Detection Of Specific Sequences Research Articles

Programmable DNA-based nanostructure sensors for mutation detection coupled with asymmetric amplification in precision genome profiling

We introduce a DNA-based nanostructure sensor for the rapid, precise identification of single-nucleotide polymorphisms (SNPs), a key factor in determining disease susceptibility and tailoring medical treatment. The proposed two-state sensor design facilitates multiplexed, sequence-specific detection of double-stranded genomic DNA (gDNA), overcoming traditional obstacles presented by base pairing interference. Utilizing detector strands with binding penalties, the sensor achieves high SNP specificity, discernible by distinct gel electrophoresis band positions. Coupled with asymmetric PCR (asy-PCR), our integrated system amplifies genetic profiling effectiveness, especially in clinical biopsies, enhancing gDNA detection sensitivity and specificity for concurrent multiple mutation identifications. This cost-effective method ($0.41 per test) streamlines mutation screening, enabling high-throughput analysis with limited equipment—requiring only a single staining agent and a unified electrophoresis apparatus. Validated clinically with 70 colorectal cancer samples, the system boasts a 98.57% sensitivity and 95.71% specificity for KRAS mutations, surpassing traditional PCR detection and uncovering low-frequency mutations untraceable by direct sequencing. Our findings suggest this asy-PCR enhanced DNA-based nanostructure sensor system has the potential to revolutionize genetic diagnostics, offering expansive applications in detecting viral variants and microbial strains, thus advancing personalized genetic testing.

Fluorescent sensing platform based on two-dimensional layered double hydroxides and exonuclease I for DNA and microRNA detection

Interfacial and interlayer interactions between layered double hydroxides (LDHs) and nucleic acids offer LDHs great potential to absorb and detect nucleic acids. Herein, we demonstrate a simple, rapid, and versatile fluorescent sensing platform based on two-dimensional LDHs and exonuclease I (Exo I) for the selective analysis of DNA and microRNA. ZnNiAl-LDHs were utilized as quenchers for FAM labelled on single-stranded DNA (ssDNA) probes via dynamic quenching with high fluorescence quenching ability. The quenching mechanism was explored by means of spectral analysis, characterization experiments, DNA adsorption and desorption studies, zeta potential analysis, and molecular dynamics simulations. The driving forces for the adsorption of LDHs toward fluorophore-labelled nucleic acids were confirmed to be electrostatic interaction, van der Waals force, and anion exchange for the first time. In order to distinguish the formed target/probe duplexes from FAM-ssDNA probes, Exo I was introduced to specifically hydrolyze FAM-ssDNA probes. The fabricated fluorescent sensing platform for nucleic acids detection shows a low detection limit of 0.08 nM. Finally, this sensor was effectively utilized for the detection of DNA and microRNA in actual biological and environmental samples, achieving recoveries ranging from 96.4 % to 105 %, indicating high accuracy. This work proposed a promising sensing platform for sequence-specific nucleic acids detection, and provides a new perspective on two-dimensional LDHs as structural supports and biomolecular reservoirs for biomedical applications.

Ligation-driven hyperbranched DNA assembly for label-free and sequence-specific measurement of RNA N6-methyladenosine in human breast tissues

N6-methyladenosine (m6A) is a critical post-transcriptional regulator, and it is linked to various biological functions (e.g., degradation, export, and translation) and multiple human diseases (e.g., cancers, immune disorders, and neurodegenerative diseases). The current methods for sequence-specific detection of m6A usually suffer from radioactive risk, large sample consumption, washing/separation steps, and false-positive signal. Herein, we develop a ligation-driven hyperbranched DNA assembly-based fluorescent biosensor for label-free and sequence-specific detection of m6A. In this assay, the unmethylated RNA can be identified and cleaved by m6A-sensitive endoribonuclease MazF. The retained intact m6A-RNA is complementary to the padlock probe to form a circular DNA, subsequently initiating ligation-driven hyperbranched rolling circle amplification (HRCA). The obtained HRCA products can be simply quantified with SYBR Gold as a fluorescent indicator. This method can be homogeneously carried out in an isothermal, label-free and antibody-free manner. The limit of detection can reach 0.079 pM. Moreover, this method can differentiate even 0.01 % m6A level in excess coexisting interferences, monitor cellular m6A RNA level with single-cell sensitivity, and differentiate m6A level in breast tumor tissues and normal adjacent tissues, with promising applications in m6A-releated epigenetic research and clinical diagnosis.

Multiplexing LAMP Assays: A Methodological Review and Diagnostic Application.

