Molecular surgery of DNA based on electrostatic micromanipulation

Abstract

A novel method for the space-resolved dissection (molecular surgery) of deoxyribonucleic acid (DNA) using electrostatic molecular manipulation is proposed and demonstrated. In conventional biochemistry, DNA-cutting enzymes and DNA are mixed in water, so the cutting reactions occur only by stochastic chances. In contrast, the present method is based upon a physical manipulation and enables the reproducible cutting of DNA at any desired position along 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) field created in microfabricated electrodes. It is found that, for the enzymatic cutting to occur, the DNA strand must be immobilized in such a way as to allow the enzyme to bind and interact with DNA. For 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-/spl mu/m diameter, and optical tweezers are used to hold it and press it against the stretched and immobilized DNA. The enzymes used are: (1) DNaseI (cuts 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 instantaneously. 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 finally cut. The authors' interpretation for this enzyme dependence is that the restriction enzyme has to get into the grooves of DNA to find the restriction sites, so the condition for the molecular contour fitting of the DNA and the enzyme are stricter compared with the case of the simple backbone-cutting enzyme DNaseI. The technique presented in this paper is expected to realize space-resolved molecular surgical operations, not just limited to dissections, but also for chemical modifications, or even insertion of genes.