Hybridization Of DNA Probes Research Articles

A sensitive fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies to detect the H1N1 virus.

We propose a sensitive H1N1 virus fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies. Products are generated via the hybridization of single-stranded DNA (ssDNA) probes containing T7 promoter and crRNA templates to a target RNA sequence using SplintR ligase. This generates large crRNA quantities in the presence of T7 RNA polymerase. At such crRNA quantities, ternary Cas13a, crRNA, and activator complexes are successfully constructed and activate Cas13a to enhance fluorescence signal outputs. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM-1 µM. Biosensor specificity was also excellent. Importantly, our biosensor may be used to detect other viral RNAs by altering the sequences of the two probe junctions, with potential applications for the clinical diagnosis of viruses and other biomedical studies.

Plug-and-Play Module for Reversible and Continuous Control of DNA Strand Displacement Kinetics.

Regulatory modules for controlling the kinetics of toehold-mediated strand displacement (TMSD) play critical roles in designing dynamic and dissipative DNA chemical reaction networks (CRNs) but are hardwired into sequence designs. Herein, we introduce antitoehold (At), a plug-and-play module for reversible and continuous tuning of TMSD kinetics by temporarily occupying the toehold domain via a metastable duplex and base stacking. We demonstrate that kinetic control can be readily activated or deactivated in real time for any TMSD by simply adding At or anti-At. Continuous tuning of TMSD kinetics can also be achieved by altering the concentration of At. Moreover, the simple addition of At could readily reprogram existing TMSDs into a pulse-generation DNA CRN with continuous tunability. Our At approach also offers a new way for engineering continuously tunable DNA hybridization probes, which may find practical uses for discriminating clinically important mutations. Because of the simplicity, we anticipate that At will find wide applications for engineering DNA CRNs with diverse dynamic and dissipative behaviors, and DNA hybridization probes with tunable affinity and selectivity.

A novel universal fluorescence detection platform based on target binding-mediated primer exchange reaction strategy for sensitive and accurate detection of multiple biomolecules

This study introduces a universal fluorescent detection platform based on the binding-mediated primer exchange reaction strategy, designed for highly sensitive and accurate detection of multiple biomolecules. This method uses an intact DNA hybridization probe as the initial target recognition site, forming a stable probe-target complex upon binding to the target. This complex drives the primer exchange reaction to amplify the signal, and then to achieve signal output through fluorescence resonance energy transfer (FRET). The reaction process is initiated directly by the catalytic target binding medium, ensuring method specificity. The versatile platform, equipped with various recognition elements such as antisense nucleic acids, biotin, and split aptamers, demonstrates exceptional universality in detecting a range of biomolecules, including virus nucleic acid fragments, streptavidin, and ATP. The method exhibits strong linear relationships with detection limits of 1.02 pM for virus nucleic acid fragments, 1.08 nM for streptavidin, and 15.7 μM for ATP, respectively. Moreover, the method displays excellent selectivity and outstanding performance in complex biological samples. This promising biosensing platform is believed to find numerous applications spanning bioanalysis, disease diagnosis, and biomedicine.

Coding Intrinsic Disorder into DNA Hybridization Probes Enables Discrimination of Single Nucleotide Variants over Wide and Tunable Temperature Ranges

AbstractDNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy‐compensate probe (Sprobe) design by coding intrinsic disorder into a stem‐loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine‐tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics.

Coding Intrinsic Disorder into DNA Hybridization Probes Enables Discrimination of Single Nucleotide Variants over Wide and Tunable Temperature Ranges.

DNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy-compensate probe (Sprobe) design by coding intrinsic disorder into a stem-loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine-tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics.

