Papers by Dmitry Kolpashchikov
Journal of the American Chemical Society, Jul 8, 2004
We report the first examples of modular aptameric sensors, which transduce recognition events int... more We report the first examples of modular aptameric sensors, which transduce recognition events into fluorescence changes through allosteric regulation of noncovalent interactions with a fluorophore. These sensors consist of: (a) a reporting domain, which signals the binding event of an analyte through binding to a fluorophore; (b) a recognition domain, which binds the analyte; and (c) a communication module, which serves as a conduit between recognition and signaling domains. We tested recognition regions specific for ATP, FMN, and theophylline in combinations with malachite green binding aptamer as a signaling domain. In each case, we were able to obtain a functional sensor capable of responding to an increase in analyte concentration with an increase in fluorescence. Similar constructs that consist only of natural RNA could be expressed in cells and used as sensors for intracellular imaging.
Chemical Communications, 2017
Highly selective probes hybridize only to fully complementary DNA or RNA sequences and, therefore... more Highly selective probes hybridize only to fully complementary DNA or RNA sequences and, therefore, often fail to recognize mutated viral genomes. Here we designed a probe that possesses two seemingly incompatible properties: it tolerates some point mutations in genome, while it remains selective towards others. An OR deoxyribozyme logic gate was designed to fluorescently report the sequences of enterovirus 71 (EV71) covering ~90% of all known EV71 strains. Importantly, sequences of closely related coxsackieviruses that differed by single nucleotides were reliably differentiated in 7 out of 8 cases. Hybridization probes are short oligonucleotide sequences designed to be complementary to targeted RNA or DNA analytes. For differentiation of closely related target sequences, e.g. those that differed by single nucleotide substitutions (SNS), a hybridization probe should form a stable hybrid only with a fully matched analyte, while it should remain unbound even if a single base mismatch is present. This approach has become an invaluable tool in the analysis of SNS in the human genome 1 and distinguishing drug resistant from drug susceptible bacterial strains, 2 but has often failed in the analysis of viral sequences. 3 Indeed, † Electronic supplementary information (ESI) available: Detailed experimental procedures; sequence selection; RNA extraction; RT-PCR; PCR; data used for the calculation of LOD; and fluorescence responses for viral genome sequences. See
Methods in molecular biology, 2013
Analysis of single-nucleotide polymorphisms (SNPs) is important for diagnosis of infectious and g... more Analysis of single-nucleotide polymorphisms (SNPs) is important for diagnosis of infectious and genetic diseases, for environment and population studies, as well as in forensic applications. Herein is a detailed description to design an "operating cooperatively" (OC) sensor for highly specific SNP analysis. OC sensors use two unmodified DNA adaptor strands and a molecular beacon probe to detect a nucleic acid targets with exceptional specificity towards SNPs. Genotyping can be accomplished at room temperature in a homogenous assay. The approach is easily adaptable for any nucleic acid target, and has been successfully used for analysis of targets with complex secondary structures. Additionally, OC sensors are an easy-to-design and cost-effective method for SNP analysis and nucleic acid detection.
Chemistry: A European Journal, Dec 30, 2020
Supporting information for this article is given via a link at the end of the document.
ChemPhysChem, Mar 24, 2017
DNA-based computational hardware has attracted ever-growing attention due to its potential tob eu... more DNA-based computational hardware has attracted ever-growing attention due to its potential tob eu seful in the analysis of complex mixtures of biological markers. Here we report the design of self-assembling logic gates that recognize DNA inputs anda ssemble into crossovert iles when the output signal is high; the crossover structures disassemble to form separateD NA standsw hen the output is low.T he output signal can be conveniently detected by fluorescenceu sing am olecular beacon probe as ar eporter.A ND, NOT,a nd OR logic gates were designed. We demonstrate that the gates can connecttoe ach other to produce other logic functions.
Chemical Communications, 2017
Analyte-dependent association of two DNA strands results in polymerization of acrylamide which ca... more Analyte-dependent association of two DNA strands results in polymerization of acrylamide which can be sensed not only visually but also by touch.
