Modular CRISPR/Cas12a synergistic activation platform for detection and logic operations.

The revolutionary technology of CRISPR/Cas has reshaped the landscape of molecular biology and molecular engineering. This tool is of interest to researchers in multiple fields, including molecular diagnostics, molecular biochemistry circuits, and information storage. As CRISPR/Cas spreads to more niche areas, new application scenarios and requirements emerge. Developing programmability and compatibility of CRISPR/Cas becomes a critical issue in the new phase. Here, we report a redundancy-based modular CRISPR/Cas12a synergistic activation platform (MCSAP). The position, length, and concentration of the redundancy in the split DNA activators can finely regulate the activity of Cas12a. With the redundant structure as an interface, MCSAP serves as a modular plug-in to seamlessly integrate with the upstream molecular network. MCSAP successfully performs three different tasks: nucleic acid detection, enzyme detection, and logic operation. MCSAP can work as an effector for different molecular networks because of its compatibility and programmability. Our platform provides powerful yet easy-to-use tools and strategies for the fields of DNA nanotechnology, molecular engineering, and molecular biology.

Time-Controlled Authentication Strategies for Molecular Information Transfer.

Modern cryptography based on computational complexity theory is mainly constructed with silicon-based circuits. As DNA nanotechnology penetrates the molecular domain, utilizing molecular cryptography for data access protection in the biomolecular domain becomes a unique approach to information security. However, building security devices and strategies with robust security and compatibility is still challenging. Here, this study reports a time-controlled molecular authentication strategy using DNAzyme and DNA strand displacement as the basic framework. A time limit exists for authorization and access, and this spontaneous shutdown design further protects secure access. Multiple hierarchical authentications, temporal Boolean logic authentication, and enzyme authentication strategies are constructed based on DNA networks'good compatibility and programmability. This study gives proof of concept for the detection and protection of bioinformation about single nucleotide variants and miRNA, highlighting their potential in biosensing and security protection.

Label-free colorimetric detection platform based on catalytic hairpin self-assembly and G-quadruplex/hemin DNAzyme for comprehensive biomarker profiling.

The expression level of human apurinic/apyrimidinic endonuclease 1 (APE1) is closely associated with the onset of various diseases, establishing it as a crucial clinical biomarker and a target in anti-cancer efforts. This study accomplished colorimetric and visual detection of APE1 by harnessing its endonuclease activity through catalytic hairpin self-assembly (CHA) and G-quadruplex/hemin DNAzyme. Optimization of the freedom degrees of the G-rich sequence significantly improved the detection performance of the strategy by influencing DNAzyme formation. Additionally, we replaced the signal reporting system with a molecular beacon to develop a fluorescence detection strategy, which served as an extension of the signal amplification system for validation and signal readout. The fluorescent probe method achieved a detection limit of 3.37 × 10-4 U/mL, while the colorimetric method yielded a detection limit of 6.5 × 10-3 U/mL, with a linear range spanning from 0.01 to 0.25 U/mL. Subsequently, the colorimetric approach effectively assessed APE1 activity in biological samples and facilitated the screening of APE1 activity inhibitors. Furthermore, this CHA/G-quadruplex/hemin DNAzyme strategy was adapted for the colorimetric detection of adenosine, showcasing its broad applicability across various biomarkers. The developed colorimetric analytical strategy represents a pivotal biosensing platform for diagnosing and treating diseases.

Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation.

G-quadruplex (G4) selective stabilizing ligands can regulate c-MYC gene expression, but the kinetic basis remains unclear. Determining the effects of ligands on c-MYC promoter G4s' folding/unfolding kinetics is challenging due to the polymorphic nature of G4s and the high energy barrier to unfold c-MYC promoter G4s. Here, we used single-molecule magnetic tweezers to manipulate a duplex hairpin containing a c-MYC promoter sequence to mimic the transiently denatured duplex during transcription. We measured the effects of six commonly used G4s binding ligands on the competition between quadruplex and duplex structures, as well as the folding/unfolding kinetics of G4s. Our results revealed two distinct roles for G4s selective stabilization: CX-5461 is mainly acting as c-MYC G4s stabilizer, reducing the unfolding rate (ku) of c-MYC G4s, whereas PDS and 360A also act as G4s chaperone, accelerating the folding rates (kf) of c-MYC G4s. qRT-PCR results obtained from CA46 and Raji cell lines demonstrated that G4s stabilizing ligands can downregulate c-MYC expression, while G4s stabilizer CX-5461 exhibited the strongest c-MYC gene suppression. These results shed light on the potential of manipulating G4s' folding/unfolding kinetics by ligands for precise regulation of promoter G4-associated biological activities.

DNAzyme-Based Dissipative DNA Strand Displacement for Constructing Temporal Logic Gates.

