A sensitive and scalable fluorescence anisotropy single stranded RNA targeting approach for monitoring riboswitch conformational states.

The capacity of riboswitches to undergo conformational changes in response to binding their native ligands is closely tied to their functional roles and is an attractive target for antimicrobial drug design. Here, we established a probe-based fluorescence anisotropy assay to monitor riboswitch conformational switching with high sensitivity and throughput. Using the Bacillus subtillis yitJ S-Box (SAM-I), Fusobacterium nucleatum impX RFN element of (FMN) and class-I cyclic-di-GMP from Vibrio cholerae riboswitches as model systems, we developed short fluorescent DNA probes that specifically recognize either ligand-free or -bound riboswitch conformational states. We showed that increasing concentrations of native ligands cause measurable and reproducible changes in fluorescence anisotropy that correlate with riboswitch conformational changes observed by native gel analysis. Furthermore, we applied our assay to several ligand analogues and confirmed that it can discriminate between ligands that bind, triggering the native conformational change, from those that bind without causing the conformational change. This new platform opens the possibility of high-throughput screening compound libraries to identify potential new antibiotics that specifically target functional conformational changes in riboswitches.

The HpSGNi system: A compact approach for genetic suppression without sequence limitation in Escherichia coli.

Targeted gene regulation is indispensable for exploring gene functions in microbes and the development of microbial cell factories. While most loci can be regulated by CRISPRi, it cannot be used for targets lacking protospacer adjacent motifs (PAM) or protospacer flanking sequences (PFS). Here, we characterized a genetic suppression approach named the hpDNA-assisted structure-guided nuclease mediating interference system (HpSGNi). It was composed of a flap endonuclease 1 (FEN1) and mis-hairpin DNA probes (mis-hpDNA) to suppress the expression of target genes simply and efficiently in microbe without sequence restrictions. By inhibiting the initiation and elongation of the transcription, HpSGNi successfully silenced the transcription of exogenous fluorescent protein genes, ampicillin resistance gene and endogenous folP/sulA genes in Escherichia coli BL21(DE3) and K-12 MG1655. Meanwhile, aiming at optimizing the mis-hpDNA, we displayed the characteristics by detecting the tolerance to the single base mismatch and length of the guide sequence. This DNA-guided recognition platform provides a simple approach for selectively inhibiting gene expression.

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells.

The base excision repair (BER) pathway is a frontline defender of genomic integrity and plays a central role in epigenetic regulation through its involvement in the erasure of 5-methylcytosine. This biological and clinical significance has led to a demand for analytical methods capable of monitoring BER activities, especially in living cells. Unfortunately, prevailing methods, which are primarily derived from nucleic acids, are mostly incompatible with intracellular use due to their susceptibility to nuclease degradation and other off-target interactions. These limitations preclude important biological studies of BER enzymes and many clinical applications. Herein, we report a straightforward approach for constructing biostable BER probes using a unique chimeric d/l-DNA architecture that exploits the bioorthogonal properties of mirror-image l-DNA. We show that chimeric BER probes have excellent stability within living cells, where they were successfully employed to monitor relative BER activity, evaluate the efficiency of small molecule BER inhibitors, and study enzyme mutants. Notably, we report the first example of a fluorescent probe for real-time monitoring of thymine DNA glycosylase (TDG)-mediated BER of 5-formylcytosine and 5-carboxylcytosine in living cells, providing a much-needed tool for studying DNA (de)methylation biology. Chimeric probes offer a robust and highly generalizable approach for real-time monitoring of BER activity in living cells, which should enable a broad spectrum of basic research and clinical applications.

Specific multiplexed detection of mRNA splice variants based on size-coding DNA probes and universal PCR amplification.