ChemBioChem, Dec 11, 2013
Human senses of taste and smell inspired the development of artificial differential receptors for... more Human senses of taste and smell inspired the development of artificial differential receptors for the recognition and identification of a great variety of chemical species. [1] A differential receptor consists of an array of sensors. Each sensor in the array recognizes a series of analytes with different recognition rates. The receptor produces a pattern of the responses. Analysis of the pattern (the fingerprint of the analyte) reveals the presence of a particular analyte in the sample. For example, Anslyn and colleagues designed a differential receptor for the recognition of fatty acids based on serum albumins. [2] Stojanovich et al. used DNA three-way junction sensors to design a differential receptor for steroids. [3] Recently, Chou et al. used nanoscale graphene oxide to design a receptor that differentiated proteins. [4] Here, we adopt this concept for differential analysis of nucleic acid sequences.
ChemistrySelect, Jul 3, 2017
Hybridization probes have been intensively used for nucleic acid analysis in medicine, forensics ... more Hybridization probes have been intensively used for nucleic acid analysis in medicine, forensics and fundamental research. Instantaneous hybridization probes (IHPs) enable signalling immediately after binding to a targeted DNA or RNA sequences without the need to isolate the probe-target complex (e. g. by gel electrophoresis). The two most common strategies for IHP design are conformational switches and split approach. A conformational switch changes its conformation and produces signal upon hybridization to a target. Split approach uses two (or more) strands that independently or semi independently bind the target and produce an output signal only if all components associate. Here, we compared the performance of split vs switch designs for deoxyribozyme (Dz) hybridization probes under optimal conditions for each of them. The split design was represented by binary Dz (BiDz) probes; while catalytic molecular beacon (CMB) probes represented the switch design. It was found that BiDz were significantly more selective than CMBs in recognition of single base substitution. CMBs produced high background signal when operated at 55°C. An important advantage of BiDz over CMB is more straightforward design and simplicity of assay optimization.
Angewandte Chemie, Feb 28, 2019
Angabe der unten stehenden Digitalobjekt-Identifizierungsnummer (DOI) zitiert werden. Die deutsch... more Angabe der unten stehenden Digitalobjekt-Identifizierungsnummer (DOI) zitiert werden. Die deutsche Übersetzung wird gemeinsam mit der endgültigen englischen Fassung erscheinen. Die endgültige englische Fassung (Version of Record) wird ehestmöglich nach dem Redigieren und einem Korrekturgang als Early-View-Beitrag erscheinen und kann sich naturgemäß von der AA-Fassung unterscheiden. Leser sollten daher die endgültige Fassung, sobald sie veröffentlicht ist, verwenden. Für die AA-Fassung trägt der Autor die alleinige Verantwortung.
Journal of Cancer Science & Therapy, Jul 23, 2015
P rostate specific antigen (PSA) is a 33-34 kDa glycoprotein commonly used to screen prostate can... more P rostate specific antigen (PSA) is a 33-34 kDa glycoprotein commonly used to screen prostate cancer and monitor treatment progression of prostate disease. However, PSA screening test has become controversial due to high percentage of falsepositive and false-negative results partially due to the presence of PSA homologs, complex formed with enzyme inhibitor, precursor forms of PSA and degradation products of PSA. Antibodies and mass spectrometry (MS) methods have been developed to specifically quantify PSA in the presence of these variables but have yet to overcome the disadvantage of poor immunoassay reproducibility or requirement of expensive MS instrument. We focus on using DNA aptamers, oligonucleotides that bind specific target analytes with high affinity. Aptamer sensor will specifically quantify PSA with high sensitivity. Specific aptamers against 2 peptides obtained by trypsinolysis of PSA were selected using SELEX method. After 13-19 rounds of selection, five aptamers were screened from a pool of 430 random libraries. The sequenced aptamers were 21-35 nucleotides long. Fluorescence biosensor was made by attaching Quencher to the fluorophore incorporated aptamer. Incubation with the positive target analyte showed increase in fluorescence as the concentration of the target increased. Kd was measured to be 178 nM. If successful, this project will deliver simple, fast and inexpensive fluorescent test for detection of PSA in human samples. Biography Nanami Kikuchi has completed Bachelors of Science degree from Lindenwood University in 2010. She joined University of Central Florida in 2013 and completed Masters of Science degree in May 2015. She is currently pursuing PhD degree in Chemistry at University of Central Florida. She has been teaching general chemistry, organic and biochemical laboratory methods for undergraduate students.
