Stem-loop Research Articles

MicroRNA-Triggered Programmable DNA-Encoded Pre-PROTACs for Cell-Selective and Controlled Protein Degradation.

Proteolysis-targeting chimeras (PROTACs) have accelerated drug development; however, some challenges still exist owing to their lack of tumor selectivity and on-demand protein degradation. Here, we developed a miRNA-initiated assembled pre-PROTAC (miRiaTAC) platform that enables the on-demand activation and termination of target degradation in a cell type-specific manner. Using miRNA-21 as a model, we engineered DNA hairpins labeled with JQ-1 and pomalidomide and facilitated the modular assembly of DNA-encoded pre-PROTACs through a hybridization chain reaction. This configuration promoted the selective polyubiquitination and degradation of BRD4 upon miR-21 initiation, highlighting significant tumor selectivity and minimal systemic toxicity. Furthermore, the platform incorporates photolabile groups, enabling the precise optical control of pre-PROTACs during DNA assembly/disassembly, mitigating the risk of excessive protein degradation. Additionally, by introducing a secondary ligand targeting CDK6, these pre-PROTACs were used as a modular scaffold for the programmable assembly of active miRiaTACs containing two different warheads in exact stoichiometry, enabling orthogonal multitarget degradation. The integration of near-infrared light-mediated photodynamic therapy through an upconversion nanosystem further enhanced the efficacy of the platform with potent in vivo anticancer activity. We anticipate that miRiaTAC represents a significant intersection between dynamic DNA nanotechnology and PROTAC, potentially expanding the versatility of PROTAC toolkit for cancer therapy.

MicroRNA‐Triggered Programmable DNA‐Encoded Pre‐PROTACs for Cell‐Selective and Controlled Protein Degradation

Proteolysis‐targeting chimeras (PROTACs) have accelerated drug development; however, some challenges still exist owing to their lack of tumor selectivity and on‐demand protein degradation. Here, we developed a miRNA‐initiated assembled pre‐PROTAC (miRiaTAC) platform that enables the on‐demand activation and termination of target degradation in a cell type‐specific manner. Using miRNA‐21 as a model, we engineered DNA hairpins labeled with JQ‐1 and pomalidomide and facilitated the modular assembly of DNA‐encoded pre‐PROTACs through a hybridization chain reaction. This configuration promoted the selective polyubiquitination and degradation of BRD4 upon miR‐21 initiation, highlighting significant tumor selectivity and minimal systemic toxicity. Furthermore, the platform incorporates photolabile groups, enabling the precise optical control of pre‐PROTACs during DNA assembly/disassembly, mitigating the risk of excessive protein degradation. Additionally, by introducing a secondary ligand targeting CDK6, these pre‐PROTACs were used as a modular scaffold for the programmable assembly of active miRiaTACs containing two different warheads in exact stoichiometry, enabling orthogonal multitarget degradation. The integration of near‐infrared light‐mediated photodynamic therapy through an upconversion nanosystem further enhanced the efficacy of the platform with potent in vivo anticancer activity. We anticipate that miRiaTAC represents a significant intersection between dynamic DNA nanotechnology and PROTAC, potentially expanding the versatility of PROTAC toolkit for cancer therapy.

Activated single nucleotide mismatch determination through toehold-embedded hairpin-mediated strand displacement reaction alongside CRISPR-Cas12a for detection of TP53 point mutation

The CRISPR-Cas12a-based diagnostic platform is renowned for its high sensitivity and, when coupled with the toehold-mediated strand displacement (TMSD) reaction, enables the detection of single nucleotide mismatch (SNM). However, conventional TMSD with the CRISPR-Cas12a system suffers from signal leakage due to overlapping activator sequences and low displacement efficiency. Here, we propose a toehold-embedded hairpin-mediated strand displacement (THMSD) method and leverage the magnesium attenuation effect of CRISPR-Cas12a for selective single-point mutation detection in the TP53 gene. This approach effectively mitigates signal leakage as the hairpin DNA retains partial crRNA targeting sequences within its loop, preventing sequence overlap with the trigger and direct Cas12a activation. Additionally, the magnesium attenuation effect reduces background signal and enhances selectivity. We investigated toehold length and mismatch position to detect SNM with a high discrimination factor (DF). This high DF allows the THMSD/CRISPR-Cas12a system to differentiate between TP53 R273H mutant and TP53 wildtype genes for cancer screening at low abundances, down to 0.1 % with a concentration of 20 pM. When used for screening between H1975 and WI38 cells, the statistically significant difference was attainable with polymerase chain reaction (PCR) template loading as low as 4 ng. These results pave the way for developing CRISPR-Cas12a-based methods to effectively detect TP53 mutations as lung cancer biomarkers.

