Sequence-specific Detection Of Nucleic Acids Research Articles

(Invited, Digital Presentation) Rational Design of Nanoplasmonic Array Geometries for Biosensing

Background: Molecular diagnostics provide early and accurate diagnosis, which is essential for the prevention and treatment of infectious as well as chronic diseases. These tests are designed to detect disease-specific bioanalytes such as nucleic acid (DNA or RNA) or protein (antigens, antibodies) biomarkers. In the context of infectious disease diagnosis, nucleic acid-based detection methods are known to provide more specific and sensitive results. Here, the presence of a unique sequence belonging to the pathogenic genomic material is targeted to identify species, organism, genera and/or antimicrobial resistant gene markers. The majority of the common nucleic acid based diagnostic techniques require amplification (polymerase chain reaction, isothermal amplification etc.) of the pathogenic genetic material prior to detection impacting diagnostic speed, complexity, and cost thereby limiting ease of use. Thus, the development of simplified nucleic acid-based diagnostics that can be even used in resource-poor settings may hugely benefit patients across the globe.Nanopath is a molecular diagnostics company utilizing a solid-state nanosensor to enable sequence-specific detection of target nucleic acids without the need of amplification. These nanostructures enable ultra-sensitive biomarker detection using geometric, feature-dependent properties highly dependent on the local dielectric environment, allowing them to be sensitive to low concentration binding events. This paper describes an application of this approach to provide highly relevant clinical information within a single doctor’s office visit. Introduction: The Nanopath team is in collaboration with NASA (National Aeronautics and Space Administration) and NIST (National Institute of Standards and Technology) to push the bounds of the fundamental physics associated with their biosensing platform. The ability of metals to support electromagnetic surface waves gives rise to surface plasmons when optically illuminated. This property, and its strong sensitivity to changes in the local refractive index, allows for the use of metal nanoparticles as ultra-sensitive transducers. In prior work by members of this team, ensembles of randomly oriented nanoparticles (i.e., colloidal nanorods dispersed on chip) were employed for sequence-specific nucleic acid sensing (1-3). While these particle sensors have the advantage of rapid fabrication, they suffer from low sensitivity and quality factor due to the random particle dispersity. In contrast, in this study we employ ordered array nanoparticle ensembles which can be used to improve sensor sensitivity and figure-of-merit. Study Methods Overview: In this talk, we detail the results of sensing experiments and computational simulations to outline a rational design of the structure of these plasmonic nanoparticle arrays for biomolecular sensing. Through simulation and experiment, we iteratively tailor nanostructure dimension to provide high quality signal and large resonance shifts upon modeled nucleic acid binding.In particular, full-wave electromagnetic simulations were conducted using Lumerical photonic simulation software in which periodic boundary conditions were applied in the x- and y- dimensions for each of the nanoplasmonic sensor geometries. To simulate the resonance response to changes in the bulk solution in contact with the sensor surface, the refractive index of the surrounding media was changed appropriately. Nucleic acid hybridization events were modeled using either using spherical structures approximating the relevant radius of genomic material as estimated by polymer models, or as conformal layers with the known refractive indices for nucleic acids. On the basis of initial simulations, nanosensors were fabricated using traditional electron-beam lithography protocols at NIST. To evaluate consensus between simulations and experiments, bulk sensing experiments were carried out in which the resonance peaks were obtained by submerging the sensors in refractive index standards. Key nanosensor characteristics including resonance peak locations, resonance peak shifts as a function of refractive index, and figure of merit (FOM) of extinction curves were examined between the experimental and simulation results prior to proceeding with simulations on additional geometries and more complex solution conditions, and further device fabrication. This iterative process is repeated toward a rational design of nanoplasmonic array geometries for biosensing optimizing response for targeted disease detection.In summary, this study puts forth a methodology for rational design and characterization of regularly spaced nanoparticle arrays for optics-based biosensing. The results of this study will allow for more informed design of nanostructure geometries towards sequence-specific nucleic acid detection. These improved designs have the potential to improve clinical sensitivity and limit-of-detection across disease indication.

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid.