Accounts of Chemical Research, Jun 20, 2019
CONSPECTUS: Hybridization probes are RNA or DNA oligonucleotides or their analogs that bind to sp... more CONSPECTUS: Hybridization probes are RNA or DNA oligonucleotides or their analogs that bind to specific nucleotide sequences in targeted nucleic acids (analytes) via Watson−Crick base pairs to form probe−analyte hybrids. Formation of a stable hybrid would indicate the presence of a DNA or RNA fragment complementary to the known probe sequence. Some of the well-known technologies that rely on nucleic acid hybridization are TaqMan and molecular beacon (MB) probes, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), antisense, siRNA, and CRISPR/cas9, among others. Although invaluable tools for DNA and RNA recognition, hybridization probes suffer from several common disadvantages including low selectivity under physiological conditions, low affinity to folded single-stranded RNA and double-stranded DNA, and high cost of dye-labeled and chemically modified probes. Hybridization probes are evolving into multifunctional molecular devices (dubbed here "multicomponent probes", "DNA machines", and "DNA robots") to satisfy complex and often contradictory requirements of modern biomedical applications. In the definition used here, "multicomponent probes" are DNA probes that use more than one oligonucleotide complementary to an analyzed sequence. A "DNA machine" is an association of a discrete number of DNA strands that undergoes structural rearrangements in response to the presence of a specific analyte. Unlike multicomponent probes, DNA machines unify several functional components in a single association even in the absence of a target. DNA robots are DNA machines equipped with computational (analytic) capabilities. This Account is devoted to an overview of the ongoing evolution of hybridization probes to DNA machines and robots. The Account starts with a brief excursion to historically significant and currently used instantaneous probes. The majority of the text is devoted to the design of (i) multicomponent probes and (ii) DNA machines for nucleic acid recognition and analysis. The fundamental advantage of both designs is their ability to simultaneously address multiple problems of RNA/DNA analysis. This is achieved by modular design, in which several specialized functional components are used simultaneously for recognition of RNA or DNA analytes. The Account is concluded with the analysis of perspectives for further evolution of DNA machines into DNA robots.
ChemBioChem, Jul 15, 2016
Split spinach aptamer (SSA) probes for fluorescent analysis of nucleic acids were designed and te... more Split spinach aptamer (SSA) probes for fluorescent analysis of nucleic acids were designed and tested. In SSA design, two RNA or RNA/DNA strands hybridized to a specific nucleic acid analyte and formed a binding site for DFHBI dye, which was accompanied by up to 270-fold increase in fluorescence. The major advantage of the SSA probe over state-of-the art fluorescent probes is high selectivity: it produces only the background fluorescence in the presence of single base mismatched analyte even at room temperature. SSA is a promising tool for label-free analysis of nucleic acids at ambient temperatures.
Journal of Biomolecular Structure & Dynamics, Aug 1, 2003
Affinity labelling is a popular method used for the study of macromolecules and their interaction... more Affinity labelling is a popular method used for the study of macromolecules and their interactions with ligands. The method is based on the targeted delivery of a chemically cross-linkable group, attached to a reactive molecule with affinity for a particular site in the biopolymer of interest. In complex multicomponent systems, the applications of affinity labelling are restricted by the tendency of the reagents to randomly label nontargetted molecules. This review highlights techniques developed to minimize non-specific cross-linking and to achieve high selectivity for the labelling of target protein. Such techniques might be termed 'superselective labelling', as opposed to traditional, less selective approaches.