Development of Graphene with Tungsten Disulfide Composite Layer Based SPR Biosensor for Biomedical Application

Surface Plasmon Resonance (SPR) biosensors have been widely used for biomedical applications due to their high sensitivity and label-free detection capabilities. However, their performance can be further enhanced by using advanced materials and signal-processing techniques. The objective of this study is to develop a composite layer-based SPR biosensor using Au, WS2, and Graphene layers and signal processing with MATLAB for enhanced sensitivity and the detection of DNA-DNA Hybridization. The composite layer-based SPR biosensor was fabricated by depositing a thin layer of Au on a glass substrate, followed by the deposition of WS2 and Graphene layers using a Chemical Vapor Deposition (CVD) technique. A self-assembled monolayer of 3-Mercaptopropionic Acid (MPA) was then attached to promote DNA immobilization. The performance of the biosensor was evaluated by detecting the hybridization of a single-stranded DNA (ssDNA) probe with a complementary ssDNA target. The sensor response was analyzed using MATLAB to enhance the sensitivity of the biosensor. The developed composite layer-based SPR biosensor exhibited a high sensitivity of 592 deg./RIU for the detection of DNA-DNA hybridization. 32.74% sensitivity has been increased. The signal processing with MATLAB significantly improved the signal-to-noise ratio and allowed for real-time monitoring of the biomolecular interactions. The composite layer-based SPR biosensor developed in this study demonstrated enhanced sensitivity for the detection of DNA-DNA hybridization in biomedical applications. The use of advanced materials such as Au, WS2, and Graphene layers, coupled with signal processing with MATLAB, can significantly improve the performance of SPR biosensors. This biosensor has great potential for use in various areas, including genetic testing, drug discovery, and disease diagnosis. Detected DNA-DNA hybridization is used in the biomedical field to identify and classify microorganisms by comparing the degree of genetic similarity between their DNA sequences.

Formamide-Free Genomic In Situ Hybridization (ff-GISH).

Fluorescence in situ hybridization allows for the mapping of various sequence types in the genomes and is thus widely used in structural, functional, and evolutionary studies. One particular type of in situ hybridization that specifically allows to map whole parental genomes in diploid and polyploid hybrids is genomic in situ hybridization (GISH). The efficiency of GISH, i.e., the specificity of hybridization of genomic DNA probes to the parental subgenomes in hybrids depends, among others, on the age of the polyploids and the similarity of the parental genomes, specifically their repetitive DNA fractions. Typically, high levels of overall repeat similarity between the parental genomes result in lower efficiency of GISH. Here, we present the formamide-free GISH (ff-GISH) protocol that can be applied to diploid and polyploid hybrids of both monocots and dicots. ff-GISH allows higher efficiency of the labeling of the putative parental genomes compared to the standard GISH protocol and allows discrimination of parental chromosome sets that share up to 80-90% repeat similarity. This modified method is nontoxic, is simple, and lends itself to modifications. It can also be used for standard FISH and mapping of individual sequence types in chromosomes/genomes.

Fluorescence In Situ Hybridization of DNA Probes on Mitotic Chromosomes of the Mexican Axolotl.

Fluorescence in situ hybridization (FISH) is used extensively for visual localization of specific DNA fragments (and RNA fragments) in broad applications on chromosomes or nuclei at any stage of the cell cycle: metaphase, anaphase, or interphase. The cytogenetic slides that serve as a target for the labeled DNA probe might be prepared using any approach suitable for obtaining cells with appropriate morphology for imaging and analysis. In this chapter, we focus on the application of molecular cytogenetic methods such as DNA labeling, slide preparation, and in situ hybridization related to cells from Mexican axolotl.

Probe Capture Enrichment Methods for HIV and HCV Genome Sequencing and Drug Resistance Genotyping.

