Biology subjects

Liddle, J. A.

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

3 recordsLinked to original sources

bioRxiv2025

Complex cooperativity in DNA origami revealed via design dependent defectivity

DNA origami has become a ubiquitous platform for nanostructure fabrication because it enables straightforward design of structures that self-assemble with high yield. The interactions between the cooperative effects involved in its assembly are currently not well understood. Fortunately, the nearly infinite number of choices available to the origami designer provide a rich environment in which to explore cooperativity in a complex system. The DNA domains comprising origami have predictable energetics and the sources of cooperativity are conceptually straightforward. While the assembly of these systems is difficult to predict because of the large number of cooperative interactions, it can be measured. We are thus able to probe cooperativity by using design variations and measuring their effect on assembly yield. We employ an accelerated assembly protocol that increases the sensitivity of structural perfection, or lack thereof, to design variation, and apply this approach to survey a broad set of design features. Using the resulting dataset, we develop metrics to correlate thermal stability, beneficial cooperativity from short folds, and detrimental cooperativity from long folds, with defectivity. Surprisingly, these metrics can be combined to create a single parameter with a clear correlation to yield that serves as a useful starting place for a predictive understanding of the interplay between cooperativity and design. In doing so, we also identify qualitative trends that provide useful insight into design best practice.

biophysics↗

bioRxiv2023

Variable Gain DNA Nanostructure Charge Amplifiers for Biosensing

Electronic measurements of engineered nanostructures comprised solely of DNA (DNA nanostructures) enable new signal conditioning modalities in biosensing. Here, we demonstrate how DNA nanostructures that alter their conformation upon binding a nucleic acid analyte drastically, and reversibly, amplify the measured electrochemical signal. This amplification was controlled by the applied electrical field to achieve a response {approx} 2x104 times greater than that measured from DNA hybridization. Because the amplification is independent of the interaction between the analyte and the DNA nanostructure, our approach provides a platform for tuning the response of the system for high performance that is agnostic of the end application. These molecularly precise self-assembled DNA nanostructures when paired with scalable electronic readout can therefore lead the way to highly sensitive multiplexed biosensing.

bioengineering↗

bioRxiv2022

Temperature-dependent Thermodynamic and Photophysical Properties of SYTO-13 Dye Bound to DNA

The benefits of dyes in nucleic acid assays above room temperature are limited by a nonlinear, highdimensional relationship between fluorescence and the biophysical and chemical processes occurring in solution. To overcome these limitations, we identify an experimental regime that eliminates bias and unnecessary complexities in this relationship, and develop an experimental-computational workflow to generate the property data required to describe the dependence of fluorescence on temperature and concentration. Specifically, we exploit the temperature-cycling capabilities of real-time PCR machine, as well as the utility of numerical optimization, to determine the binding strength and molar fluorescence of the SYTO-13 dye bound to double-stranded (DS) or single-stranded (SS) DNA at more than 60 temperatures. We find that the data analysis approach is robust; it can account for significant well-to-well and plate-to-plate variation. The weak binding strength of SYTO-13 relative to SYBR Green I is consistent with previous reports of its negligible influence on PCR and melting temperature. Discriminating between molar fluorescence and binding strength clarifies the mechanism for the larger fluorescence of a DS/dye solution than a SS/dye solution; in fact, the explanation is different at high temperature than at low temperature. The temperature-dependence of the binding strength allows for ascertainment of the enthalpic and entropic contributions to the free energy, as well as the sign of the differential heat capacity of binding. The temperature-dependence of the molar fluorescence allows for calculation of the brightness (quantum yield times molar extinction coefficient) of SYTO-13 bound to DS relative to SS. The more accurate and complete description of the relationship between solution behavior and fluorescence enabled by this work can lead to more accurate selection of dyes and quantification of nucleic acids. SIGNIFICANCEFluorescent dyes are often used to quantify nucleic acids. The accuracy and precision of quantification, however, is limited by a complex and high-dimensional relationship between fluorescence and solution behavior. This is especially true for assays above room temperature, where empirical approximations are often required in the absence of available property data. In this work, we present an experimental and computational workflow that can more accurately describe this relationship and more efficiently generate the temperature-dependent thermodynamic and photophysical properties required. This approach can improve quantification and selection of high-performing dyes for particular assays.

biophysics↗