![]() We have started this process by providing Δ G° values for (1) 80 terminal fluorophore parameters, (2) 160 single-nucleotide dangle parameters and (3) 140 multinucleotide dangle parameters. We believe our native catalysis method can be used to provide an updated database of nucleic acid thermodynamic parameters for more accurate prediction and design software. The innovation that enables this approach is a DNA catalyst system that accelerates forward and reverse rate constants by orders of magnitude, allowing rapid equilibration 27, 28. A reaction is designed with Δ G° equivalent to the Δ G° of the motif of interest, and the equilibrium concentrations of the reactant and product species are measured to numerically calculate the equilibrium constant K eq. Here we present a generalized method for measuring Δ G° values of individual DNA motifs in native conditions of interest. Large numbers of experiments on different sequences statistically mitigate the latter source of unbiased error, but the former results in systematic errors that cannot be reduced via melt curve experiments. In addition, thermodynamics parameters for individual motifs need to be inferred through linear algebra decomposition or principal component analysis, because each melt curve provides the aggregate Δ H° and Δ S° of many motifs. This indicates that there is significant inaccuracy even in common motifs such as DNA base stacks and dangles, an observation confirmed by chemical probing of nucleic acid folding structures 24.Ĭurrent thermodynamic parameters are extrapolated from experiments in very different temperatures and buffer conditions than typically used (for example, melt curves in 1 M Na + (refs 25, 26). 23), corresponding to errors of roughly 3 kcal mol −1. Currently, the best DNA design and folding software exhibit errors in predicted melting temperature of ∼1.5 ☌ (ref. In both fields, accurate rational design of oligonucleotides with intended thermodynamics properties is critical to achieving desired system function.Įrrors in predicted Δ G° or melting temperature values result in undesirable non-specific interactions or unpredictable aggregation/assembly pathways. Nucleic acid nanotechnology uses engineered oligonucleotides as building blocks for constructing precisely patterned structures 13, 14, 15, 16, 17 and complex spatio-temporal circuits 18, 19, 20, 21, 22. Nucleic acid biotechnology uses engineered oligonucleotides as therapeutic agents (for example, micro RNAs) 1, 2, 3, 4, 5, 6 and diagnostics reagents (for example, PCR primers and next generation sequencing capture probes) 7, 8, 9, 10, 11, 12.
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