Sensors and Actuators B-chemical, May 1, 2019
Analysis of single nucleotide substitutions (SNS) in nucleic acids is the basis for the diagnosis... more Analysis of single nucleotide substitutions (SNS) in nucleic acids is the basis for the diagnosis of drug-resistant pathogens as well as human genetic disorders among a broad range of other applications. However, SNS are often inaccessible for the analysis by hybridization probe due to the location in double stranded regions of folded single stranded DNA or RNA analytes. Here we introduce an electrochemical sensor that is able to analyze SNS in folded nucleic acids. The electrochemical sensor takes advantage of a universal electrode-bound hairpin (UTH). It relies on the recognition of targeted nucleic acids by analyte-specific adaptor strands R and L that also hybridize with UTH. Strand L can bind several methylene blue (MeB)-probes thus placing MeB groups close to the electrode surface, which enables cathodic charge transfer. We demonstrated that the same UTH-functionalized electrode and MeB-probe can be used to analyze DNA analytes with different sequences. The sensor is highly selective toward SNS even at room temperature and can be regenerated for next round by rinse with water. The ability of the sensor to analyze SNS within secondary structure folded DNA was demonstrated. The sensing system is capable of detecting SNS in bacterial DNA, useful to differentiate drug-resistant from drug-susceptible mycobacterium tuberculosis. The proposed platform uniquely combines high SNS selectivity with ability to analyze potentially any DNA or RNA sequence including those folded in stable structures, therefore, creates a basis for a cost efficient electrochemical sensing of nucleic acids applicable both in medical diagnostics and environmental monitoring of microorganisms.
ChemBioChem, Oct 17, 2019
Scheme 3. A prototype DNA machine for cancer therapy. Dza and Dzb strands hybridize to the cancer... more Scheme 3. A prototype DNA machine for cancer therapy. Dza and Dzb strands hybridize to the cancer marker sequence (cyan) and form a catalytic core that can bind and cleave a housekeeping gene mRNA (brown).
Nanoscale, 2018
A new biomimetic nanoreactor design, MaBiDz, is presented based on a copolymer brush in combinati... more A new biomimetic nanoreactor design, MaBiDz, is presented based on a copolymer brush in combination with superparamagnetic nanoparticles. This cellular nanoreactor features two species of magnetic particles, each functionalized with two components of a binary deoxyribozyme system. In the presence of a target mRNA analyte and a magnetic field, the nanoreactor is assembled to form a biocompartment enclosed by the polymeric brush that enables catalytic function of the binary deoxyribozyme with enhanced kinetics. MaBiDz was demonstrated here as a cellular sensor for rapid detection and imaging of a target mRNA biomarker for metastatic breast cancer, and its function shows potential to be expanded as a biomimetic organelle that can downregulate the activity of a target mRNA biomarker.
Angewandte Chemie, Oct 8, 2020
This review discusses the design and applications of binary (also known as split) light-up aptame... more This review discusses the design and applications of binary (also known as split) light-up aptameric sensors (BLAS). BLAS consist of two RNA or DNA strands and an fluorogenic organic dye added as a buffer component. When associate, the two strands form a dye-binding site, followed by increase in fluorescence of the aptamer-bound dye. The design is cost-efficient since it uses short oligonucleotides and does not require conjugation of organic dyes with nucleic acids. In some applications, BLAS design is preferable over monolith sensors due to simpler assay optimization and improved selectivity. RNA-based BLAS can be expressed in cells and used for intracellular monitoring of biological molecules. BLAS have been used as reporters of nucleic acid association events in RNA nanotechnology and nucleic acid-based molecular computation. Other applications of BLAS include detection of nucleic acids, proteins and cancer cells, and potentially they can be tailored to report a broad range of biological analytes in a cost-efficient, highly selective and easy-to-optimize format. This review summarizes the state of the art in BLAS development and outlines perspectives and applications.
Journal of the American Chemical Society, Feb 19, 2008
Supporting Information Available: Details of the experimental procedure and the detection format ... more Supporting Information Available: Details of the experimental procedure and the detection format using ABTS as an oxidizable substrate as well as the results with dithymidine linker containing probes. This material is available free of charge via the Internet at http://pubs.acs.org.
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Papers by Dmitry Kolpashchikov