RESUMO
Spatiotemporal regulation of clustered regularly interspaced short palindromic repeats (CRISPR) system is attractive for precise gene editing and accurate molecular diagnosis. Although many efforts have been made, versatile and efficient strategies to control CRISPR system are still desirable. Here, we proposed a universal and accessible acylation strategy to regulate the CRISPR-Cas12a system by efficient acylation of 2'-hydroxyls (2'-OH) on crRNA strand with photolabile agents (PLGs). The introduction of PLGs confers efficient suppression of crRNA function and rapid restoration of CRISPR-Cas12a reaction upon short light exposure regardless of crRNA sequences. Based on this strategy, we constructed a universal PhotO-Initiated CRISPR-Cas12a system for Robust One-pot Testing (POIROT) platform integrated with recombinase polymerase amplification (RPA), which showed two orders of magnitude more sensitive than the conventional one-step assay and comparable to the two-step assay. For clinical sample testing, POIROT achieved high-efficiency detection performance comparable to the gold-standard quantitative PCR (qPCR) in sensitivity and specificity, but faster than the qPCR method. Overall, we believe the proposed strategy will promote the development of many other universal photo-controlled CRISPR technologies for one-pot assay, and even expand applications in the fields of controllable CRISPR-based genomic editing, disease therapy, and cell imaging.
Assuntos
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Acilação , Humanos , Processos Fotoquímicos , Edição de Genes/métodos , Ácidos Nucleicos/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
Sensitive and specific assay of microRNAs (miRNAs) is beneficial to early disease screening. Herein, we for the first time proposed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-mediated photoelectrochemical biosensors for the direct assay of miRNA-21. In this study, compared with traditional nucleic acid-based signal amplification strategies, the CRISPR/Cas13a system can greatly improve the specificity and sensitivity of target determination due to its accurate recognition and high-efficient trans-cleavage capability without complex nucleic acid sequence design. Moreover, compared with the CRISPR/Cas12a-based biosensing platform, the developed CRISPR/Cas13a-mediated biosensor can directly detect RNA targets without signal transduction from RNA to DNA, thereby avoiding signal leakage and distortion. Generally, the proposed biosensor reveals excellent analysis capability with a wider linear range from 1 fM to 5 nM and a lower detection limit of 1 fM. Additionally, it also shows satisfactory stability in the detection of human serum samples and cell lysates, manifesting that it has great application prospects in the areas of early disease diagnosis and biomedical research.
Assuntos
Pesquisa Biomédica , Técnicas Biossensoriais , MicroRNAs , Humanos , Bioensaio , Transdução de SinaisRESUMO
CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.
Assuntos
Vírus da Febre Suína Africana , COVID-19 , Vírus da Febre Suína Africana/genética , Animais , Sistemas CRISPR-Cas/genética , DNA/genética , Glicerol , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases , SARS-CoV-2 , Sensibilidade e Especificidade , SuínosRESUMO
DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.
Assuntos
Vírus da Febre Suína Africana , Infecções por Vírus Epstein-Barr , Vírus da Febre Suína Africana/genética , Animais , Sistemas CRISPR-Cas , DNA/genética , Herpesvirus Humano 4 , SuínosRESUMO
Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.
Assuntos
Sistemas CRISPR-Cas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vírus da Febre Suína Africana/isolamento & purificação , Animais , Humanos , RNA Guia de Cinetoplastídeos , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , SuínosRESUMO
Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25â µL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.
