Stem-loop Research Articles

Origin of Type II tRNA Variable Loops, Aminoacyl-tRNA Synthetase Allostery from Distal Determinants, and Diversification of Life

The three 31 nucleotide minihelix tRNA evolution theorem describes the evolution of type I and type II tRNAs to the last nucleotide. In databases, type I and type II tRNA V loops (V for variable) were improperly aligned, but alignment based on the theorem is accurate. Type II tRNA V arms were a 3′-acceptor stem (initially CCGCCGC) ligated to a 5′-acceptor stem (initially GCGGCGG). The type II V arm evolved to form a stem–loop–stem. In Archaea, tRNALeu and tRNASer are type II. In Bacteria, tRNALeu, tRNASer, and tRNATyr are type II. The trajectory of the type II V arm is determined by the number of unpaired bases just 5′ of the Levitt base (Vmax). For Archaea, tRNALeu has two unpaired bases, and tRNASer has one unpaired base. For Bacteria, tRNATyr has two unpaired bases, tRNALeu has one unpaired base, and tRNASer has zero unpaired bases. Thus, the number of synonymous type II tRNA sets is limited by the possible trajectory set points of the arm. From the analysis of aminoacyl-tRNA synthetase structures, contacts to type II V arms appear to adjust allosteric tension communicated primarily via tRNA to aminoacylating and editing active sites. To enhance allostery, it appears that type II V arm end loop contacts may tend to evolve to V arm stem contacts.

Analysis of the miR482 Gene Family in Plants.

MicroRNA482 (miR482) is a conserved microRNA family in plants, playing critical regulatory roles in different biological activities. Though the members of the miR482 gene family have been identified in plants, a systematic study has not been reported yet. In the present research, 140 mature sequences generated by 106 precursors were used for molecular characterization, phylogenetic analysis, and target gene prediction, and the competing endogenous RNA (ceRNA) network mediated by miR482 was summarized. The length of mature sequences ranged from 17 nt to 25 nt, with 22 nt being the most abundant, and the start and end of the mature sequences had a preference for uracil (U). By sequence multiplex comparison, it was found that the mature sequences of 5p were clustered into one group, and others were clustered into the other group. Phylogenetic analysis revealed that the 140 mature sequences were categorized into six groups. Meanwhile, all the precursor sequences formed a stable hairpin structure, and the 106 precursors were divided into five groups. However, the expression of miR482 varied significantly between different species and tissues. In total, 149 target genes were predicted and their functions focused on single-organism process, cellular process, and cell and cell part. The ceRNA network of miR482 in tomato, cotton, and peanut was summarized based on related publications. In conclusion, this research will provide a foundation for further understanding of the miR482 gene family.

Work distribution for unzipping processes.

A simple zipper model is introduced, representing in a simplified way, e.g., the folded DNA double helix or hairpin structures in RNA. The double stranded hairpin is connected to a heat bath at temperature T and subject to an external force f, which couples to the free length L of the unzipped sequence. The leftmost zipped position can be seen as the position of a random walker in a special external field. Increasing the force leads to a zipping-unzipping first-order phase transition at a critical force f_{c}(T) in the thermodynamic limit of a very large chain. We compute analytically, as a function of temperature T and force f, the full distribution P(L) of free lengths in the thermodynamic limit and show that it is qualitatively very different for f<f_{c},f=f_{c}, and f>f_{c}. Next we consider quasistatic work processes where the force is incremented according to a linear protocol. Having obtained P(L) already allows us to derive an analytical expression for the work distribution P(W) in the zipped phase f<f_{c} for a long chain. We compute the large-deviation tails of the work distribution explicitly. This distribution can be interpreted as work distribution for an oscillatorylike model. Our analytical result for the work distribution is compared over a large range of the support down to probabilities as small as 10^{-200} with numerical simulations performed by applying sophisticated large-deviation algorithms.

Adjusting the Energy Profile for CH-O Interactions Leads to Improved Stability of RNA Stem-Loop Structures in MD Simulations.

