Biology subjects

Punnoose, J. A.

Publications and source records attributed to Punnoose, J. A..

4 recordsLinked to original sources

bioRxiv2025

Investigating polarity effects in DNA base stacking

Nucleic acid structures are stabilized by both base pairing and base stacking. While energetics of base pairing interactions are relatively well established, our understanding of the energetic contributions of base stacking remain incomplete. Here, we use a combination of single-molecule and computational biophysics approaches to investigate the effect of strand polarity on base-stacking energetics. We designed pairs of DNA constructs with reversed stacking polarities at nick sites, along with corresponding no-stack controls to isolate stacking contributions. Performing single-molecule force-clamp assays with a Centrifuge Force Microscope (CFM), we observed polarity-dependent differences in stacking energetics. These differences were most pronounced in purine-purine and certain purine-pyrimidine interactions. Notably, a 5' purine stacked on a 3' pyrimidine was generally more stable than the reverse polarity. We employed molecular dynamics (MD) simulations to observe stacking interfaces in the DNA constructs. The simulations were qualitatively consistent with our experiments, and showed positional differences between opposite polarity stacking pairs, giving some insight into the origin of these polarity differences. Overall, these results demonstrate that base polarity can modulate stacking stability and should be considered when designing short duplex regions such as overhangs in molecular biology and biotechnology applications.

biophysics↗

bioRxiv2024

Tuning the stability of DNA tetrahedra with base-stacking interactions

DNA nanotechnology relies on programmable anchoring of regions of single-stranded DNA through base pair hybridization to create nanoscale objects such as polyhedra, tubes, sheets, and other desired shapes. Recent work from our lab measured the energetics of base-stacking interactions and suggested that terminal stacking interactions between two adjacent strands could be an additional design parameter for DNA nanotechnology. Here, we explore that idea by creating DNA tetrahedra held together with sticky ends that contain identical base pairing interactions but different terminal stacking interactions. Testing all 16 possible combinations, we found that the melting temperature of DNA tetrahedra varied by up to 10 {degrees}C from altering a single base stack in the design. These results can inform stacking design to control DNA tetrahedra stability in a substantial and predictable way. To that end, we show that a 4 bp sticky end with weak terminal stacking does not form stable tetrahedra, while strengthening the stacks confers high stability with a 46.8 {+/-} 1.2 {degrees}C melting temperature, comparable to a 6 bp sticky end with weak stacking (49.7 {+/-} 2.9 {degrees}C). The results likely apply to other types of DNA nanostructures and suggest that terminal stacking interactions play an integral role in formation and stability of DNA nanostructures.

bioengineering↗

bioRxiv2022

Quantifying individual base stacking energies with the Centrifuge Force Microscope

Base stacking interactions between adjacent bases in DNA and RNA are known to be important for many biological processes, for drug development, and in other biotechnology applications. While previous work has estimated base stacking energies between pairs of bases, the individual contributions of each base to the stacking interaction has remained unknown. Here, we developed a novel methodology using a custom Centrifuge Force Microscope to perform high-throughput single molecule experiments to measure base stacking energies between individual adjacent bases. We found stacking energies strongest between purines (G|A at -2.3 {+/-} 0.2 kcal/mol) and weakest between pyrimidines (C|T at -0.4 {+/-} 0.1 kcal/mol). Hybrid stacking with phosphorylated, methylated, and RNA bases had no measurable effect, but a fluorophore modification reduced stacking energy. The implications of the work are demonstrated with three applications. We experimentally show that base stacking design can influence assembly and stability of a DNA nanostructure, modulate kinetics of enzymatic ligation, and determine accuracy of force fields in molecular dynamics (MD) simulations. Our results provide new insights into fundamental DNA interactions that are critical in biology and can inform rational design in diverse biotechnology applications.

biophysics↗

bioRxiv2020

Programmable low-cost DNA-based platform for viral RNA detection

Viral detection is critical for controlling disease spread and progression. Recent emerging viral threats including Zika, Ebola, and the current COVID-19 outbreak highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches designed to mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show non-enzymatic detection of viral RNA to the attomole level, with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with a sample preparation step using either RNA extraction or isothermal pre-amplification. Our assay can be performed with minimal or no lab infrastructure, and is readily adaptable to detect other viruses. We demonstrate the adaptability of our method by quickly developing and testing DNA nanoswitches for detecting a fragment of SARS-CoV-2 RNA in human saliva. Given this versatility, we expect that further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.

bioengineering↗