Biology subjects

Dunn, K. E.

Publications and source records attributed to Dunn, K. E..

9 recordsLinked to original sources

bioRxiv2025

Electronic Actuation of Surface-Immobilized, pH-Responsive DNA Nanoswitches

Dynamic DNA machines exploit the specificity of base pairing and/or sensitivity to the local environment to control the reversible switching of DNA constructs between conformational states. One such example are pH-sensitive DNA nanoswitches that can be actuated by proton-mediated Hoogsteen interactions within a DNA triplex domain. To date, studies of pH-sensitive DNA nanoswitches have largely focused on DNA machines that are freely diffusing in the solution phase. For many applications, it is advantageous to integrate these dynamic DNA machines with solid-state devices, requiring immobilization on surfaces. Here, we explore the switching of a pH-sensitive DNA triplex immobilized on a surface as a dense, 2-dimensional DNA monolayer. DNA nanoswitches were assembled onto surfaces via thiol chemistry and pH-controlled conformational switching of the constructs examined using quartz crystal microbalance with dissipation monitoring (QCM-D). These QCM-D experiments indicate that despite the high density of DNA within the monolayer (1012 molecules/cm2), pH-switching between open and closed states is retained following immobilization. Moreover, conformational switching of DNA constructs within the monolayer remains highly reversible and repeatable, with negligible reduction in switching efficiency observed over 20 switching cycles. DNA switching experiments were also performed in the solution phase using single-molecular Forster resonance energy transfer (smFRET) and circular dichroism (CD) techniques to confirm their pH responsitivity. Finally, we demonstrate electrically driven, localised, and addressable switching of the DNA triplex by employing electrochemical reduction and oxidation of water at an electrode surface, further demonstrating the potential of the technology for surface-immobilized dynamic DNA machines. This study not only provides insight into the actuation of DNA machines on-surface but also supports the development of new technologies such as hybrid electronic-DNA technologies able to store and process information using both molecular and electronic inputs.

biophysics↗

bioRxiv2025

Modulating cell-free transcription electrolytically with switchable DNA triplexes

Living things self-regulate in response to various stimuli, including chemicals, light and heat [1]. In engineered biological systems, on-demand activation or repression of transcription underpins the design of genetic programs that control synthesis of useful products [2-9]. Even electrical stimuli can be used to control gene expression, but previous such electrogenetic approaches have required a combination of exogenous redox mediators and complex regulatory proteins [10,11]. Here we show that transcription can be modulated electrically in a cell-free system through the use of a DNA triplex as a molecular brake, which is opened or closed by means of electrolytic decomposition of water. Our DNA constructs comprise a double-stranded region with a single-stranded tail that folds over to form a triplex, where this construct is made using a customised synthesis protocol. Over measurements of 48 triplex-forming constructs and their corresponding controls, we observe that triplexes inhibit gene expression more often than not. Our approach, Electrically Directed Gene Expression ( EDGE), could be used to modulate in vitro synthesis of RNA therapeutics and also has applications in smart biomaterials, where the RNA transcript would act as a signal for a change of state. To test EDGE, we produced customised 96-well plates and control electronics, demonstrating how 3D-printing and in-house manufacturing techniques can be used to provide hardware for unusual experiments with specific needs. With further development the EDGE methodology could also be used to enable electrical control over protein expression.

bioengineering↗

bioRxiv2024

GelGenie: an AI-powered framework for gel electrophoresis image analysis

Gel electrophoresis is a ubiquitous laboratory method for the separation and semi-quantitative analysis of biomolecules. However, gel image analysis principles have barely advanced for decades, in stark contrast to other fields where AI has revolutionised data processing. Here, we show that an AI-based system can automatically identify gel bands in seconds for a wide range of experimental conditions, far surpassing the capabilities of current software. We used a dataset containing 500+ images of manually-labelled gels to train various U-Nets to accurately identify bands through segmentation, i.e. classifying pixels as band or background. When applied to gel electrophoresis data from other laboratories, our system generated results that quantitatively matched those of the original authors. We have publicly released our models through GelGenie, an open-source application that allows users to extract bands from gel images on their own devices, with no expert knowledge or experience required.

molecular biology↗

bioRxiv2023

Xylazine co-self-administration suppresses fentanyl consumption during self-administration and induces a unique sex-specific withdrawal syndrome that is not altered by naloxone in rats

Prescription and illicit opioid use are a public health crisis, with the landscape shifting to fentanyl use. Since fentanyl is 100-fold more potent than morphine, its use is associated with a higher risk of fatal overdose that can be remediated through naloxone (Narcan) administration. However, recent reports indicate that xylazine, an anesthetic, is increasingly detected in accidental fentanyl overdose deaths. Anecdotal reports suggest that xylazine may prolong the fentanyl "high", alter the onset of fentanyl withdrawal, and increase resistance to naloxone-induced reversal of overdose. To date no preclinical studies have evaluated the impacts of xylazine on fentanyl self-administration (SA; 2.5 g/kg/infusion) or withdrawal to our knowledge. We established a rat model of xylazine/fentanyl co-SA and withdrawal and evaluated outcomes as a function of biological sex. When administered alone, chronic xylazine (2.5 mg/kg, IP) induced unique sex-specific withdrawal symptomatology whereby females showed delayed onset of signs and a possible enhancement of sensitivity to the motor-suppressing effects of xylazine. Xylazine reduced fentanyl consumption both male and female rats regardless of whether it was experimenter-administered or added to the intravenous fentanyl product (0.05. 0.10, and 0.5 mg/kg/infusion) when compared to fentanyl SA alone. Interestingly, this effect was dose-dependent when self-administered intravenously. Naloxone (0.1 mg/kg, SC) did not increase somatic signs of fentanyl withdrawal, regardless of the inclusion of xylazine in the fentanyl infusion in either sex; however, somatic signs of withdrawal were higher across timepoints in females after xylazine/fentanyl co-SA regardless of naloxone exposure as compared to females following fentanyl SA alone. Together, these results indicate that xylazine/fentanyl co-SA dose-dependently suppressed fentanyl intake in both sexes, and induced a unique withdrawal syndrome in females which was not altered by acute naloxone treatment.

animal behavior and cognition↗

bioRxiv2022

Multiplexed Label-Free Biomarker Detection by Targeted Disassembly of Variable-Length DNA Payload Chains

Simultaneously studying different types of biomarkers (DNA, RNA, proteins, metabolites) has the potential to significantly improve understanding and diagnosis for many complex diseases. However, extracting biomarkers of different types involves using several technically complex or expensive methodologies, often requiring specialized laboratories and personnel. Streamlining detection through the use of a single multiplexed assay would greatly facilitate the process of accessing and interpreting patient biomarker data. In this work, we present a method for multiplexed biomarker detection based on variable-length DNA payload chains, which are systematically disassembled in the presence of specific biomolecular targets, leading to fragments of different sizes that yield characteristic band patterns in gel electrophoresis. This strategy has enabled us to detect with high sensitivity and specificity DNA sequences including BRCA1, an RNA sequence (miR-141) and the steroid aldosterone. We show that our assay can be multiplexed, enabling simultaneous detection of different types of biomarker. Furthermore, we show that our method suffers no loss of sensitivity when conducted in fetal bovine serum and can be applied using capillary electrophoresis, which may be more amenable to automation and integration in healthcare settings. ToC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/485867v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@12b4169org.highwire.dtl.DTLVardef@d36ebdorg.highwire.dtl.DTLVardef@10b69e7org.highwire.dtl.DTLVardef@55dc00_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Incompatibility of DFHBI based fluorescent RNA aptamers with particular commercial cell-free expression systems

Fluorescent RNA aptamers are an increasingly used tool for quantifying transcription and for visualising RNA interactions, both in vitro and in vivo. However when tested in the commercially available, E. coli extract based Expressway cell-free expression system, no fluorescence is detected. The same experimental setup is shown to successfully produce fluorescent RNA aptamers when tested in another buffer designed for in vitro transcription, and RNA purification of the Expressway reaction products show that transcription does occur, but does not result in a fluorescent product. In this paper we demonstrate the incompatibility of a narrow selection of RNA aptamers in one particular cell-free expression system, and consider that similar issues may arise with other cell-free expression systems, RNA aptamers, and their corresponding fluorophores.

synthetic biology↗

bioRxiv2021

Using ultraviolet absorption spectroscopy to study nanoswitches based on non-canonical DNA structures

Non-canonical forms of DNA are attracting increasing interest for applications in nanotechnology. It is frequently convenient to characterize DNA molecules using a label-free approach such as ultraviolet absorption spectroscopy. In this paper we present the results of our investigation into the use of this technique to probe the folding of quadruplex and triplex nanoswitches. We confirmed that four G-quartets were necessary for folding at sub-mM concentrations of potassium and found that the wrong choice of sequence for the linker between G-tracts could dramatically disrupt folding, presumably due to the presence of kinetic traps in the folding landscape. In the case of the triplex nanoswitch we examined, we found that the UV spectrum showed a small change in absorbance when a triplex was formed. We anticipate that our results will be of interest to researchers seeking to design DNA nanoswitches based on quadruplexes and triplexes. HighlightsO_LIUltraviolet absorption spectroscopy can probe non-canonical DNA structures. C_LIO_LIAbsorbance at 295nm tends to increase as G-quadruplexes form. C_LIO_LIFour G-quartets are needed to form a quadruplex with less than 1mM potassium. C_LIO_LIFormation of DNA triplexes can also yield a small change in UV spectra. C_LIO_LIUV absorption is a cheap label-free method for studying DNA nanoswitches. C_LI

bioengineering↗

bioRxiv2020

Establishing Preclinical Withdrawal Syndrome Symptomatology Following Heroin Self-Administration in Male and Female Rats

Opioid use disorder (OUD) is a significant health problem, and understanding mechanisms of various aspects of OUD including drug use and withdrawal is important. Preclinical models provide an ideal opportunity to evaluate mechanisms underlying opioid withdrawal. Current models are limited by their reliance upon forced opioid administration, focus on the acute (and not protracted) syndrome, and exclusion of females. In this study, male and female rats self-administered heroin (maintenance dose of 12.5 g/kg/infusion) opioid withdrawal following abrupt discontinuation was measured. In Phase 1, acute withdrawal symptoms were rated in male and female rats at 0, 16, 48, and 72 hrs following the last self-administration session. Total somatic signs increased until 48 hrs (predominantly in females), and heroin intake positively correlated with total somatic signs at the 48 and 72 hr timepoints. Measures of hyperactivity and anxiety-like behavior increased by 16 and 48 hrs, respectively. In Phase 2, symptoms were assessed at baseline, acute, and protracted (168 and 312 hrs after self-administration) timepoints in a subset of male and female rats from Phase 1. The total number of somatic signs did not differ across timepoints, though females displayed significantly higher body temperature at all timepoints compared to males, indicating sex-specific protracted withdrawal symptomatology. These data provide a thorough characterization of rodent opioid withdrawal symptomatology following self-administration and abrupt discontinuation that serve as a foundation for future studies designed to mimic the human experience, and demonstrate the importance of characterizing acute and protracted withdrawal with sex-specificity in preclinical models of opioid self-administration.

animal behavior and cognition↗

bioRxiv2018

Towards a bioelectronic computer: A theoretical study of a multi-layer biomolecular computing system that can process electronic inputs

DNA molecular machines have great potential for use in computing systems. Since Adleman originally introduced the concept of DNA computing through his use of DNA strands to solve a Hamiltonian path problem, a range of DNA-based computing elements have been developed, including logic gates, neural networks, finite state machines (FSMs) and non-deterministic universal Turing machines. It has also been established that DNA molecular machines can be controlled using electrical signals and that the state of DNA nanodevices can be measured using an electrochemical readout. However, to the best of our knowledge there has as yet been no demonstration of a fully integrated biomolecular computing system that has multiple levels of information processing capacity, can accept electronic inputs and is capable of independent operation. In this paper we address the question of how such a system could work. We present simulation results showing that such an integrated hybrid system could convert electrical impulses into biomolecular signals, perform logical operations and take a decision, storing its history. We also illustrate theoretically how such a system could potentially be used to perform a task such as controlling an autonomous robot navigating through a maze.

synthetic biology↗