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- RSC Chemical Biology
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- National Science Foundation
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Abstract Nucleic acid interactions under crowded environments are of great importance for biological processes and nanotechnology. However, the kinetics and thermodynamics of nucleic acid interactions in a crowded environment remain poorly understood. We use a coarse-grained model of DNA to study the kinetics and thermodynamics of DNA duplex and hairpin formation in crowded environments. We find that crowders can increase the melting temperature of both an 8-mer DNA duplex and a hairpin with a stem of 6-nt depending on the excluded volume fraction of crowders in solution and the crowder size. The crowding induced stability originates from the entropic effect caused by the crowding particles in the system. Additionally, we study the hybridization kinetics of DNA duplex formation and the formation of hairpin stems, finding that the reaction rate kon is increased by the crowding effect, while koff is changed only moderately. The increase in kon mostly comes from increasing the probability of reaching a transition state with one base pair formed. A DNA strand displacement reaction in a crowded environment is also studied with the model and we find that rate of toehold association is increased, with possible applications to speeding up strand displacement cascades in nucleic acid nanotechnology.
Development of Cu 2+ -Based Distance Methods and Force Field Parameters for the Determination of PNA Conformations and Dynamics by EPR and MD SimulationsPeptide nucleic acids (PNAs) are a promising group of synthetic analogues of DNA and RNA that offer several distinct advantages over the naturally occurring nucleic acids for applications in biosensing, drug delivery, and nanoelectronics. Because of its structural differences from DNA/RNA, methods to analyze and assess the structure, conformations, and dynamics are needed. In this work, we develop synergistic techniques for the study of the PNA conformation. We use CuQ2, a Cu(II) complex with 8-hydroxyquinoline (HQ), as an alternative base pair and as a spin label in electron paramagnetic resonance (EPR) distance methods. We use molecular dynamics (MD) simulations with newly developed force field parameters for the spin labels to interpret the distance constraints determined by EPR. We complement these methods by UV–vis and circular dichroism measurements and assess the efficacy of the Cu(II) label on a PNA duplex whose backbone is based on aminoethylglycine and a duplex with a hydroxymethyl backbone modification. We show that the Cu(II) label functions efficiently within the standard PNA and the hydroxymethyl-modified PNA and that the MD parameters may be used to accurately reproduce our EPR findings. Through the combination of EPR and MD, we gain new insights into the PNA structure and conformationsmore »
DNA duplex stability arises from cooperative interactions between multiple adjacent nucleotides that favor base pairing and stacking when formed as a continuous stretch rather than individually. Lesions and nucleobase modifications alter this stability in complex manners that remain challenging to understand despite their centrality to biology. Here, we investigate how an abasic site destabilizes small DNA duplexes and reshapes base pairing dynamics and hybridization pathways using temperature-jump infrared spectroscopy and coarse-grained molecular dynamics simulations. We show how an abasic site splits the cooperativity in a short duplex into two segments, which destabilizes small duplexes as a whole and enables metastable half-dissociated configurations. Dynamically, it introduces an additional barrier to hybridization by constraining the hybridization mechanism to a step-wise process of nucleating and zipping a stretch on one side of the abasic site and then the other.
Peptide nucleic acids (PNAs) are high-affinity synthetic nucleic acid analogs capable of hybridization with native nucleic acids. PNAs synthesized having amino acid sidechains installed at the γ-position along the backbone provide a template for a single biopolymer to simultaneously encode nucleic acid and amino acid sequences. Previously, we reported the development of “bilingual” PNAs through the synthesis of an amphiphilic sequence featuring separate blocks of hydrophobic and hydrophilic amino acid functional groups. These PNAs combined the sequence-specific binding activity of nucleic acids with the structural organization properties of peptides. Like other amphiphilic compounds, these γ-PNAs were observed to assemble spontaneously into micelle-like nanostructures in aqueous solutions and disassembly was induced through hybridization to a complementary sequence. Here, we explore whether assembly of these bilingual PNAs is possible by harnessing the nucleic acid code. Specifically, we designed an amphiphile-masking duplex system in which spontaneous amphiphile assembly is prevented through hybridization to a nucleic acid masking sequence. We show that the amphiphile is displaced upon introduction of a releasing sequence complementary to the masking sequence through toehold mediated displacement. Upon release, we observe that the amphiphile proceeds to assemble in a fashion consistent with our previously reported structures. Our approach represents amore »
Hybridization and strand displacement kinetics determine the evolution of the base paired configurations of mixtures of oligonucleotides over time. Although much attention has been focused on the thermodynamics of DNA and RNA base pairing in the scientific literature, much less work has been done on the time dependence of interactions involving multiple strands, especially in RNA. Here we provide a study of oligoribonucleotide interaction kinetics and show that it is possible to calculate the association, dissociation and strand displacement rates displayed by short oligonucleotides (5nt–12nt) that exhibit no expected secondary structure as simple functions of oligonucleotide length, CG content, ΔG of hybridization and ΔG of toehold binding. We then show that the resultant calculated kinetic parameters are consistent with the experimentally observed time dependent changes in concentrations of the different species present in mixtures of multiple competing RNA strands. We show that by changing the mixture composition, it is possible to create and tune kinetic traps that extend by orders of magnitude the typical sub-second hybridization timescale of two complementary oligonucleotides. We suggest that the slow equilibration of complex oligonucleotide mixtures may have facilitated the nonenzymatic replication of RNA during the origin of life.