The role of ribonucleic acid (RNA) in biology continues to grow, but insight into important aspects of RNA behavior is lacking, such as dynamic structural ensembles in different environments, how flexibility is coupled to function, and how function might be modulated by small molecule binding. In the case of proteins, much progress in these areas has been made by complementing experiments with atomistic simulations, but RNA simulation methods and force fields are less mature. It remains challenging to generate stable RNA simulations, even for small systems where well-defined, thermostable structures have been established by experiments. Many different aspects of RNA energetics have been adjusted in force fields, seeking improvements that are transferable across a variety of RNA structural motifs. In this work, the role of weak CH···O interactions is explored, which are ubiquitous in RNA structure but have received less attention in RNA force field development. By comparing data extracted from high-resolution RNA crystal structures to energy profiles from quantum mechanics and force field calculations, it is shown that CH···O interactions are overly repulsive in the widely used Amber RNA force fields. A simple, targeted adjustment of CH···O repulsion that leaves the remainder of the force field unchanged was developed. Then, the standard and modified force fields were tested using molecular dynamics (MD) simulations with explicit water and salt, amassing over 300 μs of data for multiple RNA systems containing important features such as the presence of loops, base stacking interactions as well as canonical and noncanonical base pairing. In this work and others, standard force fields lead to reproducible unfolding of the NMR-based structures. Including a targeted CH···O adjustment in an otherwise identical protocol dramatically improves the outcome, leading to stable simulations for all RNA systems tested.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

Structures and genetic information of control region in mitogenomes of Odonata

Mitogenome data of Odonata is accumulating and widely used in phylogenetic analysis. However, noncoding regions, especially control region, were usually omitted from the phylogenetic reconstruction. In an effort to uncover the phylogenetic insights offered by the control region, we have amassed 65 Odonata mitogenomes and conducted an examination of their control regions. Our analysis discovered that species belonging to Anisoptera and Anisozygoptera exhibited a stem-loop structure, which was formed by a conserved polyC-polyG stretch located near the rrns gene (encoding 12S rRNA). Conversely, the polyC-polyG region was not a conserved fragment in Zygoptera. The length and number of repetitions within the control region were identified as the primary determinants of its overall length. Further, sibling species within Odonata, particularly those in the genus Euphaea, displayed similar patterns of repetition in their control region. Collectively, our research delineates the structural variations within the control region of Odonata and suggests the potential utility of this region in elucidating phylogenetic relationships among closely related species.

Advanced sampling simulations of coupled folding and binding of phage P22 N-peptide to boxB RNA

Protein-RNA interactions are crucially important for numerous cellular processes and often involve coupled folding and binding of peptide segments upon association. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes such a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics free energy simulations were used to calculate the absolute binding free energy of the N-peptide of bacteriophage P22 in complex with the boxB RNA hairpin motif at different salt concentrations and using two different water force field models. We obtained good agreement with experiment also at different salt concentrations for the TIP4P-D water model that has a stabilizing effect on unfolded protein structures. It allowed us to estimate the free energy contribution resulting from restricting the molecules’ spatial and conformational freedom upon binding, which makes a large opposing contribution to binding. In a second set of umbrella sampling simulations to dissociate/associate the complex along a separation coordinate, we analyzed the onset of preorientation of the N-peptide and onset of structure formation relative to the RNA and its dependence on the salt concentration. Peptide orientation and conformational transitions are significantly coupled to the first contact formation between peptide and RNA. The initial contacts are mostly formed between peptide residues and the boxB hairpin loop nucleotides. A complete transition to an α-helical bound peptide conformation occurs only at a late stage of the binding process a few angstroms before the complexed state has been reached. However, the N-peptide orients also at distances beyond the contact distance such that the sizable positive charge points toward the RNA’s center-of-mass. Our result may have important implications for understanding protein- and peptide-RNA complex formation frequently involving coupled folding and association processes.

Structure of Essential RNA Regulatory Elements in the West Nile Virus 3′-Terminal Stem Loop

Flaviviruses, such as West Nile and Dengue Virus, pose a significant and growing threat to global health. Central to the flavivirus life cycle are highly structured 5′- and 3′-untranslated regions (UTRs), which harbor conserved cis-acting RNA elements critical for viral replication and host adaptation. Despite their essential roles, detailed molecular insights into these RNA elements have been limited. By employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with SAXS experiments, we determined the three-dimensional structure of the West Nile Virus (WNV) 3′-terminal stem-loop core, a highly conserved element critical for viral genome cyclization and replication. Single nucleotide mutations at several sites within this RNA abolish the ability of the virus to replicate. These critical sites are located within a short 18-nucleotide hairpin stem, a substructure notable for its conformational flexibility, while the adjoining main stem-loop adopts a well-defined extended helix interrupted by three non-Watson-Crick pairs. This study enhances our understanding of several metastable RNA structures that play key roles in regulating the flavivirus lifecycle, and thereby also opens up potential new avenues for the development of antivirals targeting these conserved RNA structures. In particular, the structure we observe suggests that the plastic junction between the small hairpin and the tail of the longer stem-loop could provide a binding pocket for small molecules, for example potentially stabilizing the RNA in a conformation which hinders the conformational rearrangements critical for viral replication.

An electrochemiluminescent imaging strategy based on CRISPR/Cas12a for ultrasensitive detection of nucleic acid

BackgroundPersistent infection with human papillomavirus (HPV) significantly contributes to the development of cervical cancer. Thus, it is urgent to develop rapid and accurate methods for HPV detection. Herein, we present an ultrasensitive CRISPR/Cas12a-based electrochemiluminescent (ECL) imaging technique for the detection of HPV-18 DNA. ResultThe ECL DNA sensor array is constructed by applying black hole quencher (BHQ) and polymer dots (Pdots) co-labeled hairpin DNA (hpDNA) onto a gold-coated indium tin oxide slide (Au-ITO). The ECL imaging method involves an incubation process of target HPV-18 with a mixture of crRNA and Cas12a to activate Cas12a, followed by an incubation of the active Cas12a with the ECL sensor. This interaction causes the indiscriminate cleavage of BHQ from Pdots by digesting hpDNA on the sensor surface, leading to the restoration of the ECL signal of Pdots. The ECL brightness readout demonstrates superior performance of the ECL imaging technique, with a linear detection range of 10 fM–500 pM and a limit-of-detection (LOD) of 5.3 fM. SignificanceThe Cas12a-based ECL imaging approach offers high sensitivity and a broad detection range, making it highly promising for nucleic acid detection applications.

A bivariate fluorescence biosensor based on Janus DNA nanoarchitecture-loaded dual-emissive Ag nanoclusters as bi-responsive signaling reporters

Constructing label-free bivariate fluorescence biosensor would be intriguing and desired for the recognizable and accurate detection of two specific DNA segments, yet the design of functional DNA structures with low overlapped interference might be challenging. Herein in this work, a double-faced Janus DNA nanoarchitecture (JDNA) with bi-responsive recognition regions on opposite sides was assembled, which consisted of two substrate strands and two template strands for loading green-/red-emissive Ag nanoclusters (gAgNC and rAgNC) as bivariate signaling reporters. Of note, the hybridized double helix in the middle rationally oriented two flank faces and stabilized the rigid conformation of JDNA, while the template sequences of bicolor clusters were blocked to minimize non-specific background leakage. Upon inputting two targets, the discernible hairpins lost their hairpin structures due to forming two dsDNA complexes. They were executed to simultaneously invade JDNA for activating two individual target-recycled strand displacement (TRSD) events, guiding signal transduction and efficient amplification. Consequently, the clustering templates were unlocked via the tailored conformation switch of JDNA, in which gAgNC and rAgNC were in situ synthesized in two diagonal positions, thereby significantly emitting bi-responsive signal without cross interference. Benefited from the logic integration of double-faced JDNA and TRSD, a label-free, sensitive and specific bivariate fluorescence approach was developed, which would open a new avenue for the potential application in biosensing and bioanalysis.

