Linear Amplification Research Articles

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.

RAG-seq: A NSR Primed and Transposase Tagmentation Mediated Strand-specific Total RNA Sequencing in Single Cell.

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular diversity with unprecedented resolution. However, many current methods are limited in capturing full-length transcripts and discerning strand orientation. We present RAG-seq, an innovative strand-specific total RNA sequencing technique that combines not-so-random (NSR) primers with Tn5 transposase-mediated tagmentation. RAG-seq overcomes previous limitations by delivering comprehensive transcript coverage and maintaining strand orientation, which is essential for accurate quantification of overlapping genes and detection of antisense transcripts. Through optimized reverse transcription with oligo dT primers, rRNA depletion via Depletion of Abundant Sequences by Hybridization (DASH), and linear amplification, RAG-seq enhances sensitivity and reproducibility, especially for low-input samples and single cells. Application to mouse oocytes and early embryos highlights RAG-seq's superior performance in identifying stage-specific antisense transcripts, shedding light on their regulatory roles during early development. This advancement represents a significant leap in transcriptome analysis within complex biological contexts.

Gain-controlled taming of recurrent modulation instability

We show how the recurrence phenomenon characteristic of the nonlinear stage of induced modulational instability in a passive fiber is affected by forcing. An additional linear amplification, even if extremely weak, induces separatrix crossing corresponding to critical values of the gain around which the recurrence process considerably slows down, switching between dynamical orbits of different kind. We present evidence for such phenomenon in a fiber optics experiment where the gain is finely tuned by means of Raman amplification. A theoretical explanation is also provided that matches almost perfectly with our experimental results. Published by the American Physical Society 2024

PARN Maintains RNA Stability to Regulate Insulin Maturation and GSIS in Pancreatic β Cells.

Diabetes, a metabolic disorder characterized by hyperglycemia, underscores the importance of normal pancreatic β-cell development and function in maintaining glucose homeostasis. Poly(A)-specific ribonuclease (PARN) serves as the principal regulator of messenger RNA (mRNA) stability, yet its specific role in pancreatic β cells remains unclear. This study utilizes mice with targeted PARN deficiency in β cells to elucidate this role. Notably, Parn conditional knockout mice present unaltered β-cell development and insulin sensitivity but reduced glucose-stimulated insulin secretion (GSIS). The observed outcomes are corroborated in NIT-1 cells. Furthermore, transcriptomic analyses reveal aberrant mRNA expression of genes crucial for insulin secretion in PARN-deficient β cells. Insights from linear amplification of complementary DNA ends and sequencing and coimmunoprecipitation experiments reveal an interaction between PARN and polypyrimidine tract-binding protein 1 (PTBP1), regulating the RNA stability of solute carrier family 30, member 8 (Slc30a8) and carbohydrate sulfotransferase 3 (Chst3). Interference with either PARN or PTBP1 disrupts this stability. These data indicate that PARN deficiency hampers GSIS and insulin maturation by destabilizing Slc30a8 and Chst3 RNAs. These findings provide compelling evidence indicating that PARN is a potential therapeutic target for enhancing insulin maturation and secretion.

Full-Length Immune Repertoire Reconstruction and Profiling at the Transcriptome Level Using Long-Read Sequencing.

Due to the diversity of the immune repertoire (IR), reconstructing full-length IR using traditional short-read sequencing has proven challenging. A full-length IR sequencing (FLIRseq) work flow was developed with linear rolling circle amplification and nanopore sequencing. Its accuracy and quantification ability were verified by plasmid mixtures and commercial B-cell receptor/T-cell receptor sequencing (BCR/TCR-seq) based on short reads. IRs in tissues and the peripheral blood from 8 patients with acute lymphoblastic leukemia, 3 patients with allergic diseases, 4 patients with psoriasis, and 5 patients with prostate cancer were analyzed using FLIRseq. FLIRseq reads had lower mismatch rates and gap rates, and higher identify rates than nanopore reads (all P < 2.2 × -16). The relative quantification of components by FLIRseq was consistent with the actual quantification (P > 0.05). FLIRseq had superiority over BCR/TCR-seq, providing the long complementarity-determining region 3, B-cell isotype, and the rarely used V gene sequence. FLIRseq observed an increase in clonotype diversity (P < 0.05) and a decrease in the percentage of abnormal BCRs/TCRs in patients with leukemia in remission. For patients with allergic diseases or psoriasis, FLIRseq provided direct insights into V(D)J recombination and specific immunoglobulin classes. Compared with that in prostate cancer tissues, the full-length V segment of the biased T-cell receptor β chain from lymphocytes in psoriatic tissues showed a more consistent AlphaFold2-predicted protein structure (P < 0.05). FLIRseq enables unbiased and comprehensive analyses of direct V(D)J recombination and immunoglobulin classes, thereby contributing to characterizing pathogenic mechanisms, monitoring minimal residual disease, and customizing adoptive cell therapy.