Assuntos
COVID-19/diagnóstico , Sistemas CRISPR-Cas , Genes Virais , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/virologia , Humanos , Nasofaringe/virologia , RNA Viral , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
The recently reported freezing-based labeling method for constructing DNA-AuNP probes is rapid but still requires thiol modification. Here, we evaluated a poly(A)-tagged DNA sequence using the freezing-based labeling method, and the results demonstrated that approximately 10 A bases at the sequence ends are essential. More detailed observations revealed that some DNA sequences tend to form secondary structures and thus shield exposed A bases, resulting in inefficient or failed labeling. However, successful labeling was restored by simply increasing the poly(A)-base number. Building on these discoveries, we developed three kinds of AuNP-based bioprobes, DNA-AuNP, RNA-AuNP, and DNA-enzyme-AuNP, using the freezing-based labeling method. This method was completed in a single mixing step with no need for thiol modification, representing one of the most convenient and lowest cost AuNP bioprobe labeling techniques ever reported. In addition, the resulting AuNP bioprobes were further used to advance CRISPR-based diagnostics through the development of user-friendly colorimetric, fluorescence, and lateral flow detection strategies.
Assuntos
Técnicas Biossensoriais/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Ouro/química , Nanopartículas/química , Compostos de Sulfidrila/química , CongelamentoRESUMO
The enzyme-linked immunosorbent assay (ELISA) is a basic technique used in analytical and clinical investigations. However, conventional ELISA is still not sensitive enough to detect ultralow concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious diseases. Herein we show a mechanism utilizing the CRISPR/Cas13a-based signal export amplification strategy, which double-amplifies the output signal by T7 RNA polymerase transcription and CRISPR/Cas13a collateral cleavage activity. This process is termed the CRISPR/Cas13a signal amplification linked immunosorbent assay (CLISA). The proposed method was validated by detecting an inflammatory factor, human interleukin-6 (human IL-6), and a tumor marker, human vascular endothelial growth factor (human VEGF), which achieved limit of detection (LOD) values of 45.81 fg/mL (2.29 fM) and 32.27 fg/mL (0.81 fM), respectively, demonstrating that CLISA is at least 102-fold more sensitive than conventional ELISA.
Assuntos
Biomarcadores Tumorais/análise , Sistemas CRISPR-Cas/genética , Ensaio de Imunoadsorção Enzimática , Interleucina-6/análise , Fator A de Crescimento do Endotélio Vascular/análise , HumanosRESUMO
Gold-nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective posthybridization manipulations for genomic analysis. Here, we present a new design for a colorimetric gene-sensing platform based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the trans-ssDNA or -ssRNA cleavage. Target-induced trans-ssDNA or ssRNA cleavage triggers an aggregation behavior change for the designed AuNPs-DNA probes pair, enabling the completion of naked-eye gene detection (transgenic rice, African swine fever virus, and miRNAs as the models) within 1 h. This platform is also showing promise as a fast and inexpensive tool for bacteria identification using 16S rDNA or 16S rRNA. A CRISPR/Cas-based colorimetric platform shows superior characteristics, such as probe universality, compatibility with isothermal reaction conditions, on-site detection capability, and high sensitivity, thus, demonstrating its use as a robust next-generation gene detection platform.
Assuntos
Sistemas CRISPR-Cas/genética , Colorimetria/métodos , RNA Ribossômico 16S/análise , Vírus da Febre Suína Africana/genética , Animais , Bactérias/genética , Sondas de DNA/química , DNA Viral/análise , DNA Viral/química , Ouro/química , Nanopartículas Metálicas/química , MicroRNAs/análise , MicroRNAs/química , Regiões Promotoras Genéticas , RNA Ribossômico 16S/química , SuínosRESUMO
As an important biomarker, thrombin (TB) is a major player in thrombosis and hemostasis and has attracted increasing attention involving its determination. Herein a universal and ultrasensitive fluorescence biosensor based on a binding-induced 3D-bipedal DNA walker and catalytic hairpin assembly (CHA) strategy has been proposed for cascade signal amplification detection of thrombin. In this study, we designed two proximity probes (foot 1 and foot 2) which include a specific affinity ligand for TB binding and a Pb2+-dependent DNAzyme tail sequence. In the presence of TB, the simultaneous binding of TB to foot 1 (F1) and foot 2 (F2) via TB aptamer (TBA) brings the tail sequences into close proximity and the melting temperature for tail sequences and track DNA is increased, allowing the Pb2+-dependent DNAzyme to cleave the track DNA into two short fragments which have lower affinities for the DNAzyme and, finally, leading to the release of trigger DNA (T-DNA) for subsequent CHA reaction. In the meantime, the dissociated DNA walkers (F1 and F2) explore adjacent unwound track DNA, and the walking procedure is conducted. Unlike the conventional unipedal DNA walkers that anchor foot DNA and track DNA on the same sensing surface, the proposed 3D-bipedal DNA walking machine can not only increase the local concentration of track DNA but can also improve the walking efficiency and expand the range of the walkers to some extent due to the two free feet. Moreover, with the advantages of superior sensitivity and excellent specificity, this biosensing platform exhibits a huge potential in practical application in biomedical research and clinical diagnosis.