The loop-mediated isothermal amplification (LAMP) technique is a great alternative to PCR-based methods, as it is fast, easy to use and works with high sensitivity and specificity without the need for expensive instruments. However, one of the limitations of LAMP is difficulty in achieving the simultaneous detection of several targets in a single tube, as the methodologies that allow this rely on fluorogenic probes containing specific target sequences, complicating their adaptation and the optimization of assays. Here, we summarize different methods for the development of multiplex LAMP assays based on sequence-specific detection, illustrated with a schematic representation of the technique, and evaluate their practical application based on the real-time detection and quantification of results, the possibility to visualize the results at a glance, the prior stabilization of reaction components, promoting the point-of-care use, the maximum number of specific targets amplified, and the validation of the technique in clinical samples. The various LAMP multiplexing methodologies differ in their operating conditions and mechanism. Each methodology has its advantages and disadvantages, and the choice among them will depend on specific application interests.

Sulfur incorporation into nucleic acids accelerates enzymatic activity

DNAzymes play a crucial role in biosensors for signal detection, but specific structures are required for diagnostic applications, complicating their design. This study reports a novel approach to the development of DNAzymes by incorporating sulfur atoms into nucleic acids via phosphorothioate (PS) bonds. Unlike traditional DNAzymes that rely on specific structures such as G-quadruplexes, this approach leads to high enzymatic activity without structural constraints. Computational analysis reveals that the change in the electron density of the nucleobases due to PS modification enhances interactions within the DNAzyme-H2O2-hemin complex, accelerating the rate-determining step and improving enzymatic activity. Systematic guidelines for the development of non-sequence constrained DNAzymes are provided by investigating the effect of the number of PS modifications, the length of the DNA, and various nucleobase combinations. α-thio-dNTP, a monomer containing PS, exhibits no observable enzymatic activity, but enzymatic activity is recorded for single-stranded DNA (ssDNA) containing PS. However, when the ssDNA is transformed into double-stranded DNA (dsDNA), the bases that react with hemin are blocked by hydrogen bonding, reducing enzymatic activity. An enzymogenic signaling system that differentiates between ssDNA and dsDNA is subsequently developed for sequence-specific colorimetric detection, demonstrating significant promise for overcoming the limitations of conventional DNAzymes in molecular diagnosis.

Bioluminescent Intercalating Dyes for Ratiometric Nucleic Acid Detection.

Rapid and sensitive DNA detection methods that can be conducted at the point of need may aid in disease diagnosis and monitoring. However, translation of current assays has proven challenging, as they typically require specialized equipment or probe-specific modifications for every new target DNA. Here, we present Luminescent Multivalent Intercalating Dye (LUMID), off-the-shelf bioluminescent sensors consisting of intercalating dyes conjugated to a NanoLuc luciferase, which allow for nonspecific detection of double-stranded DNA through a blue-to-green color change. Through the incorporation of multiple, tandem-arranged dyes separated by positively charged linkers, DNA-binding affinities were improved by over 2 orders of magnitude, detecting nanomolar DNA concentrations with an 8-fold change in green/blue ratio. We show that LUMID is easily combined with loop-mediated isothermal amplification (LAMP), enabling sequence-specific detection of viral DNA with attomolar sensitivity and a smartphone-based readout. With LUMID, we have thus developed a tool for simple and sensitive DNA detection that is particularly attractive for point-of-need applications.

DNA-Aptamer-Based qPCR Using Light-Up Dyes for the Detection of Nucleic Acids.

Quantitative polymerase chain reaction (qPCR) is widely used in detection of nucleic acids, but existing methods either lack sequence-specific detection or are costly because they use chemically modified DNA probes. In this work, we apply a DNA aptamer and light-up dye-based chemistry for qPCR for nucleic acid quantification. In contrast to the conventional qPCR, in our method, we observe an exponential decrease in fluorescence upon DNA amplification. The qPCR method we developed produced consistent Ct vs log10 (DNA amount) standard curves, which have a linearfit with R2 value > 0.99. This qPCR technique was validated by quantifying gene targets from Streptococcus zooepidemicus (SzhasB) and Mycobacterium tuberculosis (MtrpoB). We show that our strategy is able to successfully detect DNA at as low as 800 copies/μL. To the best of our knowledge, this is the first study demonstrating the application of light-up dyes and DNA aptamers in qPCR.