Single-Stranded Hairpin Loop RNAs (loopmeRNAs) Potently Induce Gene Silencing through the RNA Interference Pathway.

Synthetic small interfering RNAs conjugated to trivalent N-acetylgalactosamine (GalNAc) are clinically validated drugs for treatment of liver diseases. Incorporation of phosphorothioate linkages and ribose modifications are necessary for stability, potency, and duration of pharmacology. Although multiple alternative siRNA designs such as Dicer-substrate RNA, shRNA, and circular RNA have been evaluated in vitro and in preclinical studies with some success, clinical applications of these designs are limited as it is difficult to incorporate chemical modifications in these designs. An alternative siRNA design that can incorporate chemical modifications through straightforward synthesis without compromising potency will significantly advance the field. Here, we report a facile synthesis of GalNAc ligand-containing single-stranded loop hairpin RNAs (loopmeRNAs) with clinically relevant chemical modifications. We evaluated the efficiency of novel loopmeRNA designs in vivo and correlated their structure-activity relationship with the support of in vitro metabolism data. Sequences and chemical modifications in the loop region of the loopmeRNA design were optimized for maximal potency. Our studies demonstrate that loopmeRNAs can efficiently silence expression of target genes with comparable efficacy to conventional double-stranded siRNAs but reduced environmental and regulatory burdens.

Cooperative dynamics of PARP-1 zinc-finger domains in the detection of DNA single-strand breaks

The DNA single-strand break (SSB) repair pathway is initiated by the multifunctional enzyme PARP-1, which recognizes the broken DNA ends by its two zinc-finger domains, Zn1 and Zn2. Despite a number of experiments performed with different DNA configurations and reduced fragments of PARP-1, many details of this interaction that is crucial to the correct initiation of the repair chain are still unclear. We performed Molecular Dynamics (MD) computer simulations of the interaction between the Zn1/Zn2 domains of PARP-1 and a DNA hairpin including a missing nucleotide to simulate the presence of an SSB, a construct used in recent experiments. The role of Zn1 and Zn2 interacting with the SSB ends is studied in detail, both independently and cooperatively. We also explored, PARP-1 operating as a dimer, with the two Zn-fingers coming from two separate copies of the enzyme. By an extensive set of all-atom molecular simulations employing state-of-the art force fields, assisted by empirical docking and free-energy calculations, we conclude that the particular conformation of the DNA hairpin can indeed spontaneously open up by thermal fluctuations, up to extremely kinked deformations. However, such extreme localized deformations are rarely observed in free, long DNA fragments. Protein side-loops make contact with the DNA hairpin grooves, and help Zn2 to penetrate deep in the SSB gap. In this way, Zn2 can interact with the nucleotides opposite to the missing base. Overall, Zn1 plays a secondary role: the crucial factor for the interaction is rather the relative arrangement of the Zn1/Zn2 couple, and their mutual orientation with respect to the 3′\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$3'$$\\end{document} and 5′\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$5'$$\\end{document} SSB end terminals. This helps to obtain an early interacting configuration, which ultimately leads to molecular PARP-1-DNA structures similar to those observed experimentally. Such findings represent an important step toward defining the detailed function of PARP-1 in the early stages of SSB recognition.

Sandwich-Type Electrochemical Aptasensor with Supramolecular Architecture for Prostate-Specific Antigen.

