8 research outputs found
Enhancement of the Electrical Properties of DNA Molecular Wires through Incorporation of Perylenediimide DNA Base Surrogates
No full textCMMI-1246762; Air Force Office of Scientific Research. Grant Number: FA9550-13-1-0096; Office of Naval Research. Grant Number: N00014-16-1-2741.Due to copyright restrictions and/or publisher's policy full text access from Treasures at UT Dallas is not available. UTD affiliates may be able to acquire a copy by using the link below to contact university Interlibrary Loan.DNA has long been viewed as a promising material for nanoscale electronics, in part due to its well-ordered arrangement of stacked, pi-conjugated base pairs. Within this context, a number of studies have investigated how structural changes, backbone modifications, or artificial base substitutions affect the conductivity of DNA. Herein, we present a comparative study of the electrical properties of both well-matched and perylene-3,4,9,10-tetracarboxylic diimide (PTCDI)-containing DNA molecular wires that bridge nanoscale gold electrodes. By performing current-voltage measurements for such devices, we find that the incorporation of PTCDI DNA base surrogates within our macromolecular constructs leads to an approximately 6-fold enhancement in the observed current levels. Together, these findings suggest that PTCDI DNA base surrogates may enable the preparation of designer DNA-based nanoscale electronic components. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimSchool of Natural Sciences and MathematicsErik Jonsson School of Engineering and Computer Scienc
Electrochemical Measurements on Self-Assembled Monolayers of DNA to Follow Anti-Cancer Drug Activity and Helicase Interactions
No full textBiological study would benefit greatly from techniques that detect cellular processes typically constrained within cells. Electrochemistry using DNA-mediated charge transport enables studies of DNA interactions with proteins and drugs. We developed DNA-based multiplexed chips to mimic cellular environments or operate in cellular extracts. In this dissertation, electrochemical measurements were performed with chips bearing monolayers of DNA to follow anticancer drug activity and DNA-helicase interactions. Many cancer treatments involve DNA damage, and understanding these drugs involves controlling activation pathways and precisely following DNA damage repair. We designed an electrochemical chip of DNA modified electrodes to follow DNA damage, offering benefits over gel electrophoresis assays. We used chips to study the anticancer agent β-lapachone (ß-lap), which generates DNA damaging peroxide in the presence of overexpressed NAD(P)H: quinone oxidoreductase 1 (NQO1), a hallmark of many cancer cells. Initially, ß-lap was studied in a model system reproducing certain pathways of drug activation, DNA damage repair, and drug abrogation. We observed drug-specific changes from these chips and demonstrated a high correlation with the ß-lap-induced redox cycle.Our study revealed significant signal changes at clinically relevant levels and sub-lethal concentrations. Catalase, an enzyme decomposing peroxide, suppressed signal changes under conditions specific to cancer. Thus, this chip-based platform enabled unique tracking of ß-lap-induced DNA damage repair. Subsequently, we followed ß-lap activity in cellular lysates with these devices to correlate cell death activity with DNA damage. Cells were prepared to be proficient or deficient in NQO1 to mimic cancerous and healthy cells. Cells were lysed and added to chips, and β-lap activity was followed by signal changes arising from DNA damage. Devices showed an approximate fourfold difference in electrochemical response to NQO1+ over NQO1− cells, as well as great selectivity to controls deactivating the drug-induced DNA damage pathways. Saturation of DNA damage on the chip correlated with the onset of cell death from viability assays. These devices could be applied for screening of multiple anticancer drugs from small samples to guide cancer treatment.