Alternative messenger RNA (mRNA) splicing is a vital regulatory process during the gene expression of higher eukaryotes. The specific and sensitive quantification of disease-related mRNA splice variants in biological and clinical samples is becoming particularly important. Reverse transcription polymerase chain reaction (RT-PCR), the most classical strategy for the assay of mRNA splice variants, cannot avoid false positive signals, which poses a challenge to the specificity of mRNA splice variant detection. In this paper, by rationally designing two DNA probes with double recognition at the splice site and different lengths, different mRNA splice variants could generate amplification products of unique lengths. Combined with capillary electrophoresis (CE) separation, the product peak of the corresponding mRNA splice variant can be specifically detected, which can avoid false-positive signals caused by non-specific amplification of PCR, greatly improving the specificity of the mRNA splice variant assay. In addition, universal PCR amplification eliminates amplification bias caused by different primer sequences and improves quantitative accuracy. Furthermore, the proposed method can simultaneously detect multiple mRNA splice variants as low as 100 aM in a one-tube reaction and has been successfully applied to the assay of variants in cell samples, which will provide a new strategy for mRNA splice variant-based clinical diagnosis and research.

Mitochondria MicroRNA Spatial Imaging via pH-Responsive Exonuclease-Assisted AIE Nanoreporter.

Mitochondrial microRNAs (mitomiRs) critically orchestrate mitochondrial functions. Spatial imaging of mitomiRs is essential to understand its clinical value in diagnosis and prognosis. However, the direct monitoring of mitomiRs in living cells remains a key challenge. Herein, we report an AIE nanoreporter strategy for mitomiRs imaging in living cells through pH-controlled exonuclease (Exo)-assisted target cycle signal amplification. The AIE-labeled DNA detection probes are conjugated on Exo III encapsulated polymeric nanoparticles (NPs) via consecutive adenines (polyA). The amplified sensing functions are off during the cytoplasm delivery process, and it can be spatially switched from off to on when in the alkaline mitochondria (about pH 8) after triphenylphosphonium (TPP)-mediated mitochondrial targeting. Where the NPs degraded to release Exo III and cancer-specific mitomiRs hybridize with AIE-labeled DNA detection probes to expose the cleavage site of released Exo III, enabling spatially restricted mitomiRs imaging. The mitomiRs expression fluctuation was also realized. This study contributes to a facile strategy that could easily extend to a broad application for the understanding of mitomiRs-related pathological processes.

High-Throughput DNA FISH (hiFISH).

High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.

Ultrasensitive detection of tumor-derived small extracellular vesicles based on nonlinear hybridization chain reaction fluorescence signal amplification and immunomagnetic separation.

Small extracellular vesicles (sEVs) have attracted wide attention as a promising tumor biomarker. However, sensitive and selective detection of sEVs is challenging due to the low levels of sEVs in the early stage of cancers. Herein, a novel fluorescent sensor was developed for the detection of sEVs with high sensitivity and selectivity based on nonlinear hybridization chain reaction (nHCR) signal amplification and immunomagnetic separation. Firstly, sEVs were captured and enriched by CD63 antibody conjugated magnetic beads via antibody-antigen reactions. Then, cholesterol-modified DNA probes were anchored spontaneously on lipid membranes of sEVs through efficient hydrophobic interactions between the cholesterol moiety and the phospholipid bilayer of sEVs. The simultaneous recognition of the transmembrane protein and the phospholipid bilayer structure of the sEVs could effectively eliminate interferences from free proteins. The sticky ends of the cholesterol-modified DNA probes acted as the initiator to trigger nHCR to form a hyperbranched network of DNA structure that could recruit more fluorescent signal molecules for signal amplification. Under the optimal conditions, the nHCR-based strategy showed high sensitivity for the detection of sEVs with a limit of detection of 80 particles per µL. In addition, the as-constructed method was successfully applied for the analysis of clinical samples. It provides a sensitive and selective platform for the isolation and detection of sEVs in the early diagnosis of cancers.

Exploring Mitochondrial Localization of SARS-CoV-2 RNA by Padlock Assay: A Pilot Study in Human Placenta.