Toehold-mediated DNA strand displacement is the foundation of dynamic DNA nanotechnology, encompassing a wide range of tools with diverse functions, dynamics, and thermodynamic properties. However, a majority of these tools are limited to unidirectional reactions driven by thermodynamics. In response to the growing field of dissipative DNA nanotechnology, we present an approach: DNAzyme-based dissipative DNA strand displacement (D-DSD), which combines the principles of dynamic DNA nanotechnology and dissipative DNA nanotechnology. D-DSD introduces circular and dissipative characteristics, distinguishing it from the unidirectional reactions observed in conventional strand displacement. We investigated the reaction mechanism of D-DSD and devised temporal control elements. By substituting temporal components, we designed two distinct temporal AND gates using fewer than 10 strands, eliminating the need for complex network designs. In contrast to previous temporal logic gates, our temporal storage is not through dynamics control or cross-inhibition but through autoregressive storage, a more modular and scalable approach to memory storage. D-DSD preserves the fundamental structure of toehold-mediated strand displacement, while offering enhanced simplicity and versatility.

Gene point mutation information translation and detection: Leveraging single base extension and CRISPR/Cas12a.

Gene point mutations play a significant role in the development of cancer. Therefore, developing a sensitive, specific, and universally applicable method for detecting gene point mutation is crucial for clinical diagnosis, prognosis, and cancer treatment. Recently, gene point mutation detection methods based on CRISPR/Cas12a detection have emerged. However, existing methods generally lack universality and specificity. In this study, we have developed a CRISPR/Cas12a-based method that combines improved allele-specific polymerase chain reaction and single base extension to translate the point mutation information in the target dsDNA into length information in ssDNA activators to overcome the limitations associated with PAM sequences in the CRISPR/Cas12a system. Our method achieved a detection limit of 0.002% for clinically significant EGFR T790M mutation. The CRISPR/Cas12a system we constructed demonstrates high sensitivity, specificity, and universality in detecting gene point mutations, making it a promising tool for clinical cancer screening.

Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity.

The trans-cleavage activity of Cas12a has been widely used with various applications. Here, we report that the trans-cleavage activity of Cas12a can be significantly affected by the fluorescent probe length and reaction buffer. The optimal probe length for Cas12a is found to be 15 nucleotides, and the optimal buffer is NEBuffer 4. Compared to the popularly used reaction conditions, the activity of Cas12a is improved by about 50-fold. In addition, the detection limit of Cas12a for DNA targets has been reduced by nearly three orders of magnitude. Our method provides a powerful tool for Cas12a trans-cleavage activity applications.

Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences.

The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.

A multifunctional monolithic interfacial sensor based on gold nanoparticle.

The spatial and temporal uneven distribution of complex biochemical reactions creates the diversity of biological systems. And the microenvironment confers fine regulation of these reactions, a stunning example of which is liquid-liquid phase separation (LLPS). LLPS can form a separate compartment without the physical separation formed by conventional membrane structures, and the reactions within the interface have specific reaction dynamics. Inspired by this, we report an interfacial sensor based on gold nanoparticles showing that interfacial factors have similar properties operating in natural biological environments and sensors. It repels molecules outside the interface and adjusts the DNA conformation within the interface to produce unique dynamics. The sensor adopts a modular design, allowing functional modules assembled on a single nanoparticle to avoid complex designs. We demonstrate the functionality of logical operations, using apurinic/apyrimidinic endonuclease 1 and micro RNA as inputs, showing that the sensor has the ability and potential to become a multifunctional platform with clear interface nature.

Allosteric Nucleic Acid Enzyme: A Versatile Stimuli-Responsive Tool for Molecular Computing and Biosensing Nanodevices.

Allostery is a naturally occurring mechanism in which effector binding induces the modulation and fine control of a related biomolecule function. Deoxyribozyme (DNAzyme) with catalytic activity and substrate recognition ability is ideal to be regulated by allosteric strategies. However, the current regulations frequently confront various obstacles, such as severe activity decay, signal leakage, and limited effectors. In this work, a rational regulation strategy for developing versatile effectors-responsive allosteric nucleic acid enzyme (ANAzyme) by introducing an allosteric domain in response to diverse effectors is established. The enzyme-like activity of this re-engineered ANAzyme can be modulated in a more predictable and fine way compared with the previous DNAzyme regulation strategies. Based on the allosteric strategy, the construction of allosterically coregulatory nanodevices and a series of basic logic gates and logic circuits are achieved, demonstrating that the proposed ANAzyme-regulated strategy showed great potential in molecular computing. Given these facts, the rational design of ANAzyme with the allosteric domain presented here can expand the available toolbox to develop a variety of stimuli-responsive allosteric DNA materials, including molecular machines, computing systems, biosensing platforms, and gene-silencing tools.

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease IV-assisted exponential signal amplification.

Alkaline phosphatase (ALP) is widely expressed in human tissues. ALP plays an important role in the dephosphorylation of proteins and nucleic acids. Therefore, quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods. Terminal deoxynucleotidyl transferase (TdT) catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template. In this study, we developed a highly sensitive and selective method based on TdT and endonuclease IV (Endo IV) to quantify ALP activity. After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH, TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate (dATP) and produce a poly A tail, which can be detected by the poly T probes. Endo IV digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end, leading to the generation of exponential fluorescence signal amplification. The substrate for TdT elongation was optimized, and a limit of detection of 4.3 × 10-3 U/L was achieved for ALP by the optimized substrate structure. This method can also detect ALP in the cell lysate of a single cell. This work has potential applications in disease diagnosis and biomedical detection.