Enzymatic beacons, or E-beacons, are 1 : 1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming ssDNA equipped with a dark quencher. We prepared E-beacons biocatalytically using HhC, the promiscuous Hedgehog C-terminal protein-cholesterol ligase. HhC attached nanoluciferase site-specifically to mono-sterylated hairpin oligonucleotides, called steramers. Three E-beacon dark quenchers were evaluated: Iowa Black, Onyx-A, and dabcyl. Each quencher enabled sensitive, sequence-specific nucleic acid detection through enhanced E-beacon bioluminescence upon target hybridization. We assembled prototype dabcyl-quenched E-beacons specific for SARS-CoV-2. Targeting the E484 codon of the virus Spike protein, E-beacons (80×10-12 M) reported wild-type SARS-CoV-2 nucleic acid at ≥1×10-9 M by increased bioluminescence of 8-fold. E-beacon prepared for the SARS-CoV-2 E484K variant functioned with similar sensitivity. Both E-beacons could discriminate their target from the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with mismatch specificity, E-beacons are two to three orders of magnitude more sensitive than synthetic molecular beacons.

Multiplexed sensing of biomolecules with optically detected magnetic resonance of nitrogen-vacancy centers in diamond

In the past decade, a great effort has been devoted to develop new biosensor platforms for the detection of a wide range of analytes. Among the various approaches, magneto-DNA assay platforms have received extended interest for high sensitive and specific detection of targets with a simultaneous manipulation capacity. Here, using nitrogen-vacancy quantum centers in diamond as transducers for magnetic nanotags (MNTs), a hydrogel-based, multiplexed magneto-DNA assay is presented. Near-background-free sensing with diamond-based imaging combined with noninvasive control of chemically robust nanotags renders it a promising platform for applications in medical diagnostics, life science, and pharmaceutical drug research. To demonstrate its potential for practical applications, we employed the sensor platform in the sandwich DNA hybridization process and achieved a limit of detection in the attomolar range with single-base mismatch differentiation.

Single-Labeled Oligonucleotides Showing Fluorescence Changes Upon Hybridization with Target Nucleic Acids.

Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs) is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.

Synthesis of spiny metal–phenolic coordination crystals as a sensing platform for sequence-specific detection of nucleic acids

Metal–phenolic coordination crystals with a spiny surface and tunable compositions are synthesized, and can be used as DNA sensors.

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides.

In this review, we summarize the recent advances in the use of pyrene-modified oligonucleotides as a platform for functional nucleic acid-based constructs. Pyrene is of special interest for the development of nucleic acid-based tools due to its unique fluorescent properties (sensitivity of fluorescence to the microenvironment, ability to form excimers and exciplexes, long fluorescence lifetime, high quantum yield), ability to intercalate into the nucleic acid duplex, to act as a π-π-stacking (including anchoring) moiety, and others. These properties of pyrene have been used to construct novel sensitive fluorescent probes for the sequence-specific detection of nucleic acids and the discrimination of single nucleotide polymorphisms (SNPs), aptamer-based biosensors, agents for binding of double-stranded DNAs, and building blocks for supramolecular complexes. Special attention is paid to the influence of the design of pyrene-modified oligonucleotides on their properties, i.e., the structure-function relationships. The perspectives for the applications of pyrene-modified oligonucleotides in biomolecular studies, diagnostics, and nanotechnology are discussed.

Electrochemical monitoring of biointeraction by graphene-based material modified pencil graphite electrode

Recently, the low-cost effective biosensing systems based on advanced nanomaterials have received a key attention for development of novel assays for rapid and sequence-specific nucleic acid detection. The electrochemical biosensor based on reduced graphene oxide (rGO) modified disposable pencil graphite electrodes (PGEs) were developed herein for electrochemical monitoring of DNA, and also for monitoring of biointeraction occurred between anticancer drug, Daunorubicin (DNR), and DNA. First, rGO was synthesized chemically and characterized by using UV–Vis, TGA, FT-IR, Raman Spectroscopy and SEM techniques. Then, the quantity of rGO assembling onto the surface of PGE by passive adsorption was optimized. The electrochemical behavior of rGO–PGEs was examined by cyclic voltammetry (CV). rGO-PGEs were then utilized for electrochemical monitoring of surface-confined interaction between DNR and DNA using differential pulse voltammetry (DPV) technique. Additionally, voltammetric results were complemented with electrochemical impedance spectroscopy (EIS) technique. Electrochemical monitoring of DNR and DNA was resulted with satisfying detection limits 0.55µM and 2.71µg/mL, respectively.

Sequence-specific DNA detection at 10 fM by electromechanical signal transduction.

Target DNA fragments at 10 fM concentration (approximately 6 × 105 molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences.