The ongoing COVID-19 pandemic dictated new priorities in biomedicine research. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a single-stranded positive-sense RNA virus. In this pilot study, we optimized our padlock assay to visualize genomic and subgenomic regions using formalin-fixed paraffin-embedded placental samples obtained from a confirmed case of COVID-19. SARS-CoV-2 RNA was localized in trophoblastic cells. We also checked the presence of the virion by immunolocalization of its glycoprotein spike. In addition, we imaged mitochondria of placental villi keeping in mind that the mitochondrion has been suggested as a potential residence of the SARS-CoV-2 genome. We observed a substantial overlapping of SARS-CoV-2 RNA and mitochondria in trophoblastic cells. This intriguing linkage correlated with an aberrant mitochondrial network. Overall, to the best of our knowledge, this is the first study that provides evidence of colocalization of the SARS-CoV-2 genome and mitochondria in SARS-CoV-2 infected tissue. These findings also support the notion that SARS-CoV-2 infection can reprogram mitochondrial activity in the highly specialized maternal-fetal interface.

DNA Tetrahedron-Based MNAzyme for Sensitive Detection of microRNA with Elemental Tagging.

Heterogeneous immunoassay based on magnetic separation is commonly used in inductively coupled plasma-mass spectrometry (ICP-MS)-based biomedical analysis with elemental labeling. However, the functionalized magnetic beads (MBs) often suffer from non-specific adsorption and random distribution of the functional probes. To overcome these problems, DNA tetrahedron (DT)-functionalized MBs were designed and further conjugated with substrate modified Au NPs (Sub-AuNP). Based on the prepared MB-DT-AuNP probes, an MB-DT based multicomponent nucleic acid enzyme (MNAzyme) system involving Au NPs as the elemental tags was proposed for highly sensitive quantification of miRNA-155 by ICP-MS. Target miRNA would trigger the assembly of MNAzyme, and Sub-AuNP would be cleaved from the MB-DT-AuNP probe, resulting in a cyclic amplification. Single-stranded DNA-functionalized MB (MB-ssDNA)-AuNP probes were prepared as well. Comparatively, the amount of Au NPs grafted onto MB-ssDNA-AuNP probes was higher than that grafted onto MB-DT-AuNP probes. Meanwhile, a higher signal-to-noise ratio was obtained by using MB-DT-AuNP probes over MB-ssDNA-AuNP probes in the MNAzyme system. Under the optimal experimental conditions, the limit of detection for target miRNA obtained by using MB-DT-AuNP probes was 1.15 pmol L-1, improved by 23 times over that obtained by the use of MB-ssDNA-AuNP probes. The proposed MB-DT-MNAzyme-ICP-MS method was applied to the analysis of miRNA-155 in serum samples, and recoveries of 86.7-94.6% were obtained. This method is featured with high sensitivity, good specificity, and simple operation, showing a great application potential in biomedical analysis.

Synthesis of Adenine-based Fluorescent and Naked-eye Chemosensors: Specific DNA Probes for The Detection of Bacterial Pathogens.

A rapid and confident tool to identify and diagnose bacterial pathogens with more accuracy using DNA as fingerprints is necessary. Herein, we report a smart chemosensor having a terminal adenine sticking to the thymine of single-stranded DNA (ssDNA) through supramolecular interactions and, which leaves ssDNA when the same ssDNA matches with the targeting desired DNA. We have synthesized a naked-eye coloured chemosensor with carbazole. As a model genetic material, DNA of Clavibacter michiganensis subsp. michiganensis was hybridized to ssDNA and immobilized over nitrocellulose membrane. The prepared adenine-chemosensor, by passing through the nitrocellulose-ssDNA membrane caused the formation of ssDNA nitrocellulose-ssDNA-adenine-chemosensor. FTIR results of the immobilized ssDNAs showed that the matching of same ssDNA releases the adenine-chemosensor from the surface of nitrocellulose-ssDNA that results in formation of the double stranded DNA. The selectivity of chemosensor was also confirmed with different bacterial DNA (Bacillus subtilis) as control. These data highlights accurate and reliable results of a new diagnostic kit prototype promising for further studies, which is able to diagnose DNA quickly and precisely.

Padlock Probe-Based Generation of DNAzymes for the Colorimetric Detection of Antibiotic Resistance Genes.

The increasing emergence of multidrug- and pan-resistant pathogens requires rapid and cost-efficient diagnostic tools to contain their further spread in healthcare facilities and the environment. The currently established diagnostic technologies are of limited utility for efficient infection control measures because they are either cultivation-based and time-consuming or require sophisticated assays that are expensive. Furthermore, infectious diseases are unfortunately most problematic in countries with low-resource settings in their healthcare systems. In this study, we developed a cost-efficient detection technology that uses G-quadruplex DNAzymes to convert a chromogenic substrate resulting in a color change in the presence of antibiotic resistance genes. The assay is based on padlock probes capable of high-multiplex reactions and targets 27 clinically relevant antibiotic resistance genes associated with sepsis. In addition to an experimental proof-of-principle using synthetic target DNA, the assay was evaluated with multidrug-resistant clinical isolates.

Epigenetic Quantification of DNA 5-Hydroxymethylcytosine Using DNA Hybridization-Based Single-Molecule Immunofluorescent Imaging.

5-Hydroxymethylcytosine (5hmC) is a deoxyribonucleic acid (DNA) epigenetic modification that has an important function in embryonic development and human diseases. However, the numerous methods that have been developed to detect and quantify 5hmC, require large amounts of DNA sample to be modified via chemical reactions, which considerably limits their application with cell-free DNA (cfDNA). Meanwhile, other antibody-based methods of detecting 5hmC do not offer information about the DNA sequence. Here, in this article DNA hybridization-based single-molecule immunofluorescent imaging is presented, an ultrasensitive method of detecting 5hmC modification in DNA. Via using the probe DNA to capture the DNA fragment of interest and the 5hmC antibody to detect the 5hmC modification in DNA, the fluorescent response signal of the 5hmC modification from the secondary antibody at the single-molecule level is successfully detected. Using the method, one could determine the quantity of 5hmC in the gene of interest within 6 h. In addition, it requires only 3 pg of the DNA sample and minimal experience and training for operation and analysis.

Quantifying and Adjusting Plasmon-Driven Nano-Localized Temperature Field around Gold Nanorods for Nucleic Acids Amplification.

Fast nucleic acid (NA) amplification has found widespread biomedical applications, where high thermocycling rate is the key. The plasmon-driven nano-localized thermocycling around the gold nanorods (AuNRs) is a promising alternative, as the significantly reduced reaction volume enables a rapid temperature response. However, quantifying and adjusting the nano-localized temperature field remains challenging for now. Herein, a simple method is developed to quantify and adjust the nano-localized temperature field around AuNRs by combining experimental measurement and numerical simulation. An indirect method to measure the surface temperature of AuNRs is first developed by utilizing the temperature-dependent stability of Authiol bond. Meanwhile, the relationship of AuNRs' surface temperature with the AuNRs concentration and laser intensity, is also studied. In combination with thermal diffusion simulation, the nano-localized temperature field under the laser irradiation is obtained. The results show that the restricted reaction volume (≈aL level) enables ultrafast thermocycling rate (>104  °C s-1 ). At last, a duplex-specific nuclease (DSN)-mediated isothermal amplification is successfully demonstrated within the nano-localized temperature field. It is envisioned that the developed method for quantifying and adjusting the nano-localized temperature field around AuNRs is adaptive for various noble metal nanostructures and will facilitate the development of the biochemical reaction in the nano-localized environment.

Development of Rapid and Visual Nucleic Acid Detection Methods towards Four Serotypes of Human Adenovirus Species B Based on RPA-LF Test.

OBJECTIVES: Human adenoviruses (HAdV) are classified as 7 HAdV species, and some serotypes in species B like HAdV 3, HAdV 7, HAdV 21, and HAdV 55 caused severe symptoms, even fatalities. Patients may be misdiagnosed and inadequately treated without reliable and practical methods for HAdV serotyping. Developing rapid, sensitive, and specific diagnostic methods for HAdV is critical. METHODS: Detection methods were established based on a recombinase polymerase amplification (RPA) assay and lateral flow (LF) test. Specific target sequence was screened, targeting which, primers and probes were designed, synthesized, and screened for establishing assay with high amplification efficiency. Primer or probe concentrations and amplification time were optimized. Detection limit, sensitivity, and specificity were evaluated. Results and Conclusions. Simple, sensitive, and specific RPA-LF methods for detection of four serotypes of HAdV together or separately were established, which had detection limits of 10 to 280 copies/reaction comparable to real-time PCR without recognizing other pathogens. The sensitivity and specificity were >92% and >98%, respectively, evaluated by limited clinical samples. The detection can be completed in 25 min without requirement of any instrument except a constant temperature equipment, showing superior detection performance and promising for a wide use in the field and resource-limited area.

Highly multiplexed rapid DNA detection with single-nucleotide specificity via convective PCR in a portable device.

Assays for the molecular detection of nucleic acids are typically constrained by the level of multiplexing (this is the case for the quantitative polymerase chain reaction (qPCR) and for isothermal amplification), turnaround times (as with microarrays and next-generation sequencing), quantification accuracy (isothermal amplification, microarrays and nanopore sequencing) or specificity for single-nucleotide differences (microarrays and nanopore sequencing). Here we show that a portable and battery-powered PCR assay performed in a toroidal convection chamber housing a microarray of fluorescently quenched oligonucleotide probes allows for the rapid and sensitive quantification of multiple DNA targets with single-nucleotide discrimination. The assay offers a limit of detection of 10 DNA copies within 30 min of turnaround time and a dynamic range spanning 4 orders of magnitude of DNA concentration, and we show its performance by detecting 20 genomic loci and 30 single-nucleotide polymorphisms in human genomic DNA samples, and 15 bacterial species in clinical isolates. Portable devices for the fast and highly multiplexed detection of nucleic acids may offer advantages in point-of-care diagnostics.

A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps.

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

A reversible shearing DNA probe for visualizing mechanically strong receptors in living cells.

In the last decade, DNA-based tension sensors have made significant contributions to the study of the importance of mechanical forces in many biological systems. Albeit successful, one shortcoming of these techniques is their inability to reversibly measure receptor forces in a higher regime (that is, >20 pN), which limits our understanding of the molecular details of mechanochemical transduction in living cells. Here, we developed a reversible shearing DNA-based tension probe (RSDTP) for probing molecular piconewton-scale forces between 4 and 60 pN transmitted by cells. Using these probes, we can easily distinguish the differences in force-bearing integrins without perturbing adhesion biology and reveal that a strong force-bearing integrin cluster can serve as a 'mechanical pivot' to maintain focal adhesion architecture and facilitate its maturation. The benefits of the RSDTP include a high dynamic range, reversibility and single-molecule sensitivity, all of which will facilitate a better understanding of the molecular mechanisms of mechanobiology.

Intracellular enzyme-powered DNA circuit with a tunable amplifier for miRNA imaging.

We describe an intracellular enzyme-powered DNA circuit probe with a tunable amplifier for sensitive and selective detection of miRNA. This approach has been successfully applied for in situ miRNA-21 fluorescence imaging in live cells. Also, we used chemicals to elevate the APE1 expression level rendering a tunable amplification strength for more flexible imaging applications.

Northern Blot Detection of Tiny RNAs.

Successful detection of very small RNAs (tiny RNAs, ~8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50-400 nt).

[Rapid diagnostics of novel coronavirus infection by loop-mediated isothermal amplification].

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.