An invitation to resolvent analysis

AbstractResolvent analysis is a powerful tool that can reveal the linear amplification mechanisms between the forcing inputs and the response outputs about a base flow. These mechanisms can be revealed in terms of a pair of forcing and response modes and the associated energy gains (amplification magnitude) at a given frequency. The linear relationship that ties the forcing and the response is represented through the resolvent operator (transfer function), which is constructed through spatially discretizing the linearized Navier–Stokes operator. One of the unique strengths of resolvent analysis is its ability to analyze statistically stationary turbulent flows. In light of the increasing interest in using resolvent analysis to study a variety of flows, we offer this guide in hopes of removing the hurdle for students and researchers to initiate the development of a resolvent analysis code and its applications to their problems of interest. To achieve this goal, we discuss various aspects of resolvent analysis and its role in identifying dominant flow structures about the base flow. The discussion in this paper revolves around the compressible Navier–Stokes equations in the most general manner. We cover essential considerations ranging from selecting the base flow and appropriate energy norms to the intricacies of constructing the linear operator and performing eigenvalue and singular value decompositions. Throughout the paper, we offer details and know-how that may not be available to readers in a collective manner elsewhere. Towards the end of this paper, examples are offered to demonstrate the practical applicability of resolvent analysis, aiming to guide readers through its implementation and inspire further extensions. We invite readers to consider resolvent analysis as a companion for their research endeavors.

Nonlinear Nonmodal Analysis of Hypersonic Flow over Blunt Cones

The linear amplification of modal disturbances that lead to boundary-layer transition in two-dimensional/axisymmetric hypersonic configurations is strongly reduced by the presence of a blunt nose tip, and the mechanisms underlying the low Mack’s second-mode N-factor values at the observed onset of transition over the cone frustum are currently unknown. Linear nonmodal analysis has shown that both planar and oblique traveling disturbances that peak within the entropy-layer experience appreciable energy amplification for moderate to large nose-tip bluntness. The present study extends the previous linear analysis by including the nonlinear effects. Specifically, the harmonic Navier–Stokes equations (HNSE) are solved with a fully implicit formulation and the Newton–Raphson method. The increased number of degrees of freedom for the nonlinear system presents difficulties for solution strategies based on direct solution of the linearized system. Such difficulties are overcome by using the generalized minimal residual method (GMRES) iterative method with a preconditioner corresponding to a simplified Jacobian without the cross-derivative terms. The HNSE solver is verified by comparing with nonlinear parabolized stability equation results for the nonlinear evolution of planar waves in an incompressible Blasius boundary layer and in a Mach 6 flow over a blunt cone. Finally, nonlinear nonmodal results are presented for planar traveling disturbances over a blunt cone configuration with reduced transition N factor as measured in wind-tunnel experiments. The nonmodal analysis demonstrates that entropy-layer disturbances generated close to the nose tip can seed the amplification of higher-frequency Mack’s second-mode instabilities farther downstream and hence can lead to a reduction in the transition N factor.

Multiplex competitive annealing mediated isothermal amplification with high fidelity DNA polymerase (HiFi-CAMP)