Sensitive detection of abasic sites in double-stranded DNA based on the selective reaction of enzymes.

The apurinic/apyrimidinic (AP) site is one of the most common DNA lesions and a critical intermediate during the base excision repair pathway. Therefore, AP sites are essential in clinical diagnosis, treatment and detection. However, the existing detection methods are complicated in design and synthesis and have high instrument requirements, limiting their wide application. Therefore, there is an urgent need for a sensitive and straightforward detection method without time-consuming and heterogeneous reactions. Herein, we developed two compatible detection methods for AP sites in long and short dsDNA. For long and short dsDNA, the background signal was successfully suppressed by the affinity difference of Terminal deoxynucleotidyl transferase (TdT) and 3' -end blocking, respectively, thus achieving high detectability and specificity. The detection limit was 13 pM in 20 µL, meaning that the LOD was 0.26 fmol for AP site amount and 0.05% for AP site abundance. The method has been successfully applied to detect AP sites in various biological samples quickly. Therefore, it has broad clinical application prospects, catering for the need for a point of care.

DNA circuits compatible encoder and demultiplexer based on a single biomolecular platform with DNA strands as outputs.

A series of multiple logic circuits based on a single biomolecular platform is constructed to perform nonarithmetic and arithmetic functions, including 4-to-2 encoder, 1-to-2 demultiplexer, 1-to-4 demultiplexer, and multi-input OR gate. The encoder to a DNA circuit is the equivalent of a sensory receptor to a reflex arc. They all function to encode information from outside the pathway (DNA circuit or reflex arc) into a form that subsequent pathways can recognize and utilize. Current molecular encoders are based on optical or electrical signals as outputs, while DNA circuits are based on DNA strands as transmission signals. The output of existing encoders cannot be recognized by subsequent DNA circuits. It is the first time the DNA-based encoder with DNA strands as outputs can be truly applied to the DNA circuit, enabling the application of DNA circuits in non-binary biological environments. Another novel feature of the designed system is that the developed nanodevices all have a simple structure, low leakage and low crosstalk, which allows them to implement higher-level encoders and demultiplexers easily. Our work is based on the idea of complex functionality in a simple form, which will also provide a new route for developing advanced molecular logic circuits.

A structure change-induced fluorescent biosensor for uracil-DNA glycosylase activity detection based on the substrate preference of Lambda exonuclease.

As one of the initiating DNA glycosylases in the base excision repair pathway, Uracil-DNA glycosylase (UDG) plays a pivotal role in maintaining genomic integrity. The abnormal expression of UDG in the organism is highly relevant to multiple diseases. Thus, rapid and sensitive detection of UDG activity is essential to aid early clinical diagnosis and biomedical research. Here we developed a rapid, sensitive and selective biosensor for UDG activity detection based on the substrate preference of Lambda exonuclease (λ exo). The protruding end in the substrate produced by UDG could be digested at a markedly high rate by λ exo, generating a detectable fluorescence signal. This proposed strategy for UDG detection exhibited high selectivity and high sensitivity (0.0001 U/mL) in a short time. It has also been successfully applied to detect UDG in real biological samples and the screening of UDG inhibitors.

Evaluation of Sperm DNA Integrity by Mean Number of Sperm DNA Breaks Rather Than Sperm DNA Fragmentation Index.

BACKGROUND: Sperm DNA integrity is crucial for normal fertilization, implantation, and embryo development. Several assays are available to assess sperm DNA fragmentation but are limited by high price, complicated processes, and low accuracy. METHODS: We developed a secondary amplification detection system based on terminal deoxynucleotidyl transferase and endonuclease IV, which could efficiently measure the number of 3'-OH (equivalent to the number of breakpoints). We applied this detection system in single stranded DNA with standard concentrations to obtain the standard curve. We then broke the double stranded genomic DNA by ultrasound and enzyme digestion and used the detection system to monitor the increase of DNA breakpoints. Finally, we used this method to measure the mean number of sperm DNA breakpoints (MDB) in 80 sperm samples. RESULTS: We successfully measured the number of 3'-OH in single stranded DNA with standard concentration and obtained the standard curve. The linear range for the number of DNA breakpoints was from 0.1 nM to 15 nM. The detection method was successfully validated on λ DNA and 80 human sperm samples. The results of real clinical samples revealed that the mean number of DNA breakpoints (MDB) had a stronger relevance with the sperm motility and clinical pregnancy outcomes than the commonly used parameter of DNA fragmentation index (DFI). CONCLUSION: We have developed a straight-forward method for direct measurement of the mean number of DNA breakpoints in sperms. The method has advantages of short time-consumption, simