Single-base Mismatch Research Articles

Label-Free Quantification of DNA Hybridization Kinetics and Affinity Enabled by an Electro-Optical Modulation on Single Nanoparticles.

Since the formation of a DNA duplex plays a vital role in gene expression and regulation across all kinds of organisms, quantifying the interaction during DNA hybridization is essential for understanding various biological processes in living systems on the molecular scale of nucleic acids. Here, we developed a label-free method to measure the binding kinetics and affinity of DNA hybridization with an electro-optical modulation on the individual gold nanorods based on a total internal reflection dark-field imaging microscopy. Under the electrochemical modulation, a Fourier transform filter was utilized to extract the optical scattering of the nanorods, which varies with the DNA binding due to their impedance change. We validated the imaging principle and established an analytical model to monitor the optical response during the DNA hybridization. Using the presented platform, we measured the binding kinetics and affinities of different DNA pairs and demonstrated its capability to distinguish the DNA hybridization with only single-base mismatch, which may provide guidance to explore the etiology and pathogenesis of diseases associated with single point gene mutations. Furthermore, the method allows for simultaneous imaging of multiple nanoparticles, thus enabling a high-throughput detection and opening possibilities for the study of the interfacial heterogeneity.

A sensitive electrochemical genosensor for measuring gastric cancer biomarker, microRNA-106b, based on asparagine polymerization on carbon quantum dots modified electrode

The aim of this work is to develop an electrochemical genosensor to measure microRNA 106b (miR-106b) as a gastric cancer biomarker. To construct the miR-106b genosensor, asparagine (As) was electropolymerized on the surface of carbon quantum dots modified glassy carbon electrode (CQDs/GCE). In the next step, ssDNA was immobilized on the modified electrode surface (As/CQDs/GCE) and then hybridized with miR-106b. Finally, differential pulse voltammograms of the accumulated chlorogenic acid (CGA), as an electroactive label, on the surface of immobilized ssDNA was recorded before (ssDNA/As/CQDs/GCE) and after hybridization with miR-106b (miR-106b/BSA/ssDNA/As/CQDs/GCE). The difference in the peak current of these two voltammograms was used as an analytical signal to measure of miR-106b. Using this genosensor, a linear concentration range from 1.0 fM to 1.0 µM and a detection limit of 0.39 fM were obtained for the measurement of miR-106b. The obtained data indicate that it is possible to recognize miR-106 from a non-complementary and a single-base mismatch sequence using the genosensor. In addition, the measurement of miR-106b in a human serum sample was successfully performed using the genosensor.

Digital Magnetic Detection of Biomolecular Interactions with Single Nanoparticles.

Biomolecular interactions compose a fundamental element of all life forms and are the biological basis of many biomedical assays. However, current methods for detecting biomolecular interactions have limitations in sensitivity and specificity. Here, using nitrogen-vacancy centers in diamond as quantum sensors, we demonstrate digital magnetic detection of biomolecular interactions with single magnetic nanoparticles (MNPs). We first developed a single-particle magnetic imaging (SiPMI) method on 100 nm-sized MNPs with negligible magnetic background, high signal stability, and accurate quantification. The single-particle method was performed on biotin-streptavidin interactions and DNA-DNA interactions in which a single-base mismatch was specifically differentiated. Subsequently, SARS-CoV-2-related antibodies and nucleic acids were examined by a digital immunomagnetic assay derived from SiPMI. In addition, a magnetic separation process improved the detection sensitivity and dynamic range by more than 3 orders of magnitude and also the specificity. This digital magnetic platform is applicable to extensive biomolecular interaction studies and ultrasensitive biomedical assays.

Synthesis and Evaluation of MGB Polyamide-Oligonucleotide Conjugates as Gene Expression Control Compounds.

