Biology subjects

Vilcapoma, J.

Publications and source records attributed to Vilcapoma, J..

4 recordsLinked to original sources

bioRxiv2023

The role of size in biostability of DNA tetrahedra

The potential for using DNA nanostructures for drug delivery applications requires understanding and ideally tuning their biostability. Here we investigate how biological degradation varies with size of a DNA nanostructure. We designed DNA tetrahedra of three edge lengths ranging from 13 to 20 bp and analyzed nuclease resistance for two nucleases and biostability in fetal bovine serum. We found that DNase I had similar digestion rates across sizes but appeared to incompletely digest the smallest tetrahedron, while T5 exonuclease was notably slower to digest the largest tetrahedron. In fetal bovine serum, the 20 bp tetrahedron was degraded ~four times faster than the 13 bp. These results show that DNA nanostructure size can influence nuclease degradation, but suggest a complex relationship that is nuclease specific.

synthetic biology↗

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

DNA nanoswitch barcodes for multiplexed biomarker profiling

The detection of molecular biomarkers plays a key role in the clinic, aiding in diagnostics and prognostics, and in the research laboratory, contributing to our basic understanding of diseases. The ability to detect multiple and diverse molecular biomarkers within a single accessible assay would have great utility, providing a more comprehensive picture for clinical evaluation and research, but is a challenge with standard methods. One promising approach is the use of dynamic DNA nanostructures that can respond to molecular biomarkers, which have recently been used in a variety of biosensing strategies. In this work, we report the use of programmable DNA nanoswitches for the multiplexed detection of up to 6 biomarkers within a single pot through the use of a barcoded gel-based readout. We demonstrate the barcoding capability using gene fragments that correspond to 6 different diseases, with each fragment or combination of fragments producing a unique barcode signature. As a defining feature of our method, we show "mixed multiplexing" for simultaneous barcoded detection of different types of biomolecules - DNA, RNA, antibody and protein in a single assay. To demonstrate clinical potential, we show multiplexed detection of a prostate cancer biomarker panel in serum that includes two microRNA sequences and prostate specific antigen (PSA). This strategy holds promise in clinical diagnostics for profiling complex and diverse biomarker panels.

bioengineering↗

bioRxiv2019

Exceptional biostability of paranemic crossover (PX) DNA, crossover-dependent nuclease resistance, and implications for DNA nanotechnology

Inherent nanometer-sized features and molecular recognition properties make DNA a useful material in constructing nanoscale objects, with alluring applications in biosensing and drug delivery. However, DNA can be easily degraded by nucleases present in biological fluids, posing a considerable roadblock to realizing the full potential of DNA nanotechnology for biomedical applications. Here we investigated the nuclease resistance and biostability of the multi-stranded motif called paranemic crossover (PX) DNA and discovered a remarkable and previously unreported resistance to nucleases. We show that PX DNA has more than an order of magnitude increased resistance to degradation by DNase I, serum, and urine compared to double stranded DNA. We further demonstrate that the degradation resistance decreases monotonically as DNA crossovers are removed from the structure, suggesting that frequent DNA crossovers disrupt either the binding or catalysis of nucleases or both. Further, we show using mouse and human cell lines that PX DNA does not affect cell proliferation or interfere with biological processes such as myogenesis. These results have important implications for building DNA nanostructures with enhanced biostability, either by adopting PX-based architectures or by carefully engineering crossovers. We contend that such crossover-dependent nuclease resistance could potentially be used to add "tunable biostability" to the many features of DNA nanotechnology.

bioengineering↗