Various isothermal amplification methods have been developed for point-of-care testing (POCT) of various infectious diseases. Here, we proposed a novel isothermal amplification method, named as 5′-half complementary primers mediated isothermal amplification (HCPA). Because of the similarity of our method to the previous method competitive annealing mediated isothermal amplification (CAMP) in primer design, we also use the name CAMP for our method. We demonstrated that CAMP is mediated by both a linear isothermal amplification pattern and a loop-mediated isothermal amplification pattern. To improve the specificity and enable multiplex detection, we further developed HiFi-CAMP method that uses a small amount of high-fidelity DNA polymerase to cut HFman probe to release fluorescent signal. The HiFi-CAMP method was demonstrated to have a good specificity and sensitivity, and fast amplification speed in detection of three human respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus A (RSV-A) and influenza A viruses (IAV). When compared with gold standard RT-qPCR assays, the HiFi-CAMP assays showed sensitivities of 90.0 %, 71.4 % and 78.1 %, specificities of 100 %, 100 % and 95.5 %, and consistencies of 93.0 %, 93.3 % and 88.2 % for SARS-CoV-2, RSV-A and IAV, respectively. Furthermore, a duplex HiFi-CAMP assay was also developed to simultaneously detect RSV-A and SARS-CoV-2. The HiFi-CAMP will provide a promising candidate for POCT diagnosis in resource-limited settings.

Enzyme-free fluorescent DNA detection based on nucleic acid-templated click reaction via controllable synthesis of Cu2O as heterogeneous nanocatalyst

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide–alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min−1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

Onset of turbulence in rotor–stator cavity flows

Numerous studies have indicated that turbulence typically initiates along the boundary layer of the stationary disk within a rotor–stator cavity. To describe the transition process to turbulence on the stationary side of a closed rotor–stator cavity, a comprehensive approach combining global linear stability analysis with direct numerical simulation was adopted in the present study. The proposed model aligns with that of Yim et al. (J. Fluid Mech., vol. 848, 2018, pp. 631–647), who investigated the stability characteristics of the rotating-disk boundary layer in a rotor–stator cavity. In order to achieve a stable inflow for the stationary-disk boundary layer, we rotate the shroud together with the rotating disk. Through careful global stability analysis, the predominant spiral mode exhibiting the highest instability in the boundary layer of the stationary disk was discerned, corroborating observations from simulations. Initially, the spiral mode undergoes linear amplification, reaches a state of linear saturation and enters the nonlinear regime. Following nonlinear saturation in the flow field, a circular wave mode arises due to the influence of mean flow distortion. As the Reynolds number attained a sufficiently high level, the interplay between the downstream-propagating circular mode and spiral mode amplified disturbances in the boundary layer of the stationary disk, ultimately leading to the development of localised turbulence at the mid-radius of the rotor–stator cavity. Notably, the present study is the first to elucidate the coexistence of laminar–transitional–turbulent flow states in the stationary-disk boundary layer through direct numerical simulations.

Dye‐based recombinase‐aided amplification assay with enhanced sensitivity and specificity

AbstractObjectiveFluorescent recombinase‐aided amplification (RAA) assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity, compared with real‐time PCR (qPCR) assays, but they require a complex probe design. To eliminate the addition of fluorescent probes for RAA, an EvaGreen dye‐based recombinase‐aided amplification (EvaGreen‐RAA) assay using self‐avoiding molecular recognition system (SAMRS) primers was developed.MethodsThe SAMRS primers effectively avoided the production of primer dimers, thus improving the detection sensitivity, while EvaGreen dye was used to quantitatively measure the amplified products in real time. Using Staphylococcus aureus (SA) and Listeria monocytogenes (LM) as examples, EvaGreen‐RAA with SAMRS primers was developed. As a reference and comparison, a traditional fluorescence probe RAA method and a RAA with SAMRS primers (SAMRS‐RAA) for detecting SA and LM were also investigated. Serial dilutions of recombinant plasmids were used to evaluate the sensitivity of the assays. Unenriched and enriched simulated milk samples were used to evaluate the limits of detection (LOD) of these methods. Using high‐resolution melting (HRM) was used to explore the sensitivity of the dual EvaGreen‐RAA assay.ResultsThe sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS‐RAA and EvaGreen‐RAA for detecting SA and LM plasmids was 1 copies/μL. The LOD values of the EvaGreen‐RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL, respectively, and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL. EvaGreen‐RAA had linear amplification in real time in the range of 1–105 copies/μL of the plasmids of SA and LM. The sensitivity of the dual EvaGreen‐RAA assay for SA and LM was estimated to be 102 CFU/mL.ConclusionA real‐time quantitative EvaGreen‐RAA method for detecting SA and LM was developed, which eliminates the need to design complex RAA probes. This dye‐based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens, which has many potential applications.

