The primary objective of this proposal is to investigate the feasibility of using micro-scale electrochemical approaches to rapidly characterize individual polynucleotide molecules. In preliminary work to be described here, it has been found that voltages of greater than 100 mV can draw RNA and DNA molecules into single channels of alpha-hemolysin (alpha-HL) formed in planar bilayers separating two compartments. As a result, distinct blockades of ionic current on the millisecond time scale are produced that give information about concentration and chain length of the polynucleotides. Because the polymers pass through the channel as linear molecules, the further possibility exists that it may be possible to detect individual bases of the polymer by their specific effects on channel conductance. This means that, in principle, single strands of polynucleotides up to 1000 bases in length could be sequenced in a few seconds.
The aims of the application are the following: (1) An improved, miniaturized detector will be developed that can sample microliter volumes of DNA or RNA solutions. (2) The relationship between the number of blockades observed per unit time and the molar concentration of polynucleotides will be determined and (3) that polynucleotides physically traverse the bilayer through the channel will be demonstrated. (4) The relationship between the duration of a blockade and the chain length of the polynucleotide that is traversing the channel will be quantified. (5) Whether the alpha-HL channel can discriminate between purines and pyrimidines and between two different purines or two different pyrimidines will be determined. (6) Conditions that reduce polynucleotide self-structure and that reduce the velocity at which the polynucleotide traverses the channel will be established.