Assuntos
Técnicas Biossensoriais , Sondas de DNA/química , DNA Catalítico/química , DNA/química , Técnicas de Amplificação de Ácido Nucleico , Trombina/análise , Sítios de Ligação , Biocatálise , Biomarcadores/análise , Biomarcadores/metabolismo , DNA/metabolismo , Sondas de DNA/metabolismo , DNA Catalítico/metabolismo , Fluorescência , Humanos , Ligantes , Técnicas de Sonda Molecular , Trombina/metabolismoRESUMO
In this paper, an ultrasensitive electrochemical immunoassay was constructed for the determination of carcinoembryonic antigen (CEA) based on a proximity hybridization-triggered three-layer cascade signal amplification strategy. In the presence of CEA and two antibody-labeled DNA strands (Ab-DNA1 and Ab-DNA2), a sandwich proximate complex was formed which could hybridize with the hairpin DNA (HP1) and open it, initiating the degradation process of exonuclease III (Exo III) accompanied by the release of a sandwich proximate complex used for Cycle I and autogenous yield of a DNA fragment. The fragment was then employed as a ST-DNA (secondary target DNA fragment) for the subsequent binding to the other hairpin DNA (HP2) on the gold (Au) electrode surface to trigger the catalytic hairpin assembly (CHA, Cycle II) and rolling circle amplification (RCA). After the immersion of the above final electrode into methylene blue (MB) solution, an amplified signal was obtained. The proposed electrochemical immunoassay had a good linear relationship in the CEA concentration from 10 fg mL-1 to 100 ng mL-1, and the limit of detection (LOD) was found to be 4.2 fg mL-1. Moreover, with the excellent sensitivity and selectivity based on a three-layer cascade signal amplification strategy, the developed electrochemical immunoassay can pave the way for analysis of other biomarkers.
Assuntos
Anticorpos/química , Antígeno Carcinoembrionário/análise , DNA/química , Imunoensaio/métodos , Anticorpos/imunologia , Antígeno Carcinoembrionário/sangue , Antígeno Carcinoembrionário/imunologia , DNA/metabolismo , Técnicas Eletroquímicas , Eletrodos , Exodesoxirribonucleases/metabolismo , Ouro/química , Humanos , Limite de Detecção , Azul de Metileno/química , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , Reprodutibilidade dos TestesRESUMO
The sensitive and specific determination of nucleic acids is very important in clinical diagnosis and biological studies. In this work, an ultrasensitive photoelectrochemical (PEC) biosensor has been developed for DNA detection based on a "signal-on" sensing strategy and a three-stage cascade signal amplification method (catalytic hairpin assembly (CHA), hybridization chain reaction (HCR) and alkaline phosphatase (ALP)-triggered in situ generation of ascorbic acid (AA)). Here, CHA hairpin 1 (CHA-HP1) is opened by the target DNA (T-DNA) owing to the hybridization between T-DNA and CHA-HP1, and then the opened CHA-HP1 hybridizes with CHA hairpin 2 (CHA-HP2) to displace the T-DNA, generating a CHA-HP1/CHA-HP2 complex. The displaced T-DNA triggers the next cycle of CHA, resulting in the generation of numerous CHA-HP1/CHA-HP2 complexes. Subsequently, one end of the CHA-HP1/CHA-HP2 complex hybridizes with the capture DNA immobilized on the indium tin oxide/TiO2/CdS : Mn electrode. After the introduction of dual-biotin labeled HCR hairpin 1 (HCR-HP1-Bio) and dual-biotin labeled HCR hairpin 2 (HCR-HP2-Bio), the other end of the CHA-HP1/CHA-HP2 complex opens HCR-HP1-Bio. The opened HCR-HP1-Bio triggers the HCR reaction between HCR-HP1-Bio and HCR-HP2-Bio, leading to the formation of long nicked duplex DNA structures. The dual-biotin modified HCR-hairpins can anchor more streptavidin-ALP to catalyze 2-phospho-l-ascorbic acid trisodium salt to yield more AA, leading to a larger PEC response. The proposed PEC biosensor shows superior analytical performance for T-DNA detection with a linear response ranging from 0.1 fM to 100 pM and a detection limit of 0.052 fM, and may provide a powerful biosensing platform for bioanalysis and early disease diagnosis.
