A method to measure molecular hybridization.

Fluorescence-based oligonucleotide probes have a great importance in research of molecular interactions. Molecular beacons (MBs) are special case of fluorescent probes that form a stem-loop shape, bringing together a fluorophore and quencher, thus emitting fluorescence only when hybridized to a complementary target. Here we describe a new method for the quantitation of MB hybridization based on the measurement of changes in free energy instead of the fluorescence intensity. The MB energy state can be measured by micro-fluorescence detection. The approach allowed to determine hybridization energy of the MB with target nucleotide directly from fluorescence spectra and distinguish the MB in unfolded and hybridized states. Moreover, the method enabled us to discriminate between DNA duplexes with perfect complementarity or a single-nucleotide mismatch, based on the first direct experimental prove of enthalpy-entropy compensation.

Development and Optimization of Oligonucleotide Ligation Assay (OLA) Probes for Detection of HIV-1 Resistance to Dolutegravir.

The WHO currently recommends dolutegravir (DTG)-based ART for persons living with HIV infection in resource-limited-settings (RLS). To expand access to testing for HIV drug resistance (DR) to DTG in RLS, we developed probes for use in the oligonucleotide ligation assay (OLA)-Simple, a near-point of care HIV DR kit. Genotypic data from clinical trials and case reports were used to determine the mutations in HIV-1 integrase critical to identifying individuals with DTG-resistance at virologic failure of DTG-based ART. Probes to detect G118R, Q148H/K/R, N155H and R263K in HIV-1 subtypes A, B, C, D and CRF01_AE were designed using sequence alignments from the Los Alamos database and validated using 61 clinical samples of HIV-1 subtypes A, B, C, D, CRF01_AE genotyped by PacBio (n = 15) or Sanger (n = 46). Initial OLA probes failed to ligate for 16/244 (6.5%) codons (9 at G118R and 7 at Q148H/K/R). Probes revised to accommodate polymorphisms interfering with ligation at codons G118R and Q148R reduced indeterminates to 3.7% (5 at G118R and 4 at Q148H/K/R) and detected DTG-mutations with a sensitivity of 96.5% and 100% specificity. These OLA DTG resistance probes appear highly sensitive and specific across HIV-1 subtypes common in RLS with high burden of HIV infection.

Visualisation of Mycobacterium avium subsp. paratuberculosis in cultured cells, infected sheep and human tissue sections using fluorescent in situ hybridization (FISH).

We describe the development, testing and specificity of a modified oligonucleotide probe for the specific detection of Mycobacterium avium subsp. paratuberculosis (MAP) in culture and in infected tissue using fluorescent in situ hybridisation and confocal microscopy. The detection of MAP in both animal and human tissue using our modified probe allows for a more rapid diagnosis of MAP infection compared to the more often applied detection methods of culture and PCR and has the potential for quantification of cellular abundance. This approach would enable earlier treatment intervention and therefore the potential for reduced morbidity.

RNA Analysis Using Immunoassay Detection Format.

Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.

Development of a novel Colorimetric Assay for the rapid diagnosis of Coronavirus disease 2019 from nasopharyngeal samples.

Emergence of Coronavirus disease 2019 (COVID-19) pandemic has posed a huge threat to public health. Rapid and reliable test to diagnose infected subjects is crucial for disease spread control. We developed a colorimetric test for COVID-19 detection using a Colorimetric Assay based on thiol-linked RNA modified gold nanoparticles (AuNPs) and oligonucleotide probes. This method was conducted on RNA from 200 pharyngeal swab samples initially tested by Real-Time polymerase chain reaction (RT-PCR) as gold standard. A specific oligonucleotide probe designed based on ORF1ab of COVID-19 was functionalized with AuNPs-probe conjugate. The exposure of AuNP-probe to isolated RNA samples was tested using hybridization. In this comparative study, the colorimetric functionalized AuNPs assay exhibited a detection limit of 25 copies/µL. It was higher in comparison to the RT-PCR method, which could only detect 15 copies/µL. The results demonstrated 100% specificity and 96% sensitivity for the developed method. Herein, we developed an incredibly rapid, simple and cost-effective Colorimetric Assay lasting approximately 30 min which could process considerably higher number of COVID-19 samples compared to the RT-PCR. This AuNP-probe conjugate colorimetric method could be considered the optimum alternatives for conventional diagnostic tools especially in over-populated and/or low-income countries.

Designing Oligonucleotide-Based FISH Probe Sets with PaintSHOP.

Fluorescent in situ hybridization (FISH) enables the visualization of the position and abundance of nucleic acid molecules in fixed cell and tissue samples. Many FISH-based methods employ sets of synthetic, computationally designed DNA oligonucleotide (oligo) FISH probes, including massively multiplexed imaging spatial transcriptomics and spatial genomics technologies. Oligo probes can either be designed de novo or accessed from an existing database of pre-discovered probe sequences. This chapter describes the use of PaintSHOP, a user-friendly, web-based platform for the design of sets of oligo-based FISH probes. PaintSHOP hosts large collections of pre-discovered probes from many model organisms and also provides collections of functional sequences such as primers and readout domains and interactive tools to add these functional sequences to selected probes. Detailed examples are provided for three common experimental scenarios.