MGB polyamide-oligonucleotide conjugates ON 1-4 with linked MGB polyamides at the 2-exocyclic amino group of a guanine base using aminoalkyl linkers were synthesized and evaluated in terms of binding affinity for complementary DNA containing the MGB polyamide binding sequence using T m and CD analyses. The MGB polyamides comprised pyrrole polyamides (Py4- and Py3-), which possess binding affinity for A-T base pairs, and imidazole (Im3-) and pyrrole-γ-imidazole (Py3-γ-Im3-) polyamide hairpin motifs, which possess binding affinity for C-G base pairs. It was found that the stability of modified dsDNA was greatly influenced by the linker length. Py4- and Py3-oligonucleotide conjugates (ON 1 (n = 4) and ON 2 (n = 4)) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA. Although Im3-oligonucleotide conjugate ON 3 (n = 4) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 (n = 4) was lower compared with the pyrrole amide moiety of ON 2 (n = 4). The Py3-γ-Im3-oligonucleotide conjugate ON 4 (n = 2), which possesses binding affinity for C-G base pairs via a pyrrole/imidazole combination and contains a 2-aminoethyl linker, showed high binding ability for complementary DNA. Furthermore, the DNA sequence recognition of MGB polyamide-oligonucleotide conjugates was investigated using single-base mismatch DNAs, which possess a mismatch base in the MGB polyamide binding sequence. The Py3-γ-Im3-oligonucleotide conjugate ON 4 (n = 2) showed high sequence recognition ability for complementary DNA.

Fast genotyping of K-RAS codons 12 and 13 based on streptavidin magnetic microbeads equipped with biotin-coupled single base-pair mismatch PCR (SM-PCR)

In this study, a single base-pair mismatch polymerase chain reaction (SM-PCR) combined with streptavidin magnetic microbeads (MB) for genotyping of codons 12 and 13 of K-RAS gene was established. It was performed by adding three primers including codon 12 forward single-base mismatch primer, codon 13 forward single-base mismatch primer and one universal reverse primer to differentiate single nucleotide polymorphisms. The both forward primers were designed to carry a single-base mismatch nucleotide to either codons 12 and 13. This design was included to increase the primer specificity and to enhance the discrimination power of the point mutation. Two restriction enzymes (StyD4I and HaeIII) were available for double digestion of codons 12 and 13 at the single tube. The wild type of codon 12 and 13 was digested, but the mutant type was not. And then, using the magnetic microbeads for purifying. Finally, wild type of codon 12 and 13 have different emission wavelength, but mutant type did not due to no digestion. This method showed a good consistency between genotyping results and direct sequencing results. The SM-PCR combined with MB method was feasible and efficient for screening of K-RAS codons 12 and 13 in populations.

A universal and specific RNA biosensor via DNA circuit-mediated PAM-independent CRISPR/Cas12a and PolyA-rolling circle amplification

Point of care testing (POCT) has important clinical significance for the diagnosis and prognosis evaluation of diseases. At present, the biosensor based on CRISPR/Cas12a has become a powerful diagnostic tool due to its high sensitivity. However, CRISPR/Cas12a requires PAM sequence to recognize target double strand and only can recognize specific sequence, so it is not universal. The current RNA detection techniques either lack consideration for specificity and universality, are expensive and difficult, or both. Therefore, it is crucial to create a CRISPR/Cas12a-based RNA detection system that is easy to use, cheap, specific, and universal in order to further its use in molecular diagnostics. Here, we established a DNA circuit-mediated PAM-independent CRISPR/Cas12a coupled PolyA-rolling circle amplification for RNA detection biosensor, namely DCPRBiosensor. The DCPRBiosensor not only functions as a simple, inexpensive, and highly sensitive RNA detection sensor, but it also boasts innovative specificity and universality features. More importantly, DCPRBiosensor removes the PAM restriction of CRISPR/Cas12a. The DCPRBiosensor's detection limit reached 100 aM and it had a linear relationship between 100 aM and 10 pM. We detected four piRNAs to verify the universality and stability of DCPRBiosensor. Then, we verified that DCPRBiosensor has good discrimination ability for single-base mismatch. Finally, we successfully detected piRNA in DLD-1 and HCT-116 cells and urine mixed samples within 4.5 h. In conclusion, we believe that DCPRBiosensor will have a substantial impact on both the development of CRISPR/as12a′s applications and the investigation of the clinical value of piRNA.

Recognition of recombination intermediates by the mismatch repair protein Msh2-Msh6.