N7-Ended Walker PCR: An Efficient Genome-Walking Tool.

As a tool for acquiring uncharacterized genomic DNA adjacent to characterized DNA, genome-walking is integral to bioscience-related research works. Herein, a new genome-walking tool known as N7-ended walker PCR (polymerase chain reaction) is presented. The key aspect for this method is the use of a degenerate walker primer in secondary/tertiary PCR. The 7 nt 5' tail of this primer completely degenerates to N7 relative to its corresponding primary walker primer. The degeneracy reduces the efficiency of annealing this primer to its predecessor site. Clearly, primary nontarget DNA defined by the primary walker primer prefers to form a hairpin structure via the inverted ends rather than hybridizing with the degenerate primer. As a result, N7-ended walker PCR achieves genome-walking by selectively boosting the DNA of interest. The feasibility of the N7-ended walker PCR method was proven by acquiring uncharacterized DNAs flanking several characterized DNAs.

Quantitative Effects of the Loop Region on Topology, Thermodynamics, and Cation Binding of DNA G-quadruplexes.

The thermal stability of G-quadruplexes is important for their biological roles. G-quadruplexes are stable in the presence of cations such as K+ and Na+ because these cations coordinate in the G-quartet of four guanine bases. It is well known that the number of G-quartets and the configuration of the guanine bases affect the binding affinity of the cation. Recently, structures formed in the loop regions connecting the guanine stretches have attracted significant attention, because the loop region affects G-quadruplex properties, such as topology, thermal stability, and interactions with proteins and small molecules. Considering these effects, the loop region can also affect the binding affinity of the cations. Here, we designed a series of G-quadruplex-forming DNA sequences that contain a hairpin in a loop region and investigated the effects of the sequence and structure of the loop region on the cation binding affinity as well as the thermal stability of the G-quadruplex as a whole. First, structural analysis of the DNA sequences showed that the hairpin at the loop plays a key role in determining G4 topology (strand orientation). Second, in the case of the G-quadruplexes with the hairpin-forming loop region, it was found that a longer loop length led to a higher thermodynamic stability of the G-quadruplex as well as higher cation binding affinity. In contrast, an unstructured loop region did not lead to such effects. Interestingly, the cation binding affinity was correlated to the thermodynamic stability of the hairpin structure at the loop region. It was quantitatively demonstrated that the stable loop region stabilized the whole G-quadruplex structure, which induced higher cation binding affinity. These systematic and quantitative results showed that the loop region is one of the determinants of cation binding and expanded the possibilities of drug development targeting G4s by stabilizing the loop region.

Bidirectional hybridized hairpin DNA fluorescent aptasensor based on Au-Pd NPs and CDs for ratiometric detection of AFB1.

Anovel and simple ratiometric fluorescent aptasensor was developed for the sensitive detection of aflatoxin B1 (AFB1). A hairpin DNA (h-DNA) was independently synthesized as the basic skeleton, and the bidirectional hybridization of h-DNA can increase the load of aptamer and signal probes, thereby realizing signal amplification. The high-efficiency fluorescence resonance energy transfer interaction between gold-palladium nanoparticles (Au-Pd NPs) and the self-synthesized fluorescent probe carbon dots (CDs) was utilized. Moreover, the label-free probe SYBR Green I (SG I) dye was introduced to form a double-signal probe with CDs, and a ratiometric sensor with FCDs/FSG I as a response signal was constructed. The ratio strategy can eliminate the fluctuation of external factors, thus improving the accuracy and reliability of the sensor. The quenching effect of Au-Pd NPs on CDs was 1.4 times that of AuNPs and 3.4 times that of Pd NPs, respectively. In the range 1-100ng/mL, FCDs/FSG I showed a good linear relationship with the logarithm of the concentration of AFB1, and the limit of detection was as low as 0.07ng/mL. The sensor was used to detect AFB1 in spiked peanuts and wine samples, and the recovery was between 91 and 115%, indicating that the sensor has high application potential in real sample analysis.

Molecular basis for the activation of human spliceosome

The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (Bact complex) and the catalytically activated spliceosome (B* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B* complexes: pre-Bact, Bact-I, Bact-II, Bact-III, Bact-IV, and post-Bact. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5′-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.

Develop Tandem Tension Sensor to Gauge Integrin-Transmitted Molecular Forces.

DNA-based tension sensors have innovated the imaging and calibration of mechanosensitive receptor-transmitted molecular forces, such as integrin tensions. However, these sensors mainly serve as binary reporters, only indicating if molecular forces exceed one predefined threshold. Here, we have developed tandem tension sensor (TTS), which comprises two consecutive force-sensing units, each with unique force detection thresholds and distinct fluorescence spectra, thereby enabling the quantification of molecular forces with dual reference levels. With TTS, we revealed that vinculin is not required for transmitting integrin tensions at approximately 10 pN (piconewtons) but is essential for elevating integrin tensions beyond 20 pN in focal adhesions (FAs). Such high tensions have emerged during the early stage of FA formation. TTS also successfully detected changes in integrin tensions in response to disrupted actin formation, inhibited myosin activity, and tuned substrate elasticity. We also applied TTS to examine integrin tensions in platelets and revealed two force regimes, with integrin tensions surpassing 20 pN at cell central regions and 13-20 pN integrin tensions at the cell edge. Overall, TTS, especially the construct consisting of a hairpin DNA (13 pN opening force) and a shearing DNA (20 pN opening force), stands as a valuable tool for the quantification of receptor-transmitted molecular forces within living cells.

Light-activated CRISPR-Cas12a for amplified imaging of microRNA in cell cycle phases at single-cell levels.

An ortho-nitrobenzyl phosphate ester-caged nucleic acid hairpin structure coupled to the CRISPR-Cas12a complex is introduced as a functional reaction module for the light-induced activation of the CRISPR-Cas12a (LAC12a) machinery toward the amplified fluorescence detection of microRNA-21 (miRNA-21). The LAC12a machinery is applied for the selective, in vitro sensing of miRNA-21 and for the intracellular imaging of miRNA-21 in different cell lines. The LAC12a system is used to image miRNA-21 in different cell cycle phases of MCF-7 cells. Moreover, the LAC12a machinery integrated in cells enables the two-photon laser confocal microscopy-assisted, light-stimulated spatiotemporal, selective activation of the CRISPR-Cas12a miRNA-21 imaging machinery at the single-cell level and the evaluation of relative expression levels of miRNA-21 at distinct cell cycle phases. The method is implemented to map the distribution of cell cycle phases in an array of single cells.

Comparison of Double-Stranded DNA at the 5' and 3' Ends of the G-Triplex and Its Application in the Detection of Hg(II).

Leveraging the fluorescence enhancement effect of the G-triplex (G3)/thioflavin T (ThT) catalyzed by the adjacent double-stranded DNA positioned at the 5' terminus of the G3, the G3-specific oligonucleotide (G3MB6) was utilized to facilitate the rapid detection of mercury (Hg(II)) through thymine-Hg(II)-thymine (T-Hg(II)-T) interactions. G3MB6 adopted a hairpin structure in which partially complementary strands could be disrupted with the presence of Hg(II). It prompted the formation of double-stranded DNA by T-Hg(II)-T, inducing the unbound single strand of G3MB6 to spontaneously form a parallel G3 structure, producing a solid fluorescence signal by ThT. Conversely, fluorescence was absent without Hg(II), since no double strand and formation of G3 occurred. The fluorescence intensity of G3MB6 exhibited a positive correlation with Hg(II) concentrations from 17.72 to 300 nM (R2 = 0.9954), boasting a notably low quality of limitation (LOQ) of 17.72 nM. Additionally, it demonstrated remarkable selectivity for detecting Hg(II). Upon application to detect Hg(II) in milk samples, the recovery rates went from 100.3% to 103.2%.