Xeroderma pigmentosum group B (XPB) is an essential helicase involved in both DNA repair and transcription. Significant characteristics of XPB binding and activity remains to be established. We utilized DNA electrochemistry to sense the DNA-helicase interactions of three distinct XPB helicases. Changes in DNA duplex stability were quantified upon helicase binding. Binding dissociation constants were estimated in the range from 10-50 nM. and ATP-stimulated DNA unwinding activity was followed, revealing distinct modes of operation confirmed by crystal structures. These devices provided a sensitive measure of the structural thermodynamics and kinetics of DNA-helicase interactions. Chapter 1 introduces DNA, electrochemistry, and the specific field of DNA electrochemistry. Chapter 2 relates our research of β-lap in a model system incorporating the drug activation cycle, DNA base-excision repair by a glycosylase, and a drug abrogation pathway. Chapter 3 builds on this study to investigate β-lap activity in cellular lysates with differing concentrations of NQO1 that were proficient or deficient in DNA damaging activity. Chapter 4 describes the DNA binding and unwinding activity of XPB helicases with DNA devices
Following Anticancer Drug Activity in Cell Lysates with DNA Devices
No full textThere is a great need to track the selectivity of anticancer drug activity and to understand the mechanisms of associated biological activity. Here we focus our studies on the specific NQO1 bioactivatable drug, ß-lapachone, which is in several Phase I clinical trials to treat human non-small cell lung, pancreatic and breast cancers. Multi-electrode chips with electrochemically-active DNA monolayers are used to track anticancer drug activity in cellular lysates and correlate cell death activity with DNA damage. Cells were prepared from the triple-negative breast cancer (TNBC) cell line, MDA-MB-231 (231) to be proficient or deficient in expression of the NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme, which is overexpressed in most solid cancers and lacking in control healthy cells. Cells were lysed and added to chips, and the impact of β-lapachone (β-lap), an NQO1-dependent DNA-damaging drug, was tracked with DNA electrochemical signal changes arising from drug-induced DNA damage. Electrochemical DNA devices showed a 3.7-fold difference in the electrochemical responses in NQO1+ over NQO1− cell lysates, as well as 10–20-fold selectivity to catalase and dicoumarol controls that deactivate DNA damaging pathways. Concentration-dependence studies revealed that 1.4 µM β-lap correlated with the onset of cell death from viability assays and the midpoint of DNA damage on the chip, and 2.5 µM β-lap correlated with the midpoint of cell death and the saturation of DNA damage on the chip. Results indicate that these devices could inform therapeutic decisions for cancer treatment
Combinatorial design of chemical‐dependent protein switches for controlling intracellular electron transfer
No full textApplication of Electrochemical Devices to Characterize the Dynamic Actions of Helicases on DNA
No full textMuch remains to be understood about the kinetics and thermodynamics of DNA helicase binding and activity. Here, we utilize probe-modified DNA monolayers on multiplexed gold electrodes as a sensitive recognition element and morphologically responsive transducer of helicase-DNA interactions. The electrochemical signals from these devices are highly sensitive to structural distortion of the DNA produced by the helicases. We used this DNA electrochemistry to distinguish the details of the DNA interactions of three distinct XPB helicases, which belong to the superfamily-2 of helicases. Clear changes in DNA melting temperature and duplex stability were observed upon helicase binding, shifts that could not be observed with conventional UV-visible absorption measurements. Binding dissociation constants were estimated in the range from 10 to 50 nM and correlated with observations of activity. ATP-stimulated DNA unwinding activity was also followed, revealing exponential time scales and distinct time constants associated with conventional and molecular wrench modes of operation further confirmed by crystal structures. These devices thus provide a sensitive measure of the structural thermodynamics and kinetics of helicase-DNA interactions
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Molecular wrench activity of DNA helicases: Keys to modulation of rapid kinetics in DNA repair.
No full textDNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP-stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a molecular wrench mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP-stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox-active DNA monolayers. StXPB2 ATP-stimulated activity is concentration-independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration-dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB-Bax1 inhibits rapid activity, whereas the StXPB2-Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair
Detecting Attomolar DNA-Damaging Anticancer Drug Activity in Cell Lysates with Electrochemical DNA Devices
No full textHere, we utilize electrochemical DNA devices to quantify and understand the cancer-specific DNA-damaging activity of an emerging drug in cellular lysates at femtomolar and attomolar concentrations. Isobutyl-deoxynyboquinone (IB-DNQ), a potent and tumor-selective NAD(P)H quinone oxidoreductase 1 (NQO1) bioactivatable drug, was prepared and biochemically verified in cancer cells highly expressing NQO1 (NQO1+) and knockdowns with low NQO1 expression (NQO1−) by Western blot, NQO1 activity analysis, survival assays, oxygen consumption rate, extracellular acidification rate, and peroxide production. Lysates from these cells and the IB-DNQ drug were then introduced to a chip system bearing an array of DNA-modified electrodes, and their DNA-damaging activity was quantified by changes in DNA-mediated electrochemistry arising from base-excision repair. Device-level controls of NQO1 activity and kinetic analysis were used to verify and further understand the IB-DNQ activity. A 380 aM IB-DNQ limit of detection and a 1.3 fM midpoint of damage were observed in NQO1+ lysates, both metrics 2 orders of magnitude lower than NQO1− lysates, indicating the high IB-DNQ potency and selectivity for NQO1+ cancers. The device-level damage midpoint concentration in NQO1+ lysates was over 8 orders of magnitude lower than cell survival benchmarks, likely due to poor IB-DNQ cellular uptake, demonstrating that these devices can identify promising drugs requiring improved cell permeability. Ultimately, these results indicate the noteworthy potency and selectivity of IB-DNQ and the high sensitivity and precision of electrochemical DNA devices to analyze agents/drugs involved in DNA-damaging chemotherapies
