METHYLATION OF DNA MAY BE USEFUL AS A COMPUTATIONAL TOOL: EXPERIMENTAL EVIDENCE

Previously we have explained the abstract concept we call 'aqueous computing' and illustrated it with concrete wet lab results. Here, we explore the use of methylase enzymes to 'write' on double-stranded DNA molecules at sites where restriction enzymes will cut if, and only if, the sites have not previously been methylated. A site represents the bit zero (False, F) if the site has been methylated and the bit one (True, T) if it has not been methylated. 'Reading' is done by attempting a cut at each of the sites. We found 8 commercially available methylases and 8 corresponding restriction enzymes that would not cut after the action of one of the methylases. We were able to confirm that methylation by each of these 8 enzymes individually blocked cleavage only by the restriction enzyme associated with that site and not any other enzyme. We then used these enzymes to approach a 3-variable, 4-clause satisfiability (SAT) problem using either plasmid DNA (pBluescript) or PCR product made from the region containing the restriction enzyme sites on the plasmid. Pairs of methylases were defined to represent each of the states of the operators p, q and r, one methylase for p and another for p', etc. We methylated the DNA in parallel at the two sites so either the p site was methylated (making p false) or the p' site was methylated (making p' false). We did that for the other two variables as well to create a set of logically consistent DNA fragments. Then we applied the 4 clauses using restriction enzymes to cut DNA fragments that did not satisfy them. At the end, we found evidence for intact DNA indicating an answer satisfying all of the clauses. To confirm the state of each of the Boolean operators, we used cleavage by the appropriate restriction enzyme. We found in the computation with both the plasmid and the PCR product, one site pair to show false in both sites; q and q', for instance. This should not be possible. We suspected incomplete cutting during the clauses by one of these restriction enzymes, specifically BssHII. In summary, we did successfully show the usefulness of DNA methylation in a scheme to do a mathematical computation. Thus, we have added to our arsenal of potential methods of performing DNA computing in the aqueous style.