Curcumin-capped gold nanorods as optical sensing platform for sequence specific detection of DNA based on their self-assembly

We are reporting curcumin-induced gold nanorods as an optical sensing platform for the detection of sequence-specific DNA target through their self-assembly. The combined effect of eco-friendly reducing agent (i.e., curcumin) and silver nitrate in a basic medium (i.e., pH 10) has been attributed for the formation of small gold nanorods (AuNRs) having approximate length and diameter i.e., 19.7 ± 0.8 nm and 6.0 ± 0.5 nm, respectively, and lower longitudinal surface plasmon resonance (SPR) enable to detect and analyse different biomarkers. Further, for evaluating cellular uptake of as-synthesized AuNRs, the cytotoxicity study has been carried out by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on A549 cells and HEPG2 cell lines, respectively, and shown approximately similar cytotoxicity. Interestingly, as-synthesized optically and electronically active AuNRs based nanobiosensing platform enable to detect sequence-specific DNA targets with low level of detection limit i.e., LOD 8.6 ± 0.15 pM for complimentary target (CT) DNA with higher sensitivity and better selectivity. Finally, this study is suggesting a simplistic bio-mediated approach of tuning the shape and size of AuNRs for sensitive, selective and reliable nanobiosensing platform for sequence-specific DNA detection related to cancer cells.

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC.

Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to live-cell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEAtC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson-Crick base pairing. The DEAtC-containing DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEAtC base paired with G and little fluorescence turn-on when DEAtC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern-Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA-RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEAtC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

Rational Design of Profile HMMs for Sensitive and Specific Sequence Detection with Case Studies Applied to Viruses, Bacteriophages, and Casposons.

Profile hidden Markov models (HMMs) are a powerful way of modeling biological sequence diversity and constitute a very sensitive approach to detecting divergent sequences. Here, we report the development of protocols for the rational design of profile HMMs. These methods were implemented on TABAJARA, a program that can be used to either detect all biological sequences of a group or discriminate specific groups of sequences. By calculating position-specific information scores along a multiple sequence alignment, TABAJARA automatically identifies the most informative sequence motifs and uses them to construct profile HMMs. As a proof-of-principle, we applied TABAJARA to generate profile HMMs for the detection and classification of two viral groups presenting different evolutionary rates: bacteriophages of the Microviridae family and viruses of the Flavivirus genus. We obtained conserved models for the generic detection of any Microviridae or Flavivirus sequence, and profile HMMs that can specifically discriminate Microviridae subfamilies or Flavivirus species. In another application, we constructed Cas1 endonuclease-derived profile HMMs that can discriminate CRISPRs and casposons, two evolutionarily related transposable elements. We believe that the protocols described here, and implemented on TABAJARA, constitute a generic toolbox for generating profile HMMs for the highly sensitive and specific detection of sequence classes.

Electroactive hydrolysis probe-based portable PCR platform for sequence-specific detection of nontyphoidal Salmonella drug resistance gene

Etaq-PCR, a portable real-time polymerase chain reaction platform, has been developed based on an electroactive hydrolysis probe design and immobilization-free electrochemical readout. The Etaq-PCR platform integrates cost effective commercial instruments and thermally stable cycling into a minimal device enabling real-time monitoring of PCR amplification. No complex electrode surface treatment or micro-fabrication is involved in the construction of the Etaq-PCR platform. Thus, one can easily construct a portable PCR platform via our design. Etaq-PCR achieved sufficient sensitivity, selectivity, and rapidness to meet the WHO ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid, and robust, equipment-free, and deliverable) for point-of-care diagnosis systems. It showed good tolerance to clinical sample matrix as it displayed 100% accuracy in a validation experiment testing a drug resistance gene of nontyphoidalSalmonella in 25 raw clinical isolated samples in comparison with the gold standard qPCR. All in all, Etaq-PCR is a portable, deployable, and cost-effective PCR platform suitable for clinical applications.

An amplification-free, 16S rRNA test for Neisseria gonorrhoeae in urine.

An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, 16S rRNA sequence-specific detection of Neisseria gonorrhoeae at 10-100 CFU mL-1 in human urine against background bacterial flora at 1000 CFU mL-1. Gonorrhea is a very common notifiable communicable disease, antibiotic resistant strains have emerged, and the rate of reported gonococcal infections continues to increase. Since rapid clinical identification of bacterial pathogens in clinical samples is needed to guide proper antibiotic treatment and to control disease spread, it is important to engineer rapid, sensitive, selective, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae. Our detector technology is based on straightforward conductometric detection of sustained blockage of a glass nanopore. Charge neutral, complementary peptide nucleic acid probes are conjugated to polystyrene beads to capture N. gonorrhoeae 16S rRNA selectively. In the presence of an electric field applied externally through a glass nanopore, the PNA-microbead conjugates that acquire substantial negative charge upon target hybridization are driven to the smaller diameter nanopore. At least partial blockage of the nanopore results in a sustained drop in ionic current that can be measured easily with simple electronics. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU mL-1 spiked in human urine was demonstrated successfully with estimated sensitivity and specificity of ∼98% and ∼100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU mL-1. Also, N. gonorrhoeae at 50 CFU mL-1 was successfully detected against 1000 CFU mL-1 of background flora in urine. These results suggest that this amplification-free technology may serve as the basis for rapid, inexpensive, low-power detection of pathogens in clinical samples at the POC.

