agai nst the stretched and immobilized DNA. The enzymes used are: 1) DNaseI (cut s DNA regardless of the base sequence) and 2) HindIII (a restriction enzyme ; cuts DNA at a specific sequence). It is demonstrated that, when a DNaseI- labeled bead is brought into contact with the immobilized DNA, DNA is cut i nstantaneously. On the other hand, when the restriction enzyme is used, the bead must be moved along the strand for a certain distance until it is fin ally cut. Our interpretation for this enzyme dependence is that the restric tion enzyme has to get into the grooves of DNA to find the restriction site s, so the condition for the molecular contour fitting of the DNA and the en zyme are stricter compared with the case of the simple backbone-cutting enz yme DNaseI. The technique presented in this paper is expected to realize sp ace-resolved molecular surgical operations, not just limited to dissections , but also for chemical modifications, or even insertion of genes.