Unraveling Bacterial Single-Stranded Sequence Specificities: Insights from Molecular Dynamics and MMPBSA Analysis of Oligonucleotide Probes.

We utilized molecular dynamics (MD) simulations and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) free energy calculations to investigate the specificity of two oligonucleotide probes, namely probe B and probe D, in detecting single-stranded DNA (ssDNA) within three bacteria families: Enterobacteriaceae, Pasteurellaceae, and Vibrionaceae. Due to the limited understanding of molecular mechanisms in the previous research, we have extended the discussion to focus specifically on investigating the binding process of bacteria-probe DNA duplexes, with an emphasis on analyzing the binding free energy. The role of electrostatic contributions in the specificity between the oligonucleotide probes and the bacterial ssDNAs was investigated and found to be crucial. Our calculations yielded results that were highly consistent with the experimental data. Through our study, we have successfully exhibited the benefits of utilizing in-silico approaches as a powerful virtual-screening tool, particularly in research areas that demand a thorough comprehension of molecular interactions.

Tigerfish designs oligonucleotide-based in situ hybridization probes targeting intervals of highly repetitive DNA at the scale of genomes.

Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate > 100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.

Identification of chromosomes by fluorescence in situ hybridization in Gossypium hirsutum via developing oligonucleotide probes.

Discrimination of chromosome is essential for chromosome manipulation or visual chromosome characterization. Oligonucleotide probes can be employed to simplify the procedures of chromosome identification in molecular cytogenetics due to its simplicity, fastness, cost-effectiveness, and high efficiency. So far, however, visual identification of cotton chromosomes remains unsolved. Here, we developed 16 oligonucleotide probes for rapid and accurate identification of chromosomes in Gossypium hirsutum: 9 probes, of which each is able to distinguish individually one pair of chromosomes, and seven probes, of which each distinguishes multiple pairs of chromosomes. Besides the identification of Chrs. A09 and D09, we first find Chr. D08, which carries both 45S and 5S rDNA sequences. Interestingly, we also find Chr. A07 has a small 45S rDNA size, suggesting that the size of this site on Chr. A07 may have reduced during evolution. By the combination of 45S and 5S rDNA sequences and oligonucleotide probes developed, 10 chromosomes (Chrs. 3-7, and 9-13) in A subgenome and 7 (Chrs. 1-2, 4-5, and 7-9) in D subgenome of cotton are able to be recognized. This study establishes cotton oligonucleotide fluorescence in situ hybridization technology for discrimination of chromosomes, which supports and guides for sequence assembling, particularly, for tandem repeat sequences in cotton.

Bioinformatic Prediction of Bulked Oligonucleotide Probes for FISH Using Chorus2.

Fluorescence in situ hybridization (FISH) provides great conveniences for detection and visualization of specific genomic segments. Oligonucleotide (Oligo)-based FISH further broadened the applications in plant cytogenetics researches. High-specific single-copy oligo probes are essential for successful oligo-FISH experiments. Here, we introduce the bioinformatic pipeline to design genome-scaled single-copy oligos and filter repeat-related probes with Chorus2 software. Robust probes are accessible for both well-assembled genome and species without a reference genome based on this pipeline.

Visualization of Oligonucleotide-Based Probes Along Pseudochromosomes Using RIdeogram, KaryoploteR, and Circlize (Circos).

Fluorescence in situ hybridization (FISH) using oligonucleotide-based probes is an innovative modification of classic FISH techniques, enabling karyotypic identifications. Here, we exemplarily describe the design and in silico visualization of oligonucleotide-based probes derived from the Cucumis sativus genome. Additionally, the probes are also plotted comparatively to the closely related Cucumis melo genome. The visualization process is realized in R using various libraries for linear or circular plots including RIdeogram, KaryoploteR, and Circlize.

Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells.

Fluorescence in situ hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. We present an improved method for FISH probe production that yields high-purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The degree of labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO, and Alexa dyes) was mostly >90%, comparable with commercial probes. The ease and low cost of production allowed the generation of probe sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs for Polr2a (RNA polymerase II subunit 2a) and Gapdh, and of the long noncoding RNAs Malat1 and Neat1 Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in the Gabbr1 and Noc2l transcripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

Mid-Infrared Photothermal-Fluorescence In Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells.

Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput are necessary to answer the question of "who eats what, when, and where" in complex microbial communities. Here, we present a mid-infrared photothermal-fluorescence in situ hybridization (MIP-FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically labeled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labeling with rRNA-targeted probes enabled the identification of the analyzed cells. In proof-of-concept experiments, we showed that the clear spectral red shift in the protein amide I region due to incorporation of 13C atoms originating from 13C-labeled glucose can be exploited by MIP-FISH to discriminate and identify 13C-labeled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure-function analyses for microbiology.

Design, Labeling, and Application of Probes for RNA smFISH.

