Design of hidden thermodynamic driving for non-equilibrium systems via mismatch elimination during DNA strand displacement

Abstract

Recent years have seen great advances in the development of synthetic self-assembling molecular systems. Designing out-of-equilibrium architectures, however, requires a more subtle control over the thermodynamics and kinetics of reactions. We propose a mechanism for enhancing the thermodynamic drive of DNA strand-displacement reactions whilst barely perturbing forward reaction rates: the introduction of mismatches within the initial duplex. Through a combination of experiment and simulation, we demonstrate that displacement rates are strongly sensitive to mismatch location and can be tuned by rational design. By placing mismatches away from duplex ends, the thermodynamic drive for a stranddisplacement reaction can be varied without significantly affecting the forward reaction rate. This hidden thermodynamic driving motif is ideal for the engineering of non-equilibrium systems that rely on catalytic control and must be robust to leak reactions.

Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes

“…For example, the use of stronger toehold sequences (i.e. higher G/C content) and/or introduction of mismatches into the PNA–DNA heteroduplex ( 60 ) could be used to increase strand displacement kinetics without further increasing toehold length, and will be the subject of future investigations.…”

“…The rate of DNA strand displacement reactions can be modulated over several orders of magnitude by introducing one or more mismatches between the input strand and the target duplex ( 48 , 60 ). Rational positioning of mismatches provides a useful control mechanism for competitive reaction networks ( 60 , 62 ) and enables the design of strand displacement-based nucleic acid probes having a high-degree of mismatch discrimination ( 63 , 64 ). A common practical application is the detection of single nucleotide polymorphisms (SNPs), which are of great diagnostic value ( 65–67 ).…”

In situgeneration of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems

“…The output of gate 2 is detected by an exogenous DNA probe. A mismatch was introduced in gates 1 and 2 and repaired by their corresponding inputs to provide thermodynamic energy drive 36 . When we tested the two-step cascade, we observed significant recovery only in the presence of the complete set of DNA templates (Figure 4b, black line vs other lines).…”

“…Doing so will enable us to transfer our setup to a more cell-like environment, in which components are continuously turned over by RNAse enzymes. Such a system would reach a dynamic, non-equilibrium steady state with the potential to respond to new input signals indefinitelya more life-like setting than traditional applications of DNA nanotechnology 36 . Once these systems are optimised, it should be possible to transfer the gate motif to living cells.…”

“…After being triggered by an input strand using the input toehold (red domain), the output strand of gate 1 triggers gate 2 (output toehold 1, black domain) which in turn activates DNA probe with its output (output toehold 2, blue domain). A mismatch is placed on gates 1 and 2 to provide a thermodynamic drive to the circuit (red dots)36 . All strands are produced from corresponding DNA templates (gate 1: D-I1-5, gate 2: D-G2 and input: d-I1-1) except for the probe DNA complex.…”