Human immunodeficiency virus (HIV) infections remain a significant public health concern worldwide. Over the years, sophisticated sequencing technologies such as next-generation sequencing (NGS) have emerged and been utilized to monitor the spread of HIV drug resistance (HIVDR), identify HIV drug resistance mutations, and characterize transmission dynamics. Similar applications also apply to the Hepatitis C virus (HCV), another bloodborne viral pathogen with significant intra-host genetic diversity. Several advantages to using NGS over conventional Sanger sequencing include increased data throughput, scalability, cost-effectiveness when batched sample testing is performed, and sensitivity for quantitative detection of minority resistant variants. However, NGS alone may fail to detect genomes from pathogens present in low copy numbers. As with all sequencing platforms, the primary determinant in achieving quality sequencing data is the quality and quantity of the initial template input. Samples containing degraded RNA/DNA and/or low copy number have been a consistent sequencing challenge. To overcome this limitation probe capture enrichment is a method that has recently been employed to target, enrich, and sequence the genome of a pathogen present in low copies, and for compromised specimens that contain poor quality nucleic acids. It involves the hybridization of sequence-specific DNA or RNA probes to a target sequence, which is followed by an enrichment step via PCR to increase the number of copies of the targeted sequences after which the samples are subjected to NGS procedures. This method has been performed on pathogens such as bacteria, fungus, and viruses and allows for the sequencing of complete genomes, with high coverage. Post NGS, data analysis can be performed through various bioinformatics pipelines which can provide information on genetic diversity, genotype, virulence, and drug resistance. This article reviews how probe capture enrichment helps to increase the likelihood of sequencing HIV and HCV samples that contain low viral loads and/or are compromised.

Development of fluorescence-based nucleic acid blot hybridization method using Cy5.5 labeled DNA probes

Near-infrared (NIR) fluorophores are widely used as fluorescent probes for bioimaging because of their minimal photodamage to biological samples, deep penetration, and low interference from background autofluorescence. Here, we employed a NIR fluorescent cyanine dye Cy5.5 to label DNA probes for nucleic acid blot hybridization. The specificity and sensitivity of fluorescent DNA probes were proven by both Southern blot and Northern blot using cellulolytic bacterium Ruminiclostridium cellulolyticum as a model. Furthermore, employing the method, we successfully identified the gene disruption of ClosTron to rule out off-target, analyzed the differential transcription of genes under different conditions, and confirmed RNA cleavage. Compared to other nonradioactive probes, the preparation and detection of Cy5.5-labeled probes are more simple, more economical, and versatile, suggesting that the Cy5.5-labeled probes are suitable for nucleic acid blot hybridization in addition to bioimaging.

An Expeditious Approach towards the Synthesis and Application of Water-Soluble and Photostable Fluorogenic Chromones for DNA Detection.

The intensive research for hybridization probes based on organic molecules with fluorogenic properties is currently attracting particular attention due to their potential to efficiently recognize different DNA conformations and the local environment. However, most established organic chromophores do not meet the requirements of this task, as they do not exhibit good brightness in aqueous buffer media, develop aggregation and/or are not easily conjugated to oligodeoxynucleotides (ODNs) while keeping their photophysics intact. Herein, an important modification strategy was employed for a well-known fluorophore, 2-(4-(diethylamino)phenyl)-3-hydroxychromone (dEAF). Although this push–pull dye absorbs intensively in the visible range and shows emission with large Stokes shifts in all organic solvents, it is strongly quenched in water. This Achilles’ heel prompted us to implement a new strategy to obtain a series of dyes that retain all the photophysical features of dEAF in water, conjugate readily with oligonucleotides, and furthermore demonstrate sensitivity to hydration, thus paving the way for a high-performance fluorogenic DNA hybridization probe.

Superresolution Microscopy for Visualization of Physical Contacts Between Chromosomes at Nanoscale Resolution.

This protocol describes the fluorescence in situ hybridization (FISH) of DNA probes on mitotic chromosome spreads optimized for two super-resolution microscopy approaches-structured illumination microscopy (SIM) and stimulated emission depletion (STED). It is based on traditional DNA FISH methods that can be combined with immunofluorescence labeling (Immuno-FISH). This technique previously allowed us to visualize ribosomal DNA linkages between human acrocentric chromosomes and provided information about the activity status of linked rDNA loci. Compared to the conventional wide-field and confocal microscopy, the quality of SIM and STED data depends a lot more on the optimal specimen preparation, choice of fluorophores, and quality of the fluorescent labeling. This protocol highlights details that make specimens suitable for super-resolution microscopy and tips for good imaging practices.

B Chromosomes in Free-Living Flatworms of the Genus Macrostomum (Platyhelminthes, Macrostomorpha).

