| Description |
The formation and dissociation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) nucleic acid duplexes is a fundamental aspect to many biological and biotech systems. Our current understanding and engineering of these systems are enabled by extensive biophysics research on how physical parameters, such as temperature, ionic strength, and sequence govern these reactions. While the behavior of DNA and RNA has been extensively studied, the dynamics of nonnatural nucleic acids and aptamers have not, even though they hold promise for many applications such as therapeutics and sensors. In this work, we studied the thermodynamics and kinetics of duplex formation for two artificial oligonucleotides, threose nucleic acid (TNA) and peptide nucleic acid (PNA), as well as the assembly kinetics of a DNA structure-switching aptamer against L-tyrosinamide (L-Tym). DNA and TNA duplexes were investigated using ultraviolet (UV) absorption spectroscopy melting analysis and single-molecule total internal reflection fluorescence (TIRF) microscopy. It was determined through UV melting, that increased TNA purine content greatly stabilized the duplex structure. Single-molecule TIRF kinetics studies on oligonucleotides containing high and low TNA purine content showed that higher TNA purine content led to slower duplex dissociation rates. The hybridization kinetics of DNA and PNA were investigated using TIRF microscopy over a range of ionic strength and temperature conditions. It was determined the PNA:DNA duplex was much more stable than an analogous DNA duplex and this was attributable to a slower duplex dissociation rate. The stability of the PNA:DNA duplex was found to be inversely proportional to ionic strength, where ionic strength primarily affected the association rate of the duplex. The competitive kinetics between L-Tym and a short DNA oligonucleotide complementary to the active site of L-Tym aptamer were investigated by monitoring how the concentration of L-Tym affected the rate of hybridization between the aptamer and short complementary DNA oligonucleotide. Increases in L-Tym concentration slowed the hybridization rate between the complementary DNA and the aptamer but did not affect the rate at which the DNA dissociated. The change in rates in response to L-Tym concentration allowed for the calculation of the binding affinity and kinetics of L-Tym association with the aptamer. iv |