Reduced Concentration Limit of Nucleic Acid Biosensor by Utilizing Long DNA

We previously described a simple, PCR-free, low cost device for sequence-specific nucleic acid detection which coupled target nucleic acids to a bead and measured the blockade of a large pore by the bead, achieving a 10 pM concentration detection limit of 20-mer DNA oligomers. Here, we describe function of our device for detection of longer DNA fragments. We prepared four different DNA fragments containing a specific target sequence 110, 235, 419, and 1613 nucleotides in length, and four different control fragments not containing the target sequence 125, 184, 309, and 1503 nucleotides in length. We examined detection of the target DNA oligomers at concentrations of 1pM - 1fM, finding that as target DNA length increased, lower concentrations were detectable, with a 10 fM detection limit for the 419 and 1613 nucleotide DNA oligomers.L. Esfandiari et al., J. Am. Chem. Soc., vol. 134, no. 38, pp. 15880-15886, 2012.

Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction

Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.

An isothermal and sensitive nucleic acids assay by target sequence recycled rolling circle amplification

Sequence-specific nucleic acid detection is playing a more and more important role in modern life sciences. Traditional rolling circle amplification (RCA) involves multiple distinct reaction steps and the experiment result is influenced by multiple factors. What's more, a main limitation of traditional RCA is that each target strand hybridizes with only one padlock probe, and this 1:1 hybridization ratio limits the sensitivity. Here we have proposed target sequence recycled rolling circle amplification (TR-RCA) to increase sensitivity by one step. We demonstrated that our method can not only make RCA occur, but also one target DNA can be reused and thus achieving self-recycle. In TR-RCA, the dumbbell probe recognizes the target DNA and hybridizes with it, and then the stem of the dumbbell probe is opened, after that the opened area anneals with the primer and triggers RCA. At the same time, after a target is displaced, it recognizes and hybridizes with another dumbbell probe, triggering the next cycle of RCA. This amplification method is achievable at a constant temperature simply by mixing dumbbell probes, target DNA, primers, and other chemical complexes together in one tube. Our method has significant advantages in ease of operation. And the results indicate that the target DNA can be detected at fM level with high specificity.

Enzyme-based E-RNA sensor array with a hairpin probe: Specific detection of gene mutation

An enzyme-based E-RNA sensor for the sequence-specific detection of nucleic acids was reported. The RNA sensor employed a “stem-loop” DNA probe which was immobilized on a 16-unit gold array surface via pre-labeled with thiol group (–SH), and pre-labeled biotin serves as an affinity tag for the enzyme (avidin-horseradish peroxidase) binding. The stem-loop structure of the sensor probes kept the biotin from being approached by a bulky avidin-horseradish peroxidase (avidin-HRP) by means of the steric hindrance. However, the probes would undergo a significant conformational change after DNA/RNA hybridization by forcing biotin away from the electrode, resulting the avidin-HRP access to the labeled biotin. Thus, target DNA/RNA hybridization event could be sensitively transduced via the enzymatically amplified electrochemical voltage and current signals, which were then measured by means of cyclic voltammetry and amperometric detection. The results showed that a single-base mismatch in target RNA could be selectively detected by the E-RNA sensor array, indicating that this E-RNA sensing strategy based on enzyme amplification is versatile with great potential in the construction of stem-loop based biosensors for the detection of nucleic acid mutations.

Sequence-Specific Nucleic Acid Detection from Binary Pore Conductance Measurement

We describe a platform for sequence-specific nucleic acid (NA) detection utilizing a micropipet tapered to a 2 μm diameter pore and 3 μm diameter polystyrene beads to which uncharged peptide nucleic acid (PNA) probe molecules have been conjugated. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pipet tip, which they obstruct, leading to an indefinite, electrically detectable, partial blockade of the pore. In the presence of noncomplementary NA, even to the level of single base mismatch, permanent pore blockade is not seen. We show application of this platform to detection of the anthrax lethal factor sequence.

Specific DNA detection using antibody mediated fluorescencequenching

First homogenous immunoassay for sequence-specific nucleic acid detection is developed. The assay bases on our finding that a fluorophore inserted into a DNA probe instead of one of the internal nucleotides may get protected from fluorescence quenching caused by an anti-fluorophore antibody, if the probe is hybridized with the target sequence. This ensures a positive signal in the antibody presence. The assay enables quantitative detection and may have potential for development of homogenous high-throughput platforms.

