Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes

Abstract

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.

Stimuli-responsive assembly of bilingual peptide nucleic acids

“…Previous reports using fluorophore-quenching and melting temperature studies show the thermodynamics of duplex stability are: RNA:RNA > DNA:DNA > hybrid duplexes, which explains the superior displacement capability of RS-R with MS-R compared to MS-D. 22,23 Additionally, predictive models and other kinetics studies show a difference in the rate of displacement depending on nucleic acid backbone, especially in heterochiral strand displacement and duplex formation, with a report demonstrating that RNA releasing strands provide faster rates of displacement. 24,25 Interestingly, our studies show the opposite trend for RNA releasing sequences, but this may be due to differences in experimental parameters such as concentration, stoichiometry, and buffer conditions.…”

“…15 While somewhat unexpected, similar results have been observed by others when investigating toehold systems. 19,25 Moreover, our melting temperature studies showed that the presence of the mismatch surprisingly increased the duplex stability with PNA-C1-FAM (Table S5). Given these results, we decided to focus the following work with PNA-A1-FAM on the MS-D system because it yielded the fastest and most complete displacement of the systems tested.…”

A high-fidelity light-powered nanomotor from a chemically fueled counterpart via site-specific optomechanical fuel control

“…A time-dependent kinetic analysis (similar to ref. 39, 50 and 51) for the minus-end fluorescence increase by the pre-operation fuel mix yields the rate constant for fuel-induced leg dissociation as follows. Following our previous studies, 10,22 the time-dependent fluorescence from the minus-end dye is I ( t ) = I 0 − I Q ( t ), in which I 0 is the dye's full emission from the track-only control experiment without any quencher, and I Q ( t ) is the dye's emission from the operation experiment with the quencher-carrying motor.…”

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…The sugar rings of different sugar-modified nucleic acids possess varied pucker conformations, which are closely related with the overall helical structures and stabilities of the nucleic acid duplexes [56][57][58] . Replacement of the entire sugar-phosphate backbone of DNA with a peptide backbone produces peptide nucleic acid (PNA), which can hybridize with DNA or RNA with enhanced melting temperatures, and is highly resistant to nuclease degradation 59,60 .…”