The mismatch repair (MMR) protein MutSα (Msh2-Msh6), known for improving the fidelity of DNA replication, also improves the fidelity of homologous recombination. Msh2-Msh6 and its associated proteins are responsible for suppressing homologous recombination when excessive base mispairing is present between the participating chromosomes. In MMR, Msh2-Msh6 recognizes small insertion/deletion loops and single base pair mismatches while scanning DNA post replication. Interestingly, Sc Msh2-Msh6 binds to Holliday Junctions (HJs) with an affinity comparable to that of mismatched duplex DNA (Kd =15 nM). We study the Msh2-Msh6-HJ binding interaction to gain insight into the mechanistic role of Msh2-Msh6 during the process of homologous recombination. In our previous study we examined the dynamics of single base mismatches and the changes associated with Msh2-Msh6 binding using the fluorescent DNA base analog 6-MI and time-resolved fluorescence methods. Here, we employ a similar strategy to study the dynamics of single bases within HJs alone and upon interaction with Msh2-Msh6. 6-MI probes incorporated into the center and arms of the junction only exhibit perturbed dynamics in the center location upon Msh2-Msh6 binding, suggesting protein binding takes place at the junction center. Successful UV-photo crosslinking of Msh2-Msh6 to HJs containing 5-bromouracil at the junction center further supports this finding. Base dynamics at the center of the junction resemble those of the mismatch, which possibly drives Msh2-Msh6 recognition. Interestingly, introduction of a mismatch in the arms of the HJ shifts the Msh2-Msh6 binding from the center of HJ to the mismatch location despite the similar binding affinities. Additionally, a molecular model of the Msh2-Msh6-HJ interaction has been created and subjected to molecular dynamics simulations to gain further structural insights with particular focus on how the DNA binding domain interacts with HJs compared to mismatched duplex DNA.

Cascade-amplified fluorescence polarization assay for miRNA based on aggregation strategy of Y-shaped DNA

MicroRNAs (miRNAs) have emerged as one of the promising biomarkers. Their aberrant levels are intimately connected to various diseases. However, Sensitive assay of low-abundance miRNAs remain a grand challenge. Herein, a novel sensitive cascade-amplified fluorescence polarization (FP) sensing platform for miRNA detection was developed based on aggregation strategy of Y-shaped DNA via streptavidin (SA). The miRNA-155 was employed as the model target analyte. In the presence of miRNA-155, the duplex-specific nuclease (DSN) cycling amplification and catalytic hairpin assembly (CHA) cycling amplification were triggered successively. Owing to the target-triggering dual cyclic amplification, numerous Y-shaped DNA nanostructure could be easily generated according to the assembly order of hairpin probes. Then, SA was added to the system, inducing the aggregation of Y-shaped DNA nanostructure to form a large-size of DNA/SA complexes. Taking advantages of cascade-amplified strategy, a significant enhancement of the FP signal was achieved and a sensitive assay for miRNA detection was realized with the detection limit of 1 × 10-13 M. Beyond that, the proposed method could distinguish single-base mismatch and achieved satisfactory result for the expression level assay of miRNA-155 in different cell lines, holding great potential in the applications in miRNA-related clinical diagnosis and biochemical researches.

Multiplexed smFRET Nucleic Acid Sensing Using DNA Nanotweezers

The multiplexed detection of disease biomarkers is part of an ongoing effort toward improving the quality of diagnostic testing, reducing the cost of analysis, and accelerating the treatment processes. Although significant efforts have been made to develop more sensitive and rapid multiplexed screening methods, such as microarrays and electrochemical sensors, their limitations include their intricate sensing designs and semi-quantitative detection capabilities. Alternatively, fluorescence resonance energy transfer (FRET)-based single-molecule counting offers great potential for both the sensitive and quantitative detection of various biomarkers. However, current FRET-based multiplexed sensing typically requires the use of multiple excitation sources and/or FRET pairs, which complicates labeling schemes and the post-analysis of data. We present a nanotweezer (NT)-based sensing strategy that employs a single FRET pair and is capable of detecting multiple targets. Using DNA mimics of miRNA biomarkers specific to triple-negative breast cancer (TNBC), we demonstrated that the developed sensors are sensitive down to the low picomolar range (≤10 pM) and can discriminate between targets with a single-base mismatch. These simple hybridization-based sensors hold great promise for the sensitive detection of a wider spectrum of nucleic acid biomarkers.

Electrochemically-gated graphene broadband microwave waveguides for ultrasensitive biosensing.