B chromosomes (Bs) or supernumerary chromosomes are extra chromosomes in the species karyotype that can vary in its copy number. Bs are widespread in eukaryotes. Usually, the Bs of specimens collected from natural populations are the object of the B chromosome studies. We applied another approach analyzing the Bs in animals maintained under the laboratory conditions as lines and cultures. In this study, three species of the Macrostomum genus that underwent a recent whole-genome duplication (WGD) were involved. In laboratory lines of M. lignano and M. janickei, the frequency of Bs was less than 1%, while in the laboratory culture of M. mirumnovem, it was nearer 30%. Their number in specimens of the culture varied from 1 to 14. Mosaicism on Bs was discovered in parts of these animals. We analyzed the distribution of Bs among the worms of the laboratory cultures during long-term cultivation, the transmission rates of Bs in the progeny obtained from crosses of worms with different numbers of Bs, and from self-fertilized isolated worms. The DNA content of the Bs in M. mirumnovem was analyzed with the chromosomal in situ suppression (CISS) hybridization of microdissected DNA probes derived from A chromosomes (As). Bs mainly consisted of repetitive DNA. The cytogenetic analysis also revealed the divergence and high variation in large metacentric chromosomes (LMs) containing numerous regions enriched for repeats. The possible mechanisms of the appearance and evolution of Bs and LMs in species of the Macrostomum genus were also discussed.

G-quadruplex microspheres-based optical RNA biosensor for arthropod-borne virus pathogen detection: A proof-of-concept with dengue serotype 2

Dengue virus (DENV) is a positive-sense single-stranded RNA virus and that the detection of viral RNA itself is highly desirable, which can be achieved by using RNA biosensor diagnostic method. Herein, acrylic micropolymer-based optical RNA biosensor was developed by binding anionic copper(II) phthalocyanine (CPC) planar aromatic ligand to the G-quadruplex DNA probe via end-stacking with π-system of the guanine (G) quartet, and a blue coloration was developed on the G-quadruplex microspheres. Hybridization of G-quadruplex DNA probe with target DENV serotype 2 (DENV2) RNA unfolded the G-quadruplex, and rendering release of the CPC planar optical label, causing discoloration of the G-quadruplex microbiosensor. Optical characterization of the RNA biosensor was performed by means of fiber optic reflectance spectrophotometer at maximum reflectance wavelength of 774 nm. The reflectance response enhancement of the RNA-responsive G-quadruplex-based reflectometric biosensor was linearly proportional to the target oligo DENV2 RNA concentration in the range of 2 zM–2 μM, with a 0.447 zM limit of detection and a rapid response time of 30 min. Heightening in the reflectance signal based on structural transition of G-quadruplex in response to target RNA was successfully implemented in real-time DENV2 detection in non-invasive human fluid samples (i.e. saliva and urine) under informed consent.

Nanoparticle-assisted detection of nucleic acids in a polymeric nanopore with a large pore size

Rapid and accurate detection of nucleic acids is of paramount importance in many fields, including medical diagnosis, gene therapy and virus identification. In this work, by taking advantage of two DNA hybridization probes, one of which was immobilized on the surface of gold nanoparticles, while the other was free in solution, detection of short length nucleic acids was successfully achieved using a large size (20 nm tip diameter) polyethylene terephythalate (PET) nanopore. The sensor was sensitive and selective: DNA samples with concentrations at as low as 0.5 nM could be detected within minutes and the number of mismatches can be discerned from the translocation frequency. Furthermore, the nanopore can be repeatedly used many times. Our developed large-size nanopore sensing platform offers the potential for fieldable/point-of-care diagnostic applications.

Cobalt Phthalocyanine-Ionic Liquid Composite Modified Electrodes for the Voltammetric Detection of DNA Hybridization Related to Hepatitis B Virus.

In this study, cobalt phthalocyanine (CoPc) and ionic liquid (IL) modified pencil graphite electrodes (PGEs) were designed and implemented to detect sequence-selective DNA hybridization related to the Hepatitis B virus (HBV). The surface characterization of CoPc-IL-PGEs was investigated by scanning electron microscopy (SEM), and the electrochemical behavior of electrodes were studied by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. The voltammetric detection of hybridization was investigated by evaluating the guanine oxidation signal, measured by differential pulse voltammetry (DPV) technique. The implementation of our biosensor to serum samples was also examined using fetal bovine serum (FBS). The detection limit was established as 0.19 µg/mL in phosphate buffer solution (PBS) (pH 7.40) and 2.48 µg/mL in FBS medium. The selectivity of our assay regarding HBV DNA hybridization in FBS medium was tested in the presence of other DNA sequences. With this aim, the hybridization of DNA probe with non-complementary (NC) or mismatched DNA sequence (MM), or in the presence of mixture samples containing DNA target NC (1:1) or DNA target MM (1:1), was studied based on the changes in guanine signal.

Long-term effects of fecal microbiota transplantation (FMT) in patients with irritable bowel syndrome.

We recently found fecal microbiota transplantation (FMT) in irritable bowel syndrome (IBS) patients to be an effective and safe treatment after 3months. The present follow-up study investigated the efficacy and safety of FMT at 1year after treatment. This study included 77 of the 91 IBS patients who had responded to FMT in our previous study. Patients provided a fecal sample and completed five questionnaires to assess their symptoms and quality of life at 1year after FMT. The dysbiosis index (DI) and fecal bacterial profile were analyzed using a 16S rRNA gene-based DNA probe hybridization. The levels of fecal short-chain fatty acids (SCFAs) were determined by gas chromatography. There was a persistent response to FMT at 1year after treatment in 32 (86.5%) and 35 (87.5%) patients who received 30-g and 60-g FMT, respectively. In the 30-g FMT group, 12 (32.4%) and 8 (21.6%) patients showed complete remission at 1year and 3months, respectively; the corresponding numbers in the 60-g FMT group were 18 (45%) and 11 (27.5%), respectively. Abdominal symptoms and the quality of life were improved at 1year compared with after 3months. These findings were accompanied by comprehensive changes in the fecal bacterial profile and SCFAs. Most of the IBS patients maintained a response at 1year after FMT. Moreover, the improvements in symptoms and quality of life increased over time. Changes in DI, fecal bacterial profile and SCFAs were more comprehensive at 1year than after 3months. www.clinicaltrials.gov (NCT03822299).

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA

Detection of cell-free DNA (cfDNA) has an impact on DNA analysis in liquid biopsies. However, current strategies to detect cfDNA have limitations that should be overcome, such as having low sensitivity and requiring much time and a specialized instrument. Thus, non-invasive and rapid detection tools are needed for disease prevention and early-stage treatment. Here we developed a device having a microheater integrated with zinc oxide nanowires (microheater-ZnO-NWs) to detect target single-stranded DNAs (ssDNAs) based on DNA probe hybridization. We confirmed experimentally that our device realized in-situ annealed DNA probes by which we subsequently detected target ssDNAs. We envision that this device can be utilized for fundamental studies related to nanobiodevice-based DNA detection.

Physical Mapping of the Anopheles (Nyssorhynchus) darlingi Genomic Scaffolds.

Simple SummaryAnopheles darlingi mosquitoes are the main vectors of malaria in the Brazilian Amazon. To assign genomic DNA sequences to chromosomes of this species, we performed fluorescence in situ hybridization of DNA probes with salivary glands polytene chromosomes. We compared the physical locations of the An. darlingi probes with homologous sequences in other Anopheles species, namely Anopheles albimanus and Anopheles gambiae. The results demonstrated that substantial genome rearrangements occurred throughout the evolutionary history of these mosquitoes. The physical mapping data can be useful for improving the structural accuracy of the An. darlingi genome assembly and for understanding the chromosomal evolution of these mosquitoes.The genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.

Chromogenic In Situ Hybridization (CISH) as a Method for Detection of C-Myc Amplification in Formalin-Fixed Paraffin-Embedded Tumor Tissue: An Update.

In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations, or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE ) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40× magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology, and the slides can be stored for a long time.