Visualization of single mRNA molecules in fixed cells can be achieved using single molecule fluorescent in situ hybridization (smFISH). This approach enables accurate quantification of mRNA numbers and localization at a single-cell level. To ensure reliable results using smFISH, it is critical to use fluorescent probes that are highly specific to their RNA target. To facilitate probe design, we have created anglerFISH, a user-friendly command-line based pipeline. In this chapter, we present how to perform a smFISH experiment using user-designed and labeled probes.

Preparation of Labeled DNA, RNA, and Oligonucleotide Probes.

Labeled nucleic acids and oligonucleotides are typically generated by enzymatic methods such as end-labeling, random priming, nick translation, in vitro transcription, and variations of the polymerase chain reaction (PCR). Some of these methods place the label in specific locations within the nucleic acid (e.g., at the 5' or 3' terminus); others generate molecules that are labeled internally at multiple sites. Some methods yield labeled single-stranded products, whereas others generate double-stranded nucleic acids. Finally, some generate probes of defined length, whereas others yield a heterogeneous population of labeled molecules. Options available for generating and detecting labeled nucleic acids, as well as advice on designing oligonucleotides for use as probes, is included here.

Facile design of multifunction-integrated linear oligonucleotide probe with multiplex amplification effect for label-free and highly sensitive GMO biosensing.

Well-defined structures and compositions of nucleic acids afford oligonucleotide probes with unique chemical properties and biological functions for various biosensing applications. Herein, a unique and special oligonucleotide probe, named multifunction-integrated linear oligonucleotide probe (MI-LOP), was facile designed and reported for label-free and turn-on fluorescent detection of the codon component of genetically modified organisms (GMOs). The MI-LOP contains four different functional regions including recognition of target, serving as polymerization template, and creating polymerization primer-linked G-quadruplex (PP-G-quadruplex). Without the aid of any other oligonucleotides, the introduction of target DNA can make each function of the MI-LOP executed one-by-one, during which the species of target DNA, target analogue, and PP-G-quadruplex can be cyclically utilized and in turn induce a multiplex signal amplification responsible for substantial collection of the G-quadruplex moieties under isothermal conditions. The stable G-quadruplexes can combine with N-methyl mesoporphyrin IX (NMM) and function as efficient fluorescence light-up probes, rapidly leading to a dramatic increase in the fluorescence intensity for the amplified detection of the target codon component. Our results strongly demonstrate that the developed MI-LOP with multiplex amplification effect confers the sensing strategy a high sensitivity and specificity for quantitative and qualitative detection of the target codon. And it has also been successfully applied for analyzing target codon in the complex extractions of soybean. The achievements highlight the significance of using oligonucleotide probes as promising analytical tools to promote the basic biochemical research and help in food and environmental analysis.

A Recent Update on Advanced Molecular Diagnostic Techniques for COVID-19 Pandemic: An Overview.

Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR-Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.

Monitoring co-cultures of Clostridium carboxidivorans and Clostridium kluyveri by fluorescence in situ hybridization with specific 23S rRNA oligonucleotide probes.

The development of co-cultures of clostridial strains which combine different physiological traits represents a promising strategy to achieve the environmentally friendly production of biofuels and chemicals. For the optimization of such co-cultures it is essential to monitor their composition and stability throughout fermentation. FISH is a quick and sensitive method for the specific labeling and quantification of cells within microbial communities. This technique is neither limited by the anaerobic fermenter environment nor by the need of prior genetic modification of strains. In this study, two specific 23S rRNA oligonucleotide probes, ClosKluy and ClosCarb, were designed for the monitoring of C. kluyveri and C. carboxidivorans, respectively. After the optimization of hybridization conditions for both probes, which was achieved at 30% (v/v) formamide, a high specificity was observed with epifluorescence microscopy using cells from different pure reference strains. The discriminating properties of the ClosKluy and ClosCarb probes was verified with samples from heterotrophic co-cultures in anaerobic flasks as well as autotrophic stirred-tank bioreactor co-cultures of C. kluyveri and C. carboxidivorans. Besides being suited to monitor defined co-cultures of these two species, the new specific FISH oligonucleotide probes for C. kluyveri and C. carboxidivorans additionally have potential to be applied in environmental studies.

Single Copy Oligonucleotide Fluorescence In Situ Hybridization Probe Design Platforms: Development, Application and Evaluation.

Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome variation, identification of allopolyploid, and deciphering of three-dimensional (3D) genome structures. Based on the demand for highly efficient oligo probes for oligo-FISH experiments, increasing numbers of tools have been developed for probe design in recent years. Obsolete oligonucleotide design tools have been adapted for oligo-FISH probe design because of their similar considerations. With the development of DNA sequencing and large-scale synthesis, novel tools have been designed to increase the specificity of designed oligo probes and enable genome-scale oligo probe design, which has greatly improved the application of single copy oligo-FISH. Despite this, few studies have introduced the development of the oligo-FISH probe design tools and their application in FISH experiments systematically. Besides, a comprehensive comparison and evaluation is lacking for the available tools. In this review, we provide an overview of the oligo-FISH probe design process, summarize the development and application of the available tools, evaluate several state-of-art tools, and eventually provide guidance for single copy oligo-FISH probe design.

Real-time monitoring of bead-based DNA hybridization in a microfluidic system: study of amplicon hybridization behavior on solid supports.

DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.