A novel sandwich-type electrochemical aptasensor based on supramolecularly immobilized affinity bioreceptor was prepared via host-guest interactions. This method utilizes an adamantane-modified, target-responsive hairpin DNA aptamer as a capture molecular receptor, along with a perthiolated β-cyclodextrin (CD) covalently attached to a gold-modified electrode surface as the transduction element. The proposed sensing strategy employed an enzyme-modified aptamer as the signalling element to develop a sandwich-type aptasensor for detecting prostate-specific antigen (PSA). To achieve this, screen-printed carbon electrodes (SPCEs) with electrodeposited reduced graphene oxide (RGO) and gold nanoferns (AuNFs) were modified with the CD derivative to subsequently anchor the adamantane-modified anti-PSA aptamer via supramolecular associations. The sensing mechanism involves the affinity recognition of PSA molecules on the aptamer-enriched electrode surface, followed by the binding of an anti-PSA aptamer-horseradish peroxidase complex as a labelling element. This sandwich-type arrangement produces an analytical signal upon the addition of H2O2 and hydroquinone as enzyme substrates. The aptasensor successfully detected the biomarker within a concentration range of 0.5 ng/mL to 50 ng/mL, exhibiting high selectivity and a detection limit of 0.11 ng/mL in PBS.

Single-Molecule adenosine detection and chiral selectivity by DNA aptamer conformational changes using α-Hemolysin nanopore

Nucleic acid aptamers, which are short single-stranded RNA or DNA molecules, undergo conformational changes upon recognizing small molecules. However, their structural characterization is challenging due to the flexibility of single-stranded oligonucleotides. Nanopores provide a sensitive method for single-molecule analysis of molecular conformational changes as they transport through the pore. In this study, we employed α-hemolysin (α-HL) nanopore with high spatiotemporal resolution to investigate the conformational changes of DNA aptamer (ADO-23) upon binding to adenosine (Ado) molecules. Unlike the events generated by the aptamer alone, the DNA-Ado complex produced two distinct new characteristic events, indicating the formation of two unique secondary structures. By analyzing current events, the structures formed by the DNA-Ado complex were speculated to be hairpin DNA and G-quadruplex (G4) DNA. Furthermore, we demonstrated the potential of the aptamer as a chiral selector within the nanopore. The results showed that the aptamer specifically bound to D-Ado and not to L-Ado. Finally, we realized specific and sensitive detection of small molecule Ado by α-HL pore, with a detection limit as low as 10.5 nM, and successfully applied it to the detection of real human serum samples. The nanopore platform provides a powerful tool for studying small molecule-biomolecule conformational changes, and we constructed sensors for detecting adenosine through the aptamer. This work also offers a promising new approach to the recognition of enantiomers at the single-molecule level.

DNA polymerase mediated CRISPR/Cas12a trans-cleavage for dual-mode quantification of uracil DNA glycosylase activity

Since an unusual expression of uracil-DNA glycosylase (UDG) is often associated with the pathogenesis of numerous disorders, the detection of UDG activity is regarded as a promising application in disease diagnosis. Here, we develop a DNA polymerase-mediated CRISPR/Cas12a trans cleavage strategy, which can achieve dual-mode determination of UDG activity. By introducing a hairpin DNA probe containing a single uracil base, the probe undergoes specific cleavage and elongation under the existence of UDG only, thus activating the trans cleavage of ssDNA regardless of its length and sequence. To accommodate different detection modes, the ssDNA was further modified by fluorophore-quencher pairs or designed for connecting magnetic microparticles (MMPs) and polystyrene microparticles (PMPs). Finally, the UDG activity is quantified by fluorescence signal and microparticle accumulation length on a microfluidic chip visible to the naked eye. This strategy provides a detectable minimum UDG concentration of 0.00047 U/mL for fluorescent mode and 0.0048 U/mL for microfluidic mode. Furthermore, we performed the UDG inhibition test and detected UDG activity in cell lysates, suggesting its potential for inhibitor screening and detection of UDG in biological samples.

Characterization of Hibiscus Chlorotic Ringspot Virus-Derived vsiRNAs from Infected Hibiscus rosa-sinensis in China.

Lots of progress have been made about pathogen system of Hibiscus rosa-sinensis and hibiscus chlorotic ringspot virus (HCRSV), however, interactions between H. rosa-sinensis and HCRSV remain largely unknown. Hereon, firstly, HCRSV infection in H. rosa-sinensis from Zhangzhou city of China was confirmed by traditional electron microscopy, modern reverse transcription polymerase chain reaction and RNA-seq methods. Secondly, sequence feature analysis showed the full-length sequence of HCRSV-ZZ was 3,909 nucleotides (nt) in length and had a similar genomic structure with other carmovirus. It contains a 5' untranslated region (UTR), followed by seven open reading frames encoding for P28, P23, P81, P8, P9, P38, and P25, and the last a 3-terminal UTR. Thirdly, HCRSV- ZZ-derived vsiRNAs were identified and characterized for the first time from disease H. rosa-sinensis through sRNA-seq to reveal interactions between pathogen ant plant host. It was shown that the majority of HCRSV-ZZ-derived vsiRNAs were 21 nt, 22 nt, and 20 nt, with 21 nt being most abundant. The 5'-terminal nucleotide of HCRSV-ZZ vsiRNAs preferred U and C. HCRSV-ZZ vsiRNAs derived predominantly (72%) from the viral genome positive-strand RNA. The distribution of HCRSV-ZZ vsiRNAs along the viral genome is generally even, with some hot spots and cold spots forming in local regions. These hot spots and cold spots could be corresponded to the regions of stem loop secondary structures forming in HCRSV-ZZ genome by nucleotide paring. Taken together, our findings certify HCRSV infection in H. rosa-sinensis and provide an insight into interaction between HCRSV and H. rosa-sinensis and contribute to the prevention and treatment of this virus.

CRISPR/Cas13a-mediated triple signal amplification strategy for viral RNA detection

CRISPR/Cas13a system has been combined with different isothermal amplification strategies for RNA analysis. Nevertheless, expensive enzymes are usually indispensable in these isothermal amplification methods, which increases the detection cost. In this study, a CRISPR/Cas13a-mediated triple signal amplification strategy has been designed for selective, sensitive and inexpensive detection of viral RNA. SARS-CoV-2 S RNA activated CRISPR/Cas13a to cut hairpin DNA, triggering catalytic hairpin assembly (CHA) and subsequent hybridization chain reaction (HCR) to form a long-stranded Y-type DNA. G-quadruplexes were distributed on the branch of the long-stranded DNA and bound with N-methyl mesoporphyrin IX (NMM) to enhance the fluorescence signal. SARS-CoV-2 S RNA was specifically detected with a limit of detection (LOD) of 285 fM. Notably, no additional expensive enzymes were required in the entire detection process. The CRISPR/Cas13a-mediated triple signal amplification strategy holds great potential for specific and sensitive viral RNA detection with low cost.

Dual-Responsive MnO2-Loaded Carbonaceous Nanobottle Motors Fabricated by Interfacial Superassembly for On-The-Fly MicroRNA Sensing.

Diverse nanomotors with advanced motion manipulation have been proposed to revolutionize the way problems in many fields are solved. However, rational and controllable synthetic methods of multifunctional nanomotor are still limited. Herein, dual-responsive MnO2-loaded carbonaceous nanobottle motors (MnO2 NBMs) are developed through an interfacial superassembly strategy. Asymmetric carbonaceous nanobottles are first synthesized, and the reductive carbonaceous shell induces an oxidation-reduction reaction with KMnO4 for in-situ growth of MnO2 nanosheets, which enables the nanomotor to perform either self-diffusiophoretic or self-thermophoretic motion in response to H2O2 and near-infrared light, respectively. Inspired by bioaffinity sensing, the nanomotors are sequentially assembled with functional nanoparticles and hairpin DNA to construct swimming functional MnO2 NBMs (MnO2 FNBMs) probes. The probes can move around complex samples to improve target miRNA transport and accelerate receptor-target interaction. Coupling with the photocurrent-signal amplification, the self-assembly of photoelectrochemical (PEC) biosensors has been achieved for sensitive microRNA detection. Trace amounts of miRNA-155 can be quickly detected with a wide detection range (100 fM to 100nM). Moreover, the direct detection of microRNA in tumor cell lysates by the biosensor is demonstrated. Given the merits of automation and miniaturization, the proposed strategy provides a promising method for fast and effective self-assembly of biosensors.

ATP-driven AP endonuclease-active Exo III@zeolitic imidazolate framework for uracil-DNA glycosylase imaging in living cells

In the currently available approaches for intracellular uracil-DNA glycosylase (UDG) detection, their signal outputs usually rely on the cleavage of the residual AP sites in DNA probes by apurinic/apyrimidinic endonuclease 1 (APE1), whose expression level varies across different types of cells. Therefore, reliable visualizing detection of UDG in living cells remains challenging. Herein, a nanoprobe for imaging of intracellular UDG activity has been designed by taking advantages of both zeolitic imidazolate framework-90 (ZIF-90) and endonuclease III. ZIF-90 is used to deliver exonuclease III (Exo III) and hairpin DNA strands into cells. One of the hairpin strands (HP1) contains a deoxyuridine (dU) base in its stem region, while the other (HP2) was modified with FAM molecule and BHQ1 at its 3′ and 5′ ends, respectively. In the presence of UDG, the dU base in HP1 is excised to form an AP site. Exo III can cleave the AP site in HP1 to generate a stem-loop fragment, which can hybridize with HP2 to initiate the recycling digestion of HP2 by Exo III, resulting in amplification of the fluorescence signal and hence sensitive detection of UDG (limit of detection: 2.0×10−6 U/mL). Experimental results have demonstrated that the nanoprobe has merits of good biocompatibility, high sensitivity and selectivity. It realizes in-situ imaging of intracellular UDG without the involvement of APE1, is a promising alternative tool for UDG detection in fields of clinical diagnosis and biomedical research.

Phylogenomic instructed target analysis reveals ELAV complex binding to multiple optimally spaced U-rich motifs.

ELAV/Hu RNA-binding proteins are gene-specific regulators of alternative pre-mRNA processing. ELAV/Hu family proteins bind to short AU-rich motifs which are abundant in pre-mRNA, making it unclear how they achieve gene specificity. ELAV/Hu proteins multimerize, but how multimerization contributes to decode degenerate sequence environments remains uncertain. Here, we show that ELAV forms a saturable complex on extended RNA. Through phylogenomic instructed target analysis we identify the core binding motif U5N2U3, which is repeated in an extended binding site. Optimally spaced short U5N2U3 binding motifs are key for high-affinity binding in this minimal binding element. Binding strength correlates with ELAV-regulated alternative poly(A) site choice, which is physiologically relevant through regulation of the major ELAV target ewg in determining synapse numbers. We further identify a stem-loop secondary structure in the ewg binding site unwound upon ELAV binding at three distal U motifs. Base-pairing of U motifs prevents ELAV binding, but N6-methyladenosine (m6A) has little effect. Further, stem-loops are enriched in ELAV-regulated poly(A) sites. Additionally, ELAV can nucleate preferentially from 3' to 5'. Hence, we identify a decisive mechanism for ELAV complex formation, addressing a fundamental gap in understanding how ELAV/Hu family proteins decode degenerate sequence spaces for gene-specific mRNA processing.

A nanopore-based label-free CRISPR/Cas12a system for portable and ultrasensitive detection of zearalenone

BackgroundFood safety has become a serious global concern. Therefore, there is a need for effective detection technologies in this field. Currently, the development of effective on-site detection techniques is extremely important for food safety. However, the traditional on-site detection methods currently lack effective signal amplification. Herein, the aim of this study was to construct a nanopore-based label-free CRISPR/Cas12a system for the detection of Zearalenone (ZEN). The method is expected to be highly sensitive for portable detection of ZEN in food. ResultsThe proposed strategy was mainly involved three steps, including the displacement of the target DNA, the triggering of the cleavage of hairpin DNA probes (probes 1) by the trans-cleavage of CRISPR/Cas12a, and the generation of a measurable nanopore current signal. The probes 1 and DNA after the cleavage of probes 1 (probes 2) produce different characteristic nanopore signals as they pass through the nanopore. The established method achieved a low limit of detection (LOD) of 6.52 fM for ZEN and a wide liner range under optimized conditions. Furthermore, the practical applicability of this method was verified in real maize samples and showed good recoveries (90.68–101.98 %) and low relative standard deviations (RSD) (9.21–9.72 %). Therefore, this method is a promising option for rapid and ultrasensitive detection of ZEN. Significance and noveltyThe study presented a portable nanopore-based CRISPR/Cas12a signal amplification detection system for the detection of ZEN in food, which had a low LOD and the advantages of rapid, portability, and on-site detection potential. In conclusion, the method presented a promising prospect and universal platform for the detection of ZEN and other mycotoxins, offering a novel insight into on-site food safety detection.

N2-methylguanosine and N2, N2-dimethylguanosine in cytosolic and mitochondrial tRNAs

Decoration of cellular RNAs with modified RNA nucleosides is an important layer of gene expression regulation. Throughout the transcriptome, RNA modifications influence the folding, stability and function of RNAs as well as their interactions with RNA-binding proteins. Although first detected more than 50 years ago, the modified nucleosides N2-methylguanosine (m2G) and N2,N2-dimethylguanosine (m22G) have recently come to the fore through the identification and characterization of the human methyltransferases (MTases) responsible for their installation. In tRNAs, m2G and m22G are present at the junctions between the acceptor stem and the D-arm, and the D-arm and the anticodon stem loop. Here, we review the current knowledge on the effects of mono- and di-methylation of N2 of guanosine on base-pairing and provide an overview of m2(2)G sites in cytosolic and mitochondrial tRNAs. We highlight key features of m2G and m22G MTases, and describe how these enzymes specifically recognize their RNA substrates and target nucleosides. We also discuss the impact of these modifications on tRNA functions, their dynamic regulation and their implications in disease.

Rational Design of Untranslated Regions to Enhance Gene Expression

How to improve gene expression by optimizing mRNA structures is a crucial question for various medical and biotechnological applications. Previous efforts focus largely on investigation of the 5′ UTR hairpin structures. In this study, we present a rational strategy that enhances mRNA stability and translation by engineering both the 5′ and 3′ UTR sequences. We have successfully demonstrated this strategy using green fluorescent protein (GFP) as a model in Escherichia coli and across different expression vectors. We further validated it with luciferase and Plasmodium falciparum lactate dehydrogenase (PfLDH). To elucidate the underlying mechanism, we have quantitatively analyzed both protein, mRNA levels and half-life time. We have identified several key aspects of UTRs that significantly influence mRNA stability and protein expression in our system: (1) The optimal length of the single-stranded spacer between the stabilizer hairpin and ribosome binding site (RBS) in the 5′ UTR is 25–30 nucleotide (nt) long. An optimal 32% GC content in the spacer yielded the highest levels of GFP protein production. (2) The insertion of a homodimerdizable, G-quadruplex structure containing RNA aptamer, “Corn”, in the 3′ UTR markedly increased the protein expression. Our findings indicated that the carefully engineered 5′ UTRs and 3′ UTRs significantly boosted gene expression. Specifically, the inclusion of 5 × Corn in the 3′ UTR appeared to facilitate the local aggregation of mRNA, leading to the formation of mRNA condensates. Aside from shedding light on the regulation of mRNA stability and expression, this study is expected to substantially increase biological protein production.

Exploration of Tertiary Structure in Sequence-Defined Polymers Using Molecular Dynamics Simulations.

Peptoids are a class of sequence-defined biomimetic polymers with peptide-like backbones and side chains located on backbone nitrogens rather than alpha carbons. These materials demonstrate a strong ability for precise control of single-chain structure, multiunit self-assembly, and macromolecular assembly through careful tuning of sequence due to the diversity of available side chains, although the driving forces behind these assemblies are often not understood. Prior experimental work has shown that linked 15mer peptoids can mimic the protein helical hairpin structure by leveraging the chirality-inducing nature of bulky side chains and hydrophobicity, but there are still gaps in our understanding of the relationship between sequence, stability, and particular secondary or tertiary structure. We present a molecular dynamics (MD) study on the folding behavior of these polymers into hairpins, discussing the differences in structure from sequences with various characteristics in water and acetonitrile, and then compare the handedness preference of common helical motifs between solvents.

Label-Free and Universal CRISPR/Cas12a-Based Detection Platform for Nucleic Acid Biomarkers.

CRISPR/Cas12a has been widely used in molecular diagnostics due to its excellent trans-cleavage activity. However, conventional reporters, such as F/Q-labeled single-stranded DNA (ssDNA) reporters, enzyme-labeled reporters, and spherical nucleic acid reporters, require complex modification or labeling processes. In this study, we have developed a rapid, universal, and label-free CRISPR/Cas12a-based biomarker detection platform via designing a G-quadruplex (G4) containing a hairpin structure as the reporter. The hairpin loop design of hairpin G4 improves the cleavage efficiency of Cas12a and the signal strength of the G4 binding ligand. Meanwhile, the incorporation of a G4 binding dye (protoporphyrin IX) eliminates the need for complex modifications. The CRISPR-hairpin G4 detection platform is capable of detecting ssDNA, double-stranded DNA, genetic RNAs, and miRNAs. Moreover, this platform achieves label-free detection in clinical samples, demonstrating its practical applicability and efficiency.

Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual-emission silver nanoclusters and catalytic hairpin amplification strategy.

Adual-emission fluorescent biosensing method was developed for simultaneous determinationof CaMV35S and NOS in genetically modified(GM) plants. Two designed hairpin DNA (H1, H2) sequences were used as templates to synthesize H1-AgNCs (λex = 570nm, λem = 625nm) and H2-AgNCs (λex = 470nm, λem = 555nm). By using H1-AgNCs and H2-AgNCs as dual-signal tags, combined with signal amplification strategy of magnetic separation to reduce background signal and an enzyme-free catalytic hairpin assembly (CHA) signal amplification strategy, a novel multi-target fluorescent biosensor was fabricated to detect multiple targets based on FRET between signal tags (donors) and magnetic Fe3O4 modified graphene oxide (Fe3O4@GO, acceptors). In the presence of the target NOS and CaMV35S, the hairpin structures of H1 and H2 can be opened respectively, and the exposed sequences will hybridize with the G-rich hairpin sequences HP1 and HP2 respectively, displacing the target sequences to participate in the next round of CHA cycle. Meanwhile, H1-HP1 and H2-HP2 double-stranded DNA sequences (dsDNA) were formed, resulting in the desorption of dsDNA from the surface of Fe3O4@GO due to weak π-π interaction between dsDNA and Fe3O4@GO and leading to the fluorescence recovery of AgNCs. Under optimal conditions, the linear ranges of this fluorescence sensor were 5 ~ 300nmol L-1 for NOS and 5 ~ 200nmol L-1 CaMV35S, and the LODs were 0.14nmol L-1 and 0.18nmol L-1, respectively. In addition, the fluorescence sensor has good selectivity for the detection of NOS and CaMV35S in GM soybean samples, showing the potential applications in GM screening.

Ultrasensitive miRNA-let-7a detection based on AgInS2 quantum dots co-sensitization and CRISPR/Cas12a induced strand displacement reaction for signal amplification

The evolution of breast cancer is usually accompanied by abnormal expression of miRNA-let-7a. Sensitive gauging of miRNA-let-7a is crucial for breast cancer monitoring and prognosis. Herein, a novel photoelectrochemical (PEC) biosensor was served as miRNA-let-7a detection on the basis of the coupling of co-sensitization and CRISPR/Cas12a-mediated strand displacement amplification (SDA). In short, AgInS2 quantum dots (QDs), characterized by a wide visible light absorption range and a stable photocurrent response, have an obvious sensitizing effect on TiO2/Bi2MoO6. Under the condition of without miRNA-let-7a, the AgInS2 fixed DNA probe was located near the surface of the TiO2/Bi2MoO6 electrode, forming a co-sensitive structure. During the detection process, hairpin DNA2 (HP2) specifically recognized miRNA-let-7a through base complementary pairing, triggering the SDA reaction to convert massive double-stranded DNA (dsDNA). The dsDNA product then triggered CRISPR/Cas12a lateral cleavage activity, causing AgInS2-sensitized DNA to leave the electrode surface and the co-sensitization effect to disappear, further reducing the photocurrent. Using a dual effect for signal amplification, the biosensor platform had linear detection capability within the scope of 0.01–10 nM, and the detection limit was 3.35 pM. The biosensor which was fabricated effectively provided an excellent platform for the effective monitoring of various tumor biomarkers in clinical detection.