Combining poly-epitope MoonTags and labeled nanobodies for signal amplification in cell-specific PET imaging in vivo

Immunorecognition provides an excellent basis for targeted imaging techniques covering a wide range from basic research to diagnostics and from single cells to whole organisms. Fluorescence- or radioisotope-labeled antibodies, antibody fragments or nanobodies enable a direct signal readout upon binding and allow for versatile imaging from microscopy to whole-body imaging. However, as the signal intensity directly correlates with the number of labeled antibodies bound to their epitopes (1:1 binding), sensitivity for low-expressing epitopes can be limiting for visualization. For the first time, we developed poly-epitope tags with multiple copies (1 to 7) of a short peptide epitope, specifically the MoonTag, that are recognized by a labeled nanobody and aimed at signal amplification in microscopy and cell-specific PET imaging. In transiently transfected HeLa cells or stably transduced A4573 cells we characterized complex formation and in vitro signal amplification. Indeed, using fluorescently and radioactively labeled nanobodies we found an approximately linear signal amplification with increasing numbers of epitope copies in vitro. To test the poly-epitope approach in vivo, A4573 tumor cells were injected subcutaneously into the shoulder of NSG mice, with A4573 tumor cells expressing a poly-epitope of 7 MoonTags on one side and WT cells on the other side. Using a [68Ga]-labeled NODAGA-conjugated MoonTag nanobody, we performed PET/CT imaging at day 8–9 after tumor implantation. Specific binding of a [68Ga]-labeled NODAGA-conjugated MoonTag nanobody was observed in 7xMoonTag tumors (1.7 ± 0.5%ID/mL) by PET imaging, showing significantly higher radiotracer accumulation compared to the WT tumors (1.1 ± 0.3%ID/mL; p < 0.01). Ex vivo gamma counter measurements confirmed significantly higher uptake in 7xMoonTag tumors compared to WT tumors (p < 0.001). In addition, MoonTag nanobody binding was detected by autoradiography which was spatially matched with histological analysis of the tumor tissues. In conclusion, we expect nanobody-based poly-epitope tag strategies to be widely applicable for multimodal imaging techniques given the advantageous properties of nanobodies and their amenability to genetic and chemical engineering.

Development of a new primer tool for quantification and identification of geosmin-producing cyanobacteria in drinking water reservoirs

Elevated geosmin concentrations in drinking water reservoirs can lead to customer complaints and increased treatment costs for utilities. Use of molecular approaches, like qPCR and targeted amplicon sequencing, can help with prediction and preventive measures, but many of the primers targeting the geosmin synthase gene suffer from limited coverage of taxa or poor specificity. Here, a set of primers (CGeo1F/R) were developed that had high specificity for geosmin synthase in cyanobacteria without the use of probes. When tested on samples from three drinking water reservoirs with elevated geosmin levels, these primers amplified the geosmin synthase gene in seven cyanobacterial genera, including Dolichospermum, Aphanizomenon, Cylindrospermum, Planktothrix, Nostoc, Coelosphaerium, and Tychonema. These primers exhibited acceptable amplification efficiency (∼ 90 %) and a linear amplification range of 6 × 101–6 × 105 copies per ml in lake water. Within the limited set of samples used to evaluate these primers, a good correlation was observed (r = 0.8) between abundance of geosmin synthase and geosmin concentration. When compared to some existing, well-utilized primers in the literature, these new primers had better specificity and amplification properties, and thus may prove valuable to other researchers.

LABS: linear amplification-based bisulfite sequencing for ultrasensitive cancer detection from cell-free DNA

Methylation-based liquid biopsies show promises in detecting cancer using circulating cell-free DNA; however, current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Additionally, underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here, we report linear amplification-based bisulfite sequencing (LABS), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis). It employs random nonamer primer based method which can amplify short, fragmented RNA with high fidelity from as low as 5 ng to obtain enough material for WTA. The two-cycle method significantly amplified RNA at ∼1000 folds (p < 0.0001) improving the mean read lengths (p < 0.05) in WTA. Overall, increased mean read length positively correlated with on-target reads (Pearson's r = 0.71, p < 0.0001) in both amplified and unamplified RNA-seq analysis. Gene expression analysis compared between unamplified and amplified group displayed substantial overlap of the differentially expressed genes (DEGs) (log2 fold change cut-off < −2 and >2, p < 0.05) identified between lung cancer and asthma cohorts validating the method developed. This method is applicable in clinical molecular pathology field for both diagnostics and elucidation of disease mechanisms.

