High-Fidelity DNA Hybridization Using Programmable Molecular DNA Devices

The hybridization of complementary nucleic acid strands is the most basic of all reactions involving nucleic acids, but has a major limitation: the specificity of hybridization reactions depends critically on the lengths of the complementary pairs of strands and can drop (in the presence of other competing strands whose sequences are close to that of a given target strand) to very low values if the strands have sufficiently long length. This reduction in specificity of hybridization reactions occurs especially in the presence of noise in the form of other competing strands that have sequence segments identical to the target. This limitation in specificity significantly limits the scale and accuracy of biotechnology and nanotechnology applications which depend on hybridization reactions. Our paper develops techniques for ensuring specific high-fidelity DNA hybridization reactions for target strands of arbitrary length. Given an in vitro solution which contains various DNA strands with differing sequences, among them a particular known target DNA sequence s of relatively long length (say at least 60 to hundreds of bases), our goal is to bind to each subsequence segment of s with high specificity and exact complementarity for a significant fraction of strands s in the solution. To do this, we develop a protocol that relies only on hybridization reactions between relatively short length (of at most 15 bases) DNA sequence segments. Our basic approach is to design DNA devices that essentially scan over strands in solution, subsegment by subsegment, and determine if one is indeed an instance of the given target strand s. This scanning is achieved by carefully designed relatively short sequences that effectively perform, in sequential order, successive verifications of subsequence identities on the target sequence, which if successfully completed indicate that the complete target sequence has been verified and completely hybridized. Our protocol is executed autonomously, without external mediation and driven by a series of conversions of single stranded DNA into duplex DNA that help overcome kinetic energy traps, similar to DNA walkers. In addition to the design of our high-fidelity DNA hybridization protocol, we also discuss the kinetics of our hybridization reactions, reducing the overall kinetics to a series of typical strand displacement reactions. Further, we describe a detailed design of an ongoing small-scale experimental demonstration of our protocols for high-fidelity hybridization. We also discuss potential applications for our protocols in molecular detection and DNA computing.