Assuntos
Técnicas Biossensoriais , DNA/análise , Hibridização de Ácido Nucleico , Biotina , Técnicas Eletroquímicas , Limite de Detecção , Processos Fotoquímicos , EstreptavidinaRESUMO
Biomolecular receptors such as nucleic acids that switch between two or more conformations upon binding to a specific target can be used to build specific and sensitive biosensors. In this work, based on the electrochemical dual-signaling ratiometric strategy and triple-helix molecular switch, we developed a selective, reusable, and simple electrochemical DNA (E-DNA) biosensor for target DNA (T-DNA) detection. A hairpin DNA capture probe labeled with methylene blue (MB-DNA) self-assembles on the surface of a gold electrode (GE) through Au-S bond, and then a single-strand DNA modified with two ferrocenes (Fc-DNA) on each end to enhance the oxidation signal hybridizes with the MB-DNA to form a triple-helix conformation. When T-DNA exists, the Fc-DNA hybridizes with T-DNA disassembling the triple-helix stem and allowing the MB-DNA to revert to its hairpin structure. Hence, the Fc tags diffuse away from the GE surface while the MB tags remain affixed close to it, resulting in a decrease in the peak current of Fc (IFc) and an increase in that of MB (IMB). The linear relationship between the value of IMB/IFc and the T-DNA concentration is observed from 0.5 to 80 pM, and the limit of detection is as low as 0.12 pM. The developed E-DNA biosensor may have great potential in the electrochemical detection of a wide range of analytes and be a biosensing platform for early clinical diagnosis and biomedical research.
Assuntos
Técnicas Biossensoriais/métodos , DNA Bacteriano/análise , Técnicas Eletroquímicas/métodos , Sondas de DNA/metabolismo , DNA Bacteriano/metabolismo , Eletrodos , Ouro/química , Limite de Detecção , Metalocenos/química , Azul de Metileno/química , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oxirredução , Reprodutibilidade dos TestesRESUMO
Because of the intrinsic importance of nucleic acids as biotargets, the simple and sensitive detection of nucleic acids is very essential for biological studies and medical diagnostics. In this work, a novel, simple, and selective electrochemical DNA biosensor for the sensitive detection of target DNA (T-DNA) has been developed based on the dual-signaling electrochemical ratiometric method and exonuclease III (Exo III)-assisted target recycling amplification strategy. The assay strategy includes both "signal-on" and "signal-off" elements. The stem-loop (hairpin) DNA capture probe (HP), which was labeled by thiolated methylene blue (MB) at the 3'-protruding termini and ferrocene (Fc) in the middle of the loop, first self-assembled on the gold electrode surface via a Au-S bond. In the presence of T-DNA, the T-DNA hybridized with HP, which triggered the Exo III cleavage process and accompanied the release of T-DNA. As a result, the MB tags were away from and the Fc tags close to the gold electrode surface, leading to the decrease of the oxidation peak current of MB (I(MB)) and the increase of that of Fc (I(Fc)). The value of ΔI(Fc)/|ΔI(MB)| (ΔI(Fc) and ΔI(MB) are the change values of the oxidation peak currents of Fc and MB, respectively) is linear with the concentration of T-DNA from 0.01 pM to 0.8 pM. The detection limit (4.16 fM) of the developed method is much lower than that of the most reported electrochemical DNA biosensors. This strategy provides a simple and sensitive approach for the detection of T-DNA and has promising applications in bioanalysis, disease diagnostics, and clinical biomedicine.