Identification of non-amplified DNA sequences and single-base mutations is essential for molecular biology and genetic diagnostics. This paper reports a novel sensor consisting of electrochemically-gated graphene coplanar waveguides coupled with a microfluidic channel. Upon exposure to analytes, propagation of electromagnetic waves in the waveguides is modified as a result of interactions with the fringing field and modulation of graphene dynamic conductivity resulting from electrostatic gating. Probe DNA sequences are immobilised on the graphene surface, and the sensor is exposed to DNA sequences which either perfectly match the probe, contain a single-base mismatch or are unrelated. By monitoring the scattering parameters at frequencies between 50 MHz and 50 GHz, unambiguous and reproducible discrimination of the different strands is achieved at concentrations as low as one attomole per litre (1 aM). By controlling and synchronising frequency sweeps, electrochemical gating, and liquid flow in the microfluidic channel, the sensor generates multidimensional datasets. Advanced data analysis techniques are utilised to take full advantage of the richness of the dataset. A classification accuracy >97% between all three sequences is achieved using different Machine Learning models, even in the presence of simulated noise and low signal-to-noise ratios. The sensor exceeds state-of-the-art sensitivity of field-effect transistors and microwave sensors for the identification of single-base mismatches.

OWL2: a molecular beacon-based nanostructure for highly selective detection of single-nucleotide variations in folded nucleic acids.

Hybridization probes have been used in the detection of specific nucleic acids for the last 50 years. Despite the extensive efforts and the great significance, the challenges of the commonly used probes include (1) low selectivity in detecting single nucleotide variations (SNV) at low (e.g. room or 37 °C) temperatures; (2) low affinity in binding folded nucleic acids, and (3) the cost of fluorescent probes. Here we introduce a multicomponent hybridization probe, called OWL2 sensor, which addresses all three issues. The OWL2 sensor uses two analyte binding arms to tightly bind and unwind folded analytes, and two sequence-specific strands that bind both the analyte and a universal molecular beacon (UMB) probe to form fluorescent 'OWL' structure. The OWL2 sensor was able to differentiate single base mismatches in folded analytes in the temperature range of 5-38 °C. The design is cost-efficient since the same UMB probe can be used for detecting any analyte sequence.

Ultrasensitive and multiplexed miRNA detection system with DNA-PAINT

MiRNAs hold great potential as biomarkers for the early detection and monitoring of diseases based on their differential expression profiles. Therefore, the sensitive, specific and accurate detection of miRNAs represents an emerging new tool to improve diagnosis and treatment of several diseases, cancer in particular. DNA origami-based miRNA detection is particularly advantageous as it allows to incorporate multiple attachment sites to capture different target miRNAs at the nanoscale. In this work, we present a DNA origami nanoarray system providing distance-dependent recognition of miRNAs by applying super-resolution microscopy technique; DNA-PAINT (point accumulation for imaging in nanoscale topography). The sensor can detect up to 4 miRNAs either separately or in combination based on the relative distance to the boundary markers on the structure using a single imager strand. The detection is highly sensitive, with a limit of detection down to the low femtomolar range (11 fM - 388 fM) and has a large dynamic range up to 10 nM without need for amplification. Moreover, our detection system can discriminate single base mismatches with low false positive rates. Using our strategy, we demonstrate the detection of endogenous miRNAs from cell extracts of cancer cell lines and plasma from breast cancer patients. Overall, we developed an ultrasensitive and amplification-free, DNA-PAINT imaging-based miRNA detection method using DNA origami nanoarray system for the detection of breast-cancer associated miRNAs which potentially provides a sensitive and accurate alternative to the current multiplexed diagnostic technologies.

Photoelectrochemical biosensor based on FTO modified with BiVO4 film and gold nanoparticles for detection of miRNA-25 biomarker and single-base mismatch

MicroRNAs (miRNAs) are of great important as non-invasive biomarkers for early diagnosis of cancer. Based on this, a photoelectrochemical (PEC) biosensor was developed to measure the concentration of microRNA-25 (miR-25), as a lung cancer biomarker. To fabricate the PEC biosensor, a suitable amino modified probe was immobilized on the surface of a florin tin oxide (FTO) electrode modified with bismuth vanadate (BiVO4) film and gold nanoparticles (AuNPs). The fabrication steps of the PEC biosensor were monitored by electrochemical impedance spectroscopy (EIS) and PEC measurements. The results indicate that the combination of BiVO4 film and AuNPs enhances the separation of photogenerated electrons and increase the photocurrent signal. Upon hybridization of the probe immobilized on the biosensor with miR-25, the photocurrent is significantly decreased. This current decrease is attributed to the fact that the hybridization processes blocks the electrode surface by creating a steric hindrance and by decrease the diffusion of AA to the electrode surface, it reduces the AA oxidation and subsequently decreases the capture photogenerated holes. Therefore, the amount of photocurrent decrease is dependent on the miR-25 concentration, and this PEC biosensor can be used to measure miR-25. The developed PEC biosensor shows good sensitivity for the determination of miR-25 in the linear concentration range of 1.0 × 10−14 M to 1.0 × 10−6 M with a detection limit of 3.4 fM, under optimal conditions. Using this biosensor, miR-25 can be detected from a non-complementary and one base mismatch sequence. It is also possible to measure miR-25 in human serum samples with satisfactory results using the PEC biosensor.

PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA.

Although CRISPR-Cas12a [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a] combiningpre-amplification technology has the advantage of high sensitivity in biosensing, its generality and specificity are insufficient, which greatly restrains its application range. Here, we discovered a new targeting substrate for LbaCas12a (Lachnospiraceae bacterium Cas12a), namely double-stranded DNA (dsDNA) with a sticky-end region (PAM-SE+ dsDNA). We discovered that CRISPR-Cas12a hadspecial enzymatic properties for this substrate DNA, including the ability to recognize and cleave it without needing a protospacer adjacent motif (PAM) sequence and a high sensitivity to single-base mismatches in that substrate. Further mechanism studies revealed that guide RNA (gRNA) formed a triple-stranded flap structure with the substrate dsDNA. We also discovered the property of low-temperature activation of CRISPR-Cas12a and, by coupling with the unique DNA hybridization kinetics at low temperature, we constructed a complete workflow for low-abundance point mutation detection in real samples, which was fast, convenient and free of single-stranded DNA (ssDNA) transformation. The detection limits were 0.005-0.01% for synthesized strands and 0.01-0.05% for plasmid genomic DNA, and the mutation abundances provided by our system for 28 clinical samples were in accordance with next-generation sequencing results. We believe that our work not only reveals novel information about the target recognition mechanism of the CRISPR-Cas12a system, but also greatly broadens its application scenarios.

An enzyme-free turn-on fluorescent strategy for nucleic acid detection based on hybridization chain reaction and transferable silver nanoclusters.

A fluorescence biosensor has been developed based on hybridisation chain reaction (HCR) amplification coupled with silver nanoclusters (AgNCs) for nucleic acid detection. The fluorescence was activated via end-to-end transfer of dark AgNCs caged within a DNA template to another DNA sequence that could enhance their red fluorescence emission at 611nm. Such cluster-transfer approach allows us to introduce fluorogenic AgNCs as external signal transducers, thereby enabling HCR to perform in a predictable manner. The resulted HCR-AgNC biosensor was able to detect target DNA with a detection limit of 3.35 fM, and distinguish the DNA target from single-base mismatch sequences. Moreover, the bright red fluorescence emission was detectable with the naked eye, with concentration of target DNA down to 1pM. The biosensor also performed well in human serum samples with good recovery. Overall, our cluster-transfer approach provides a good alternative to construct HCR-AgNC assay with less risk of circuit leakage and produce AgNCs in a controllable manner.

High-Throughput DNA Analysis Platform Based on an Optofluidic Ring Resonator Laser

We demonstrate an optofluidic laser using DNA microdroplets, an intercalating dye, and a glass capillary ring resonator. Only the target DNA emits the laser signal, while the non-target DNA, including those with single-base mismatches, exhibits zero optical signals. Subsequently, a single laser emission detected within a few milliseconds can distinguish the target DNA from others, allowing for truly digital DNA sequence analysis from an optical point of view. In addition, the microdroplets could be individually investigated using our optofluidic system. These advances in laser-based DNA analysis make our proposed scheme a promising, rapid, and high-throughput DNA sequence analysis platform.