Sequence-specific Nucleic Acid Detection from Binary Pore Conductance Measurement

We describe a platform for sequence-specific nucleic acid (NA) detection utilizing a micropipet tapered to a 2 μm diameter pore and 3 μm diameter polystyrene beads to which uncharged peptide nucleic acid (PNA) probe molecules have been conjugated. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pipet tip, which they obstruct, leading to an indefinite, electrically detectable, partial blockade of the pore. In the presence of noncomplementary NA, even to the level of single base mismatch, permanent pore blockade is not seen. We show application of this platform to detection of the anthrax lethal factor sequence.

Sequence-specific detection of nucleic acids utilizing isothermal enrichment of G-quadruplex DNAzymes

G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Various chemical sensors and biosensors have been developed, based on such DNAzymes. Here we report a novel, specific nucleic acid detection method utilizing the isothermal amplification strategy of G-quadruplex DNAzymes. In this method, an unlabeled oligonucleotide probe was used. The probing sequence of the oligonucleotide was in the form of a stem-loop structure. A G-rich sequence, containing three GGG repeats, was linked to the 5′-end of the stem-loop structure. In the presence of target, the probing sequence hybridized to the target, and a Gn (n≥2) repeat was extended from its 3′-end. This Gn repeat, together with the three GGG repeats at the 5′-end, folded into a G-quadruplex, and displayed enhanced peroxidase acitivity upon hemin binding. Utilizing the dynamic binding interaction between the probe and its target, the enrichment of G-quadruplex DNAzymes was achieved. Using this method, simple, rapid and cost-effective nucleic acid detection could be achieved. This method displayed high target-length tolerance and good detection specificity; one-base mismatch could be judged easily, even by visual inspection. This method may be used as an auxiliary tool for amplified detection of specific DNA targets in some situations, in which isothermal detection is desirable.

Metal-containing peptide nucleic acid conjugates

Peptide Nucleic Acids (PNAs) are non-natural DNA/RNA analogues with favourable physico-chemical properties and promising applications. Discovered nearly 20 years ago, PNAs have recently re-gained quite a lot of attention. In this Perspective article, we discuss the latest advances on the preparation and utilisation of PNA monomers and oligomers containing metal complexes. These metal- conjugates have found applications in various research fields such as in the sequence-specific detection of nucleic acids, in the hydrolysis of nucleic acids and peptides, as radioactive probes or as modulators of PNA·DNA hybrid stability, and last but not least as probes for molecular and cell biology.

Sequence-specific electrochemical detection of nucleic acids in real samples

This paper reviews past and current developments in the field of electrochemical biosensors with a focus on the sequence-specific detection of nucleic acids in real samples. After electrochemical hybridization sensors had been first described in 1993, it took nearly a decade until some of the many proposed protocols were indeed applied to real samples like blood or tissue. Electrochemical transduction schemes used either rely on electroactive moieties such as intercalators, groove binders, covalently attached labels, and products of enzyme markers or they are completely indicator free like impedance-based detection principles. Most detection schemes require a polymerase chain reaction amplification step to allow for sufficient selectivity and sensitivity. Today, several companies develop electrochemical microarrays able to detect dozens to many thousands of sequences in a single experiment.

A Fluorogenic, Nucleic Acid Directed “Click” Reaction

A fluorogenic, nucleic acid directed, Cu(+)-catalyzed chemical reaction was developed that allows sequence-specific detection of nucleic acids at concentrations as low as 20 nM through monitoring of the increase of the fluorescence intensity. A single nucleotide mismatch in the template strand leads to the complete inhibition of this reaction. We observed that a Cu(+) ion stabilized with a water-soluble ligand is a significantly more efficient catalyst than free Cu(+).

Integrated Microfluidic Electrochemical DNA Sensor

Effective systems for rapid, sequence-specific nucleic acid detection at the point of care would be valuable for a wide variety of applications, including clinical diagnostics, food safety, forensics, and environmental monitoring. Electrochemical detection offers many advantages as a basis for such platforms, including portability and ready integration with electronics. Toward this end, we report the Integrated Microfluidic Electrochemical DNA (IMED) sensor, which combines three key biochemical functionalities--symmetric PCR, enzymatic single-stranded DNA generation, and sequence-specific electrochemical detection--in a disposable, monolithic chip. Using this platform, we demonstrate detection of genomic DNA from Salmonella enterica serovar Typhimurium LT2 with a limit of detection of <10 aM, which is approximately 2 orders of magnitude lower than that from previously reported electrochemical chip-based methods.