Noiseless linear amplification of polarization-encoded quantum states with efficient quantum scissors

Noiseless linear amplification of polarization-encoded quantum states with efficient quantum scissors

Multiplex Asymmetric PCR by Combining the Amplification Refractory Mutation System with the Homo-Tag-Assisted Nondimer System.

Asymmetric PCR is widely used to produce single-stranded amplicons (ss-amplicons) for various downstream applications. However, conventional asymmetric PCR schemes are susceptible to events that affect primer availability, which can be exacerbated by multiplex amplification. In this study, a new multiplex asymmetric PCR approach that combines the amplification refractory mutation system (ARMS) with the homo-Tag-assisted nondimer system (HANDS) is described. ARMS-HANDS (A-H) PCR utilizes equimolar-tailed forward and reverse primers and an excess Tag primer. The tailed primer pairs initiate exponential symmetric amplification, whereas the Tag primer drives linear asymmetric amplification along fully matched strands but not one-nucleotide mismatched strands, thereby generating excess ss-amplicons. The production of ss-amplicons is validated using agarose gel electrophoresis, sequencing, and melting curve analysis. Primer dimer alleviation is confirmed by both the reduced Loss function value and a 20-fold higher sensitivity in an 11-plex A-H PCR assay than in an 11-plex conventional asymmetric PCR assay. Moreover, A-H PCR demonstrates unbiased amplification by its allele quantitative ability in correct identification of all 31 trisomy 21 samples among 342 clinical samples. A-H PCR is a new generation of multiplex asymmetric amplification approach with various applications, especially when sensitive and quantitative detection is required.

Toehold Strand Displacement-Mediated Exponential HCR for Highly Sensitive and Specific Analysis of miRNA in Living Cells.

As an important disease biomarker, the development of sensitive detection strategies for miRNA, especially intracellular miRNA imaging strategies, is helpful for early diagnosis of diseases, pathological research, and drug development. Hybridization chain reaction (HCR) is widely used for miRNA imaging analysis because of its high specificity and lack of biological enzymes. However, the classic HCR reaction exhibits linear amplification with low efficiency, limiting its use for the rapid analysis of trace miRNA in living cells. To address this problem, we proposed a toehold-mediated exponential HCR (TEHCR) to achieve highly sensitive and efficient imaging of miRNA in living cells using β-FeOOH nanoparticles as transfection vectors. The detection limit of TEHCR was as low as 92.7 fM, which was 8.8 × 103 times lower compared to traditional HCR, and it can effectively distinguish single-base mismatch with high specificity. The TEHCR can also effectively distinguish the different expression levels of miRNA in cancer cells and normal cells. Furthermore, TEHCR can be used to construct OR logic gates for dual miRNA analysis without the need for additional probes, demonstrating high flexibility. This method is expected to play an important role in clinical miRNA-related disease diagnosis and drug development as well as to promote the development of logic gates.

Coupling Exponential to Linear Amplification for Endpoint Quantitative Analysis.

Exponential DNA amplification techniques are fundamental in ultrasensitive molecular diagnostics. These systems offer a wide dynamic range, but the quantification requires real-time monitoring of the amplification reaction. Linear amplification schemes, despite their limited sensitivity, can achieve quantitative measurement from a single end-point readout, suitable for low-cost, point-of-care, or massive testing. Reconciling the sensitivity of exponential amplification with the simplicity of end-point readout would thus break through a major design dilemma and open a route to a new generation of massively scalable quantitative bioassays. Here a hybrid nucleic acid-based circuit design is introduced to compute a logarithmic function, therefore providing a wide dynamic range based on a single end-point measurement. CELIA (Coupling Exponential amplification reaction to LInear Amplification) exploits a versatile biochemical circuit architecture to couple a tunable linear amplification stage - optionally embedding an inverter function - downstream of an exponential module in a one-pot format. Applied to the detection of microRNAs, CELIA provides a limit of detection in the femtomolar range and a dynamic range of six decades. This isothermal approach bypasses thermocyclers without compromising sensitivity, thereby opening the way to applications in various diagnostic assays, and providing a simplified, cost-efficient, and high throughput solution for quantitative nucleic acid analysis.