An Ultrasensitive PCR-Based CRISPR-Cas13a Method for the Detection of Helicobacter pylori.

The rapid and simple detection of Helicobacter pylori (H. pylori) is essential for its clinical eradication. Although various methods for detecting H. pylori have been well established, such as endoscopy in combination with histology or culture, rapid urease test (RUT) and molecular tests using clinical specimens, it is of great importance to develop an ultrasensitive and accurate nucleic acid detection platform and apply it to identify H. pylori. To meet these demands, a novel method based on PCR and CRISPR-Cas13a, called PCR-Cas13a, was developed and validated using the DNA of 84 clinical strains and 71 clinical specimens. PCR primers for the pre-amplification of conservative sequence and CRISPR RNA (crRNA) for the detection of specific sequence were designed according to the principle. The designed primers and crRNA were specific to H. pylori, and the assay showed a high degree of specificity compared with other common pathogens. Our detection system can screen H. pylori with a limit of 2.2 copies/μL within 30 mins after PCR amplification. Using a coincidence analysis with traditional methods, our method exhibited 100% accuracy for the detection of H. pylori. Furthermore, its diagnostic performance was compared, in parallel with a q-PCR. The PCR-Cas13a demonstrates 98% sensitivity and 100% specificity. Moreover, our approach had a lower limit of detection (LOD) than q-PCR. Herein, we present a diagnostic system for the highly sensitive screening of H. pylori and distinguish it from other pathogens. All the results demonstrated that this PCR-based CRISPR assay has wide application prospects for the detection of H. pylori and other slow-growth pathogens.

Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. We show that both Can1 and Can2 nucleases cleave single-stranded RNA, single-stranded DNA, and double-stranded DNA in the presence of cA4. We integrate the Can2 nuclease with type III-A RNA capture and concentration for direct detection of SARS-CoV-2 RNA in nasopharyngeal swabs with 15 fM sensitivity. Collectively, this work demonstrates how type-III CRISPR-based RNA capture and concentration simultaneously increases sensitivity, limits time to result, lowers cost of the assay, eliminates solvents used for RNA extraction, and reduces sample handling.

Efficient multiplexing and variant discrimination in reverse-transcription loop-mediated isothermal amplification with sequence-specific hybridization probes.

Loop-mediated isothermal amplification (LAMP) has proven a robust and reliable nucleic acid amplification method that is well suited for simplified and rapid molecular diagnostics. Various approaches have emerged for sequence-specific detection of LAMP products, but with limitations to their widespread utility or applicability for single-nucleotide polymorphism detection and multiplexing. Here we demonstrate the use of simple hybridization probes (as used for qPCR) that enable simple multiplexing and SARS-CoV-2 variant typing in reverse-transcription LAMP. This approach requires no modification to the LAMP primers and is amenable to the detection of single-nucleotide polymorphisms and small sequence changes, which is usually difficult in LAMP. By extending LAMP's ability to be utilized for multitarget and single-base change detection, we hope to increase its potential to enable more and better molecular diagnostic testing.

(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.

RNA-targeting CRISPR-Cas systems.

CRISPR-Cas is a widespread adaptive immune system in bacteria and archaea that protects against viral infection by targeting specific invading nucleic acid sequences. Whereas some CRISPR-Cas systems sense and cleave viral DNA, type III and type VI CRISPR-Cas systems sense RNA that results from viral transcription and perhaps invasion by RNA viruses. The sequence-specific detection of viral RNA evokes a cell-wide response that typically involves global damage to halt the infection. How can one make sense of an immune strategy that encompasses broad, collateral effects rather than specific, targeted destruction? In this Review, we summarize the current understanding of RNA-targeting CRISPR-Cas systems. We detail the composition and properties of type III and type VI systems, outline the cellular defence processes that are instigated upon viral RNA sensing and describe the biological rationale behind the broad RNA-activated immune responses as an effective strategy to combat viral infection.

Sequence-specific amperometric detection based on a double-probe mode and enzyme-mediated multiple signal electrocatalysis for the double-stranded DNA of PML/RARα-related fusion gene

A new electrochemical DNA biosensor based on double-probe mode and enzyme-mediated multiple signal electrocatalysis is constructed for the highly sensitive determination of double-stranded (ds-) PML/RARα fusion gene. Through the ingenious design of two groups of detection probes, including two thiolated capture probes anchored on dual standalone detection units integrated into one customized gold electrode and four biotinylated reporter probes, hybridizing with different segments of the same target single-stranded DNA (ssDNA) simultaneously, the hybridization efficiency between the probes and target is improved by preventing the reannealing of the two separate target ssDNA. Compared with a single reporter probe, this method can dramatically increase the amount of biotin and introduce numerous streptavidin-labelled horseradish peroxidase (HRP), thereby significantly amplifying electrochemical signals with low background signals. The combination of the dual-probe mode, multiple signal amplification strategy, and the inherent electrocatalytic activity of the HRP results in the prominent electrochemical sensing performance in detecting large-fragment target dsDNA with a detection limit as low as 71 fM. Furthermore, taking advantage of the new detection strategy, polymerase chain reaction (PCR) products and enzyme-digested PCR products from NB4 cells can be effectively analysed, showing great promise for the development of a new class of point-of-care platforms for disease-/drug-related genes.

A split β-lactamase sensor for the detection of DNA modification by cisplatin and ruthenium-based chemotherapeutic drugs

Here we present a split-enzyme sensor approach for the sequence-specific detection of metal-based drug adducts of DNA. Split β-lactamase reporters were constructed using domain A of the High Mobility Group Box 1 protein (HMGB1a) in conjunction with zinc finger DNA-binding domains. As a proof of concept, the sensors were characterized with the well-known drug cisplatin, which forms 1,2-intrastrand crosslinks with DNA that are recognized by HMGB1a. After promising results with cisplatin, five ruthenium-based drugs were studied, four of which produced significant signal over background. These results highlight the utility of our approach for rapid screening of novel metal-based chemotherapeutic drug candidates and provide evidence that HMGB1a likely binds to DNA adducts formed by NAMI-A (imidazolium trans-tetrachlorodimethylsulfoxideimidazoleruthenate(III)), KP1019 (indazolium trans-tetrachlorodiindazoleruthenate(III)), KP418 (imidazolium trans-tetrachlorodiimidazoleruthenate(III)), and RAPTA-C (dichloro(η6-p-cymene)(1,3,5-triaza-7-phosphaadamantane)ruthenium(II)). These results thus imply a potential biologically relevant mode of action for the ruthenium-based drugs investigated herein.

Digital Droplet Loop-Mediated Isothermal Amplification Featuring a Molecular Beacon Assay, 3D Printed Droplet Generation, and Smartphone Imaging for Sequence-Specific DNA Detection.

Nucleic acid detection is widely used in the amplification and quantitation of nucleic acids from biological samples. While polymerase chain reaction (PCR) enjoys great popularity, expensive thermal cyclers are required for precise temperature control. Loop-mediated isothermal amplification (LAMP) enables highly sensitive, rapid, and low-cost amplification of nucleic acids at constant temperatures. LAMP detection often relies on double-stranded DNA-binding dyes or metal indicators that lack sequence selectivity. Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes whose sequence specificity in LAMP provides the capability of differentiating between single-nucleotide polymorphisms (SNPs). Digital droplet LAMP (ddLAMP) enables a large number of independent LAMP reactions to be performed and provides quantification of target DNA sequences. However, a major challenge with ddLAMP is the requirement of expensive droplet generators to form homogeneous microdroplets. In this study, we demonstrate for the first time that a three-dimensional (3D) printed droplet generation platform can be coupled to a LAMP assay featuring MBs as sequence-specific probes. The low-cost 3D printed droplet generator system was designed, and its customizability was demonstrated in the formation of monodisperse ddLAMP assay-in-oil microdroplets. Additionally, a smartphone-based imaging system is demonstrated to increase accessibility for point-of-care applications. The MB-ddLAMP assay is shown to discriminate between two SNPs at various amplification temperatures to afford a useful platform for sequence-specific, sensitive, and accurate DNA quantification. This work expands the utility of MBs to ddLAMP for quantitative studies in the detection of SNPs and exploits the customizability of 3D printing technologies to optimize the homogeneity, size, and volume of oil-in-water microdroplets.