A cytidine deaminase regulates axon regeneration by modulating the functions of the Caenorhabditis elegans HGF/plasminogen family protein SVH-1.

The pathway for axon regeneration in Caenorhabditis elegans is activated by SVH-1, a growth factor belonging to the HGF/plasminogen family. SVH-1 is a dual-function factor that acts as an HGF-like growth factor to promote axon regeneration and as a protease to regulate early development. It is important to understand how SVH-1 is converted from a protease to a growth factor for axon regeneration. In this study, we demonstrate that cytidine deaminase (CDD) SVH-17/CDD-2 plays a role in the functional conversion of SVH-1. We find that the codon exchange of His-755 to Tyr in the Asp-His-Ser catalytic triad of SVH-1 can suppress the cdd-2 defect in axon regeneration. Furthermore, the stem hairpin structure around the His-755 site in svh-1 mRNA is required for the activation of axon regeneration by SVH-1. These results suggest that CDD-2 promotes axon regeneration by transforming the function of SVH-1 from a protease to a growth factor through modification of svh-1 mRNA.

A single CAA interrupt in a DNA three-way junction containing a CAG repeat hairpin results in parity-dependent trapping.

An increasing number of human disorders are attributed to genomic expansions of short tandem repeats (STRs). Secondary DNA structures formed by STRs are believed to play an important role in expansion, while the presence of nucleotide interruptions within the pure repeat sequence isknown to delay the onset and progression of disease. We have used two single-molecule fluorescence techniques to analyse the structure and dynamics of DNA three-way junctions (3WJs) containing CAG repeat hairpin slipouts, with and without a single CAA interrupt. For a 3WJ with a (CAG)10 slipout, the CAA interrupt is preferentially located in the hairpin loop, and the branch migration dynamics are 4-fold slower than for the 3WJ with a pure (CAG)10, and 3-fold slower than a 3WJ with a pure (CAG)40 repeat. The (CAG)11 3WJ with CAA interrupt adopts a conformation that places the interrupt in or near the hairpin loop, with similar dynamics to the pure (CAG)10 and (CAG)11 3WJs. We have shown that changing a single nucleotide (G to A)in a pure repeat can have a large impact on 3WJ structure and dynamics, which may be important for the protective role of interrupts in repeat expansion diseases.

A bulged-type enzyme-free recognition strategy designed for single nucleotide polymorphisms integrating with label-free electrochemical biosensor

Compared to conventional nucleic acid detection methods, label-free single nucleotide polymorphism (SNP) detection presents challenging due to the necessity of discerning single base mismatches, especially in the field of enzyme-free detection. In this study, we introduce a novel bulged-type DNA duplex probe designed to significantly amplify single-base differences. This probe is integrated with programmable DNA-based nanostructures to develop a sensitive, label-free biosensor for nonenzymatic SNP detection. The duplex probe with one bulge could selectively identify wild-typed DNA (WT) and mutant-type DNA (MT) based on a competitive strand displacement reaction mechanism. The hyperbranched HCR (HHCR) by incorporating of hairpin DNA into the DNA tetrahedron and surface-tethering on the portable screen printing electrode (SPCE) significantly favor the formation of negatively charged DNA nanostructure. We harnessed strong repulsion of DNA nanostructure towards the electroactive [Fe(CN)₆]³⁻/⁴⁻ in combination with electrochemical technique to create a label-free biosensor. This simple, enzyme-free and label-free biosensor could detect MT with a detection limit of 56 aM, even in multiple sequence backgrounds. The study served as the proof-of-concept for the integration of enzyme-free competitive mechanism and label-free strategy, which can be extended as a powerful tool to various fields.