Assuntos
Técnicas Biossensoriais , DNA/análise , Técnicas Eletroquímicas , Exodesoxirribonucleases/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Propriedades de SuperfícieRESUMO
Single-nucleotide variants (SNVs) are the most common type variation of sequence alterations at a specific location in the genome, thus involving significant clinical and biological information. The assay of SNVs has engaged great awareness, because many genome-wide association studies demonstrated that SNVs are highly associated with serious human diseases. Moreover, the investigation of SNV expression levels in single cells are capable of visualizing genetic information and revealing the complexity and heterogeneity of single-nucleotide mutation-related diseases. Thus, developing SNV assay approaches in vitro, particularly in single cells, is becoming increasingly in demand. In this review, we summarized recent progress in the enzyme-free and enzyme-mediated strategies enabling SNV assay transition from sensing interface to the test tube and single cells, which will potentially delve deeper into the knowledge of SNV functions and disease associations, as well as discovering new pathways to diagnose and treat diseases based on individual genetic profiles. The leap of SNV assay achievements will motivate observation and measurement genetic variations in single cells, even within living organisms, delve into the knowledge of SNV functions and disease associations, as well as open up entirely new avenues in the diagnosis and treatment of diseases based on individual genetic profiles.
RESUMO
A simple electrochemical aptasensor for sensitive and selective determination of adenosine triphosphate (ATP) has been developed on the basis of a new dual-signaling amplification strategy. This aptasensor features both ''signal-on'' and ''signal-off'' elements. The ferrocene (Fc)-labeled aptamer probe (Fc-P) is designed to hybridize with the thiolated methylene blue (MB)-modified DNA probe (MB-P) on gold electrode to form rigid duplex DNA. In the presence of ATP, the interaction between ATP and the aptamer leads to the dissociation of the duplex DNA structure and thereby the release of the Fc-P from the sensing interface. The single-stranded MB-P could thus tend to form a hairpin structure through the hybridization of the complementary sequences at both its ends. Such conformational changes result in the oxidation peak current of Fc decreases and that of MB increases, and the changes of dual signals are linear with the concentration of ATP. When "ΔI = ΔI(MB) + |ΔI(Fc)|" (ΔI(MB) and ΔI(Fc) are the change values of the oxidation peak currents of MB and Fc, respectively.) is used as the response signal for quantitative determination of ATP, the detection limit (1.9 nM) is much lower than that by using either MB-P or Fc-P alone. This new dual-signaling aptasensor is readily regenerated and shows good response toward the target. It will have important applications in the sensitive and selective electrochemical determination of other small molecules and proteins.
Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Eletroquímicas/métodos , Trifosfato de Adenosina/urina , HumanosRESUMO
The ability to predict nucleic acid hybridization energies has been greatly enabling for many applications, but predictive models require painstaking experimentation, which may limit expansion to non-natural nucleic acid analogues and chemistries. We have assessed the utility of dye-based, high-resolution melting (HRM) as an alternative to UV-Vis determinations of hyperchromicity in order to more quickly acquire parameters for duplex stability prediction. The HRM-derived model for phosphodiester (PO) DNA can make comparable predictions to previously established models. Using HRM, it proved possible to develop predictive models for DNA duplexes containing phosphorothioate (PS) linkages, and we found that hybridization stability could be predicted as a function of sequence and backbone composition for a variety of duplexes, including PS:PS, PS:PO, and partially modified backbones. Individual phosphorothioate modifications destabilize helices by around 0.12 kcal/mol on average. Finally, we applied these models to the design of a catalytic hairpin assembly circuit, an enzyme-free amplification method used for nucleic acid-based molecular detection. Changes in PS circuit behavior were consistent with model predictions, further supporting the addition of HRM modeling and parameters for PS oligonucleotides to the rational design of nucleic acid hybridization.
Assuntos
DNA , Oligonucleotídeos Fosforotioatos , DNA/genética , Conformação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here, we discover that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies show that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent. Spectroscopy, test strip hybridization, and loading counting experiments indicate that low-affinity poly (T/U) tag mediates the formation of a standing-up conformation, which is distributed in the outer layer of SNA structure. In further application studies, CRISPR/Cas9-sgRNA (136 bp), SARS-CoV-2 RNA fragment (1278 bp), and rolling circle amplification (RCA) DNA products (over 1000 bp) can be successfully attached on AuNPs, which overcomes the routine methods in long-chain nucleic acid-AuNP conjugation, exhibiting great promise in biosensing and nucleic acids delivery applications. Current heating-dry strategy has improved traditional DNA/RNA-AuNP conjugation methods in simplicity, rapidity, cost, and universality.
Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Nanopartículas Metálicas/química , Biotecnologia/métodos , COVID-19/diagnóstico , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/métodos , DNA/química , Calefação/métodos , Humanos , Limite de Detecção , Micro-Ondas , Nanomedicina/métodos , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , SARS-CoV-2/genéticaRESUMO
Gold nanoparticles (AuNPs) colorimetric assays based on distance-dependent optical characteristics have been widely employed for bioanalysis. However, this assay is not effective for visually detecting low-concentration targets due to the faint color change. Here, we developed a handheld nano-centrifugal device which could separate the crosslinked and non-crosslinked AuNPs. Results showed that the handheld nano-centrifugal device could easily reach more than 6000 r/min within 10 s simply by stretching and tightening the coiled rope in an appropriate rhythm. Further, combined with the CRISPR/Cas12a nucleic acids recognition system, a field-deployable colorimetric platform termed handheld nano-centrifugal device assisted CRISPR/Cas12a (Hand-CRISPR) has been validated. Moreover, clinical diagnostics applications for Epstein-Barr virus (EBV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection with high sensitivity and accuracy (100% consistency with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) test results) have been demonstrated. Overall, the Hand-CRISPR platform showed great promise in point-of-care-test (POCT) application, expected to become a powerful supplement to the standard nucleic acid testing method in remote or poverty-stricken areas. Supplementary Information: The online version contains supplementary material available at 10.1007/s41664-022-00232-0.
RESUMO
Signal amplification is ubiquitous in biology and engineering. Protein enzymes, such as DNA polymerases, can routinely achieve >106-fold signal increase, making them powerful tools for signal enhancement. Considerable signal amplification can also be achieved using nonenzymatic, cascaded nucleic acid strand exchange reactions. However, the practical application of such kinetically trapped circuits has so far proven difficult due to uncatalyzed leakage of the cascade. We now demonstrate that strategically positioned mismatches between circuit components can reduce unprogrammed hybridization reactions and therefore greatly diminish leakage. In consequence, we were able to synthesize a three-layer catalytic hairpin assembly cascade that could operate in a single tube and that yielded 3.7 × 104-fold signal amplification in only 4 h, a greatly improved performance relative to previous cascades. This advance should facilitate the implementation of nonenzymatic signal amplification in molecular diagnostics, as well as inform the design of a wide variety of increasingly intricate nucleic acid computation circuits.