DNA Probes Research Articles

Factors impacting target-enriched long-read sequencing of resistomes and mobilomes.

We investigated the efficiency of target-enriched long-read sequencing (TELSeq) for detecting antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) within complex matrices. We aimed to overcome limitations associated with traditional antimicrobial resistance (AMR) detection methods, including short-read shotgun metagenomics, which can lack sensitivity, specificity, and the ability to provide detailed genomic context. By combining biotinylated probe-based enrichment with long-read sequencing, we facilitated the amplification and sequencing of ARGs, eliminating the need for bioinformatic reconstruction. Our experimental design included replicates of human fecal microbiota transplant material, bovine feces, pristine prairie soil, and a mock human gut microbial community, allowing us to examine variables including genomic DNA input and probe set composition. Our findings demonstrated that TELSeq markedly improves the detection rates of ARGs and MGEs compared to traditional sequencing methods, underlining its potential for accurate AMR monitoring. A key insight from our research is the importance of incorporating mobilome profiles to better predict the transferability of ARGs within microbial communities, prompting a recommendation for the use of combined ARG-MGE probe sets for future studies. We also reveal limitations for ARG detection from low-input workflows, and describe the next steps for ongoing protocol refinement to minimize technical variability and expand utility in clinical and public health settings. This effort is part of our broader commitment to advancing methodologies that address the global challenge of AMR.

Programming the Dynamic Range of Nanochannel Biosensors for MicroRNA Detection through Allosteric DNA Probes.

Solid-state nanochannel biosensors are extensively utilized for microRNA detection owing to their high sensitivity and rapid response. However, conventional nanochannel biosensors face limitations in their fixed dynamic range, restricting their versatility and efficacy. Herein, we introduce tunable tri-block DNA probes with varying affinities for target miRNA to engineer solid-state nanochannel biosensors capable of programmable dynamic range adjustment. The tri-block DNA architecture comprises a poly-adenine (polyA) block for adjustable surface density anchoring, alongside stem and loop blocks for modulating structural stability. Through systematic manipulation of these blocks, we demonstrate the ability to achieve diverse target binding affinities and detection limits, achieving an initial 81-fold dynamic range. By combining probes with different affinities, we extend this dynamic range significantly to 10,900-fold. Furthermore, by implementing a sequestration mechanism, the effective dynamic range of the nanochannel biosensor was narrowed to only a 3-fold range of target concentrations. The customizable dynamic range of these advanced nanochannel biosensors makes them highly promising for a broad spectrum of biomedical and clinical applications.

Gold–Graphene Quantum Dot Hybrid Nanoparticle for Smart Diagnostics of Prostate Cancer

Prostate cancer is one of the most prevalent cancers afflicting men worldwide, often detected at advanced stages, leading to increased mortality rates. Addressing this challenge, we present an innovative approach employing electrochemical biosensing for early-stage prostate cancer detection. This study used Indium–Tin Oxide (ITO) as a substrate and a deposited gold–graphene quantum dot (Au–GQD) nanohybrid to establish electrochemical sensing platforms for DNA-hybridization assays. A capturing DNA probe, PCA3, was covalently immobilized on the surface of the Au–GQDs and deposited electrochemically onto the ITO electrode surface. The Au–GQDs enabled the capturing of the target PCA3 biomarker probe. The sensor achieved a limit of detection (LoD) of up to 211 fM and presented a linear detection range spanning 1 µM to 100 fM. A rapid 5-min response time was also achieved. The tested shelf life of the pre-immobilized sensor was approximately 19 ± 1 days, with pronounced selectivity for its intended target amidst various interferants. The sensing device has the potential to revolutionize prostate cancer management by facilitating early-stage detection and screening with enhanced treatment efficacy.

LAMP-MS for Locus-Specific Visual Quantification of DNA 5mC and RNA m6A Using Ultra-Low Input.

Enhancing the effectiveness of utilizing circulating cell-free DNA (cfDNA) for disease screening remains a challenge, necessitating improved sensitivity, specificity, cost-efficiency, and patient adherence. We present here LAMP-MS, an innovative technology that integrates linear amplification with single-base quantitative nucleic acid mass spectrometry on silicon chips. This approach overcomes several limitations in utilizing cfDNA 5-methylcytosine (5mC) status for colorectal cancer (CRC) screening. LAMP-MS enables unbiased amplification of as little as 1 ng of cfDNA, site-specifically quantify methylation levels of tens to hundreds of 5mC sites, thereby facilitating cost-effective, high-resolution quantitative detection of cfDNA methylation markers. We have validated the accuracy of DNA methylation determination using DNA probes and cfDNA from patient plasma samples, confirmed by mass spectrometric peak areas. Additionally, we have further shown this Mass Array technology could be expanded to also quantify RNA m6A modification sites. Combining the ability to work with ultra-low input materials and a visually interpretable output, LAMP-MS stands out as a promising method for real-world applications in clinics and laboratories for nucleic acid methylation detection and quantification.

LAMP‐MS for Locus‐Specific Visual Quantification of DNA 5mC and RNA m6A Using Ultra‐Low Input

Enhancing the effectiveness of utilizing circulating cell‐free DNA (cfDNA) for disease screening remains a challenge, necessitating improved sensitivity, specificity, cost‐efficiency, and patient adherence. We present here LAMP‐MS, an innovative technology that integrates linear amplification with single‐base quantitative nucleic acid mass spectrometry on silicon chips. This approach overcomes several limitations in utilizing cfDNA 5‐methylcytosine (5mC) status for colorectal cancer (CRC) screening. LAMP‐MS enables unbiased amplification of as little as 1 ng of cfDNA, site‐specifically quantify methylation levels of tens to hundreds of 5mC sites, thereby facilitating cost‐effective, high‐resolution quantitative detection of cfDNA methylation markers. We have validated the accuracy of DNA methylation determination using DNA probes and cfDNA from patient plasma samples, confirmed by mass spectrometric peak areas. Additionally, we have further shown this Mass Array technology could be expanded to also quantify RNA m6A modification sites. Combining the ability to work with ultra‐low input materials and a visually interpretable output, LAMP‐MS stands out as a promising method for real‐world applications in clinics and laboratories for nucleic acid methylation detection and quantification.

Protein-bridged size-permeable DNAzyme@PEG hollow nanosphere reactor facilitates direct and rapid monitoring of trace circulating microRNAs in undiluted cancer patient serums

The direct monitoring of low levels of circulating microRNAs (miRNAs) in undiluted human serums is of significant clinical importance for the diagnosis of different diseases in the early stages; however, this is highly challenged by both the short lengths, low abundance and homogeneity nature of miRNAs and the complicated sample matrix of human serums. In this work, we show the synthesis of a new protein-bridged and size-permeable DNAzyme@PEG hollow nanosphere reactor (p-DPHNR) that enables direct and rapid detection of trace circulating microRNAs in undiluted serums from prostate carcinoma patients. The hydrophilic PEG shell of p-DPHNR efficiently reduces protein adsorption; its size-permeability only allows short miRNAs to enter the core cavity and protects the core DNA signal probes from digestion; and the confinement of the NIR signal amplification DNA probes in the nanosphere core accelerates the reaction kinetics and minimizes the serum background fluorescence. Such p-DPHNR exhibits a 1.8 pM detection limit with a short 20 min assay time for miRNA-141 and can realize clear discrimination of prostate carcinoma patients from healthy donors, thereby manifesting its robust clinical applications for the convenient and early diagnosis of various diseases

Aptamer-based sensitive fluorescence β-lactoglobulin food allergen bioassay via dual and cyclic bidirectional strand displacement amplifications.

β-Lactoglobulin (β-Lg) is a prevalent allergenic protein found in most dairy products, which poses great food safety risks for individuals with allergies, especially for infants. Sensitive and effective detection methods for such allergens are essential to reduce the risk of allergies in daily life. Herein, a fluorescent aptamer bioassay based on a dual and cyclic bidirectional strand displacement means is developed for thesensitive detection of β-Lg in infant rice porridge and milk. The aptamer in the duplex DNA probe binds β-Lg to release the assistance strand to further hybridize with two hairpins, which triggers the initiation of two cyclic amplification cycles through the polymerization, displacement, and nicking of the strands under the action of DNA polymerase and endonuclease restriction enzymes. The amplification cycles lead to the unfolding of many fluorescently quenched signal probes to exhibit substantially enhanced fluorescence recovery for detecting β-Lg. The assay can realize detection of β-Lg in concentrations as low as 4.41pM within the range of 0.01 to 10nM. Furthermore, our sensing method has the capability to discriminate β-Lg from other proteins with high selectivity, resulting in low levels of β-Lg detection in rice porridge and milk samples, demonstrating promising potentials of the developed sensing method for monitoring various food allergens.

Acidic Extracellular pH-Activated Allosteric DNA Nanodevice for Fluorescence Imaging of APE1 Activity in Tumor Cells.

Allostery is a phenomenon where the binding of a ligand at one allosteric site influences the affinity for another ligand at an active site. Different from orthosteric regulation, it allows for more precise control of biomolecular activity and enhances the stability of the molecules. Inspired by allosteric regulation of natural molecules, we present a Y-shaped allosteric DNA nanodevice, termed YssAP, that was pH-responsive and functionalized with the AS1411 aptamer for accurate fluorescence imaging of human apurinic/apyrimidinic endonuclease (APE1) activity in tumor cells. With rational design, YssAP could not be cut by APE1, and Cy5 was in the proximity of BHQ2, leading to suppressed signal emission. In contrast, since acidic pH acted as an allosteric effector, YssAP underwent a conformational change into an activated DNA probe (YdsAP) at acidic extracellular pH. After entering the tumor cell via the specific recognition of AS1411 aptamer, the overexpressed APE1 in the tumor cell cut the AP site on YdsAP. Cy5 moved far away from BHQ2, resulting in a strong signal output. Compared with the direct construction of the APE1 substrate, allosteric DNA nanodevices have more accurate imaging effects, which can be precisely adjusted by changing the switching state. We anticipate that this strategy will be applied in the screening of APE1 inhibitors and precise tumor diagnosis.

A Novel PCR-Free Ultrasensitive GQD-Based Label-Free Electrochemical DNA Sensor for Sensitive and Rapid Detection of Francisella tularensis

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π−π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two–five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 105 to 0.01 nM and has good specificity. The biosensor’s detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R2 = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs.

Immuno-RCA for highly sensitive detection of the antigen-antibody complex in the blood group antigen model

The problem of detecting tiny quantities of analytes by immunochemical methods has been tried for a long time. One approach is to use nucleic acid amplification methods to amplify the signal from a single antigen-antibody interaction. An amplification method suitable for microarrays is the rolling circle amplification reaction. The principle of the method is usage of a conjugate of a detecting antibody with a primer and subsequent isothermal amplification. The generation of a huge single-stranded reaction product starts after adding the necessary components for amplification reaction: circular oligonucleotides, which serves as a template for amplification and phage phi29 polymerase with the other components. This reaction product consists of a lot of repeats of a nucleotide sequence, that is complementary to the circular template. The fluorescent DNA probe can hybridize to each repeat on the product molecule, resulting in a significantly higher level of fluorescence than with fluorescently labeled antibody or streptavidin development systems. In addition, the reaction product remains immobilized on the surface, allowing usage of this approach for the detection of antigen-antibody interactions in other solid-phase analysis systems, such as microarrays. A common problem with such approaches is the nonspecific sorption of components of the immunochemical reaction or amplification reaction, leading to a high background. It is obvious that no matter how highly sensitive the analysis is in theory, a high background will reduce the entire potential of the method to nothing. Herein, we have developed a method that makes it possible to detect small amounts of antibodies to glycans in blood serum and in swabs from tumor cells in a microarray format using a model of blood group antigens. It was possible to obtain a 30 to 70-fold increase of fluorescence level from a specific interaction compared to the use of fluorescently labeled streptavidin. The method we are developing is promising, as it allows us to significantly increase the signal from the specific antigen/antibody interaction in the glycochip format, which will make it possible to detect antibodies to glycans in samples with a very low concentrations of antibodies, for example, in washes from tumor cells.

Novel metal–organic framework biosensing platform for detection of COVID-19 RNA

The latest pandemic resulting from the novel SARS-CoV-2 coronavirus has significantly affected public health, the worldwide economy, and social life. Metal organic frameworks (MOFs) are currently being implemented in biosensors for rapid and accurate detection of viruses thanks to their exceptional properties. This research aims to develop a Zeolitic Imidazolate Framework-8 (ZIF-8) based fluorescent biosensor for facile and rapid COVID-19 RNA sequence detection. ZIF-8 was characterized using several tests, such as FT-IR, TGA, and PXRD, to examine the MOF’s crystalline structure and thermal stability. The results demonstrated high crystallinity and thermal stability up to a temperature of 550 °C. The experimental study showed that ZIF-8 is an excellent fluorescence quencher, with 78.39% quenching efficiency. Analyzing the adsorption mechanism of probe DNA into ZIF-8 revealed that they can form electrostatic and π–π stacking interactions, forming a P-DNA@ZIF-8 complex and that PET is more dominant than FRET in the quenching mechanism. This ZIF-8 biosensing platform showed high sensitivity towards COVID-19 RNA with an ultra-low limit of detection of 6.24 pM, a rapid detection time of 8 min, and high selectivity to COVID-19 RNA. Indeed, ZIF-8 experienced much lower fluorescence recovery when tested on two mismatched RNAs. The experimental results show the potential use of ZIF-8 as a novel biosensor for a rapid and sensitive COVID-19 diagnosis.

A novel approach ofdifferentiation of adenoma and carcinoma in lung cancer based on biogenic in situ synthesis of gold nanostructures on various oligonucleotide motifs.

Aunique approach is introducedfor constructing gold nanocrystals (AuNCs) with RNA motif-directed morphologies in a sequence-independent manner and itsapplications in theclinical areaare described. By using this method, a label-free LSPR-based detection method for the SOX2OT transcript, long non-coding RNAs (lncRNAs), which is a prognostic indicator of poor survival in lung cancer patientsis presented. For the first time, we examined how the structural changes of RNA after the heteroduplex formation with a specific DNA probe can change the morphology and LSPR band of AuNCs. Using this method, is was possible to differentiate lung squamous cell carcinoma from adenocarcinoma samples without a need for a prior amplification of the target lncRNA. The approach of using specific DNA probe enables the in situ synthesis of nanocrystals in a different way and expands this method for future translational medicine, particularly detection of specific RNA.

The Study of Sensing Elements Parameters Optimization for Developed Biosensor of SARS-CoV-2 Detection

New advancements in developing sensitive and selective biosensors have demonstrated outstanding potential for Deoxyribonucleic Acid (DNA biosensors). The detection mode of DNA biosensors primary depends on a particular DNA hybridization that precisely occurs on the surface of the physical transducer that can only be detected using high-performance assays due to slight current changes. The analytical performance (sensitivity) of the DNA biosensor is conclusively rely on the confluence constructing of the sensing surface, which must be optimized. Thus, in this study, the sensing elements of the developed biosensors were optimized for detecting RNA of SARS-CoV-2. This optimization included concentration of nanomaterials (carbon quantum dots), probe density (concentration of DNA probe) and concentration of linker (APTES). It was observed that 0.15 % V/V of concentration CQD, 0.1μM of DNA probe and 36% V/V of APTES were the optimum parameters which provided their maximum response during electrical measurements and increased the sensitivity of the developed biosensor for SARS-CoV-2 detection.

Reduced Non-Specific Binding of Super-Resolution DNA-PAINT Markers by Shielded DNA-PAINT Labeling Protocols.

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events approximately five-fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific backgrounds.

Rapid Molecular Diagnostics in Vulvovaginal Candidosis.

Vulvovaginal candidosis (VVC) is a common condition among women, with current diagnostic methods relying on clinical evaluation and laboratory testing. These traditional methods are often limited by the need for specialized training, variable performance, and lengthy diagnostic processes, leading to delayed treatment and inappropriate antifungal use. This review evaluates the efficacy of molecular diagnostic tools for VVC and provides guidance on their application in clinical practice. A literature search was conducted using PubMed to identify studies evaluating rapid diagnostic tests specifically for vulvovaginal Candida isolates. Inclusion criteria focused on studies utilizing molecular diagnostics for the detection of Candida species in VVC. Articles discussing non-vaginal Candida infections, non-English studies, and animal or in vitro research were excluded. Twenty-three studies met the inclusion criteria, predominantly evaluating nucleid acid amplification tests/polymerase chain reaction (NAAT/PCR) assays and DNA probes. PCR/NAAT assays demonstrated high sensitivity and specificity (>86%) for VVC diagnosis, outperforming conventional diagnostic methods. Comparatively, DNA probes, while simpler, exhibited lower sensitivity. The included studies were mostly observational, with only one randomized controlled trial. Emerging diagnostic technologies, including artificial intelligence and integrated testing models, show promise for improving diagnostic precision and clinical outcomes. Molecular diagnostics offer a significant improvement in VVC management, though traditional methods remain valuable in resource-limited settings.

An Electrochemical Bioplatform Based on Acrylamide‐Induced Inhibition of DNA‐RNA Hybridization for the Determination of Acrylamide

AbstractThis study reports the development of an innovative electrochemical bioplatform for the indirect determination of acrylamide (AM) based on its inhibitory effect on the hybridization of nucleic acids through the formation of AM‐guanine single‐stranded (ssDNA) adducts. The method combines the advantages of the formation and selective immunorecognition of DNA/RNA heteroduplex, with the use of MBs and amperometric transduction using disposable SPCEs. Increasing concentrations of acrylamide prevented the efficient hybridization of a synthetic biotinylated DNA probe immobilized on streptavidin‐coated magnetic beads with its complementary RNA target and the as‐formed heteroduplex recognition with a specific antibody and a secondary HRP‐labeled antibody used for electrochemical readout. The resulting magnetic bioconjugates were magnetically trapped on the surface of the working electrode of a SPCE and the amperometric transduction was carried out in the presence of the H2O2/HQ system, recording a cathodic response inversely proportional to the AM concentration. The developed bioplatform is more competitive with other reported methods in terms of simplicity and assay time, allowing the selective detection of 1 nM AM in 60 min. In addition, the bioplatform was applied to the analysis of potato crisp water extracts.

“Grafting-from” and “Grafting-to” Poly(N-isopropyl acrylamide) Functionalization of Glass for DNA Biosensors with Improved Properties

Surface-based biosensors have proven to be of particular interest in the monitoring of human pathogens by means of their distinct nucleic acid sequences. Genosensors rely on targeted gene/DNA probe hybridization at the surface of a physical transducer and have been exploited for their high specificity and physicochemical stability. Unfortunately, these sensing materials still face limitations impeding their use in current diagnostic techniques. Most of their shortcomings arise from their suboptimal surface properties, including low hybridization density, inadequate probe orientation, and biofouling. Herein, we describe and compare two functionalization methodologies to immobilize DNA probes on a glass substrate via a thermoresponsive polymer in order to produce genosensors with improved properties. The first methodology relies on the use of a silanization step, followed by PET-RAFT of NIPAM monomers on the coated surface, while the second relies on vinyl sulfone modifications of the substrate, to which the pre-synthetized PNIPAM was grafted to. The functionalized substrates were fully characterized by means of X-ray photoelectron spectroscopy for their surface atomic content, fluorescence assay for their DNA hybridization density, and water contact angle measurements for their thermoresponsive behavior. The antifouling properties were evaluated by fluorescence microscopy. Both immobilization methodologies hold the potential to be applied to the engineering of DNA biosensors with a variety of polymers and other metal oxide surfaces.

Potential-resolved ratiometric electrochemiluminescence detection for prostate-specific antigen based on CdS nanocrystals modified on carbon nanotubes and luminol functionalized nanocomposites.

A ratiometric electrochemiluminescence (ECL) aptamer-based sensing platform was fabricated for prostate-specific antigen (PSA) determination. Activated CdS nanocrystals/multi-walled carbon nanotubes (CdS/MCNTs) and luminol-Pt/PAMAM nanocomposites (L-Pt/PAMAM NCs) were synthesized and used as cathodic and anodic ECL emitters, respectively. Amino group-modified aptamers were assembled on carboxylated magnetic beads, followed by hybridization with probe DNA functionalized L-Pt/PAMAM NCs. In the presence of PSA, the aptamer would bind specifically to the target PSA, thereby releasing L-Pt/PAMAM NCs. After magnetic separation, the separated L-Pt/PAMAM NCs would hybridize with capture DNA on CdS/MCNTs coated on glassy carbon electrode. This binding would lead to a decrease in cathodic ECL signal of CdS/MCNTs, due to the efficient energy transfer from CdS/MCNTs to L-Pt/PAMAM NCs. Meanwhile, L-Pt/PAMAM brought the anodic ECL signal from luminol. With the increase of PSA concentration, the ECL emission from luminol increased and the ECL emission from CdS/MCNTs decreased. The ratio of ECL intensity of luminol at 0.55V and CdS/MCNTs at - 1.25V could be used to quantify the concentration of PSA. This method enables sensitive and reliable detection of PSA over a wide range from 0.05 to 200ngmL-1, and the detection limit is 0.02ngmL-1.

Quantitative and spatially resolved detection of multiplexed microRNA from plant tissue via hybridization to hydrogel-bound DNA probes in nanoliter well arrays

Understanding complex regulatory networks in plant systems requires elucidating the roles of various gene regulators under a spatial landscape. MicroRNA are key regulators that impart high information value through their tissue specificity and stability when using expression patterns for evaluating network outcomes. However, current techniques that utilize spatial multiplexing and quantitation of microRNA are limited to primarily mammalian systems. Here, we present a method to spatially resolve and quantify multiple endogenous microRNA in situ using ethanol fixed, paraffin embedded model plant species. This method utilizes target-specific microRNA capture along with universal ligating and labelling, all within functionalized hydrogel posts containing DNA probes in nanoliter well arrays. We demonstrate the platform’s multiplexing capabilities through analyzing three endogenous microRNA in Arabidopsis thaliana rosettes which provide useful answers to fundamental plant growth and development from the unique expression patterns. The spatial tissue technique is also validated using non-spatial small RNA assays to demonstrate the versatility of the well array platform. Our new platform expands the toolkit of spatial omics technologies for plants.

The Escherichia coli ribB riboswitch senses flavin mononucleotide within a defined transcriptional window.

Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli. Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process.