Development of an electrochemical genosensor for quantitative determination of miR-200a based on the current response of ferrocene-functionalized graphene oxide nanosheets

The present study describes the construction of a sensitive electrochemical genosensor for detection of miR-200a using ferrocene-functionalized graphene oxide nanosheets (Fc-GO) as an electroactive label. For this purpose, a glassy carbon electrode (GCE) was modified by amino-functionalized CuO nanoparticles decorated on MnO2 nanorods (NH2-CuONPs-MnO2NRs). The process of the genosensor construction is confirmed by electrochemical impedance spectroscopy (EIS). A sensing mechanism has been proposed based on the π-stacking interaction between Fc-GO nanosheets and the aromatic bases of DNA. The basis of the assay is based on the interaction of the electroactive label of Fc-GO nanosheets with ss-DNA in the absence of the target and its release after hybridization of the ss-DNA with the target, followed by a severe decrease in the current response. Using the genosensor, miR-200a can be measured in the concentration range of 1.0 fM to 0.1 µM and with a detection limit of 0.29 fM using the differential pulse voltammetry method. Depending on the response of the genosensor, a single-base mismatch as well as a non-complementary-one can be detected from the target sequence. Furthermore, the genosensor is capable of measuring miR-200a in a real sample.

CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter

Herein, we described a washing- and label-free clustered regularly interspaced short palindromic repeats (CRISPR)/LwaCas13a-based RNA detection method utilizing a personal glucose meter (PGM), which relies on the trans-cleavage activity of CRISPR/Cas13a and kinase reactions. In principle, the presence of target RNA activates the trans-cleavage of CRISPR/Cas13a, generating 2′,3′-cyclic phosphate adenosine, which is converted to adenosine monophosphate (AMP) by the T4 polynucleotide kinase. Subsequently, the AMP is converted to adenosine diphosphate (ADP) through phosphorylation by a myokinase; ADP is then used as a substrate in the cascade enzymatic reaction promoted by pyruvate kinase and hexokinase. The overall reaction leads to the continuous conversion of glucose to glucose-6-phosphate, resulting in a reduction of glucose concentration proportional to the level of target RNA, which can therefore be indirectly measured with a PGM. By employing this novel strategy, severe acute respiratory syndrome coronavirus-2 RNA can be successfully detected with excellent specificity. In addition, we were able to overcome non-specific responses of CRISPR/Cas13a and distinguish single nucleotide polymorphisms by introducing a single-base mismatch in the complementary RNA. Our study provides an alternative coronavirus disease 2019 detection technology that is affordable, accessible, and portable with a fast turnaround time and excellent selectivity.

Sensitive microRNA detection based on bimetallic label photothermal lateral flow locked nucleic acid biosensor with smartphone readout

Fast, convenient, and highly sensitive detection of microRNA (miRNA) is vital for the early diagnosis of diseases. Lateral flow biosensor (LFB) as microdevice has been used in rapid detection, however, the sensitivity is not satisfied for low-abundant miRNA detection, and short length of the miRNA requires high affinity detection probe. To address these issues, herein, we synthesized palladium-gold (Pd-Au) bimetallic nanoplates with high photothermal effect combined locked nucleic acid (LNA) detection probe to develop a photothermal lateral flow locked nucleic acid biosensor (P-LFLNAB) for miRNA detection. The P-LFLNAB enables sensitive and selective miRNA-21 detection with detection limit down to 0.094 pM using a smartphone with external thermal imager, which is 639.5 times lower than that of the conventional gold nanoparticles based colorimetric LFB and the most sensitive enzyme-free LFB for miRNA detection. Besides, the P-LFLNAB achieved single-base mismatch discrimination and can quantify signals in various cell lysates. This highly sensitive, selective and portable P-LFLNAB with smartphone readout has great potential in point-of-care test of miRNAs-related biomedical application.

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues.

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Although two such methods have been developed for mammalian tissues, they have a low signal-noise ratio and/or poor resolution at the single-cell level. To overcome these drawbacks, we develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. We conduct several applications. First, the spatial expression profiling of osa-miR156 and OsSPL12 in rice leaves reveals their specific expression in mesophyll cells. Second, studying rice and its mutant lines with our method reveals opposite expression patterns of miRNA and its target mRNA in tissues. Third, the dynamic expression profiles of ZmGRF8 and zma-miR396 during maize leaf development provide evidence that zma-miR396 regulates the preferential spatial expression of ZmGRF8 in bundle sheath cells. Finally, our method can be scaled up to simultaneously detect multiple miRNAs and mRNAs in a tissue. Thus, it is a sensitive and versatile technique for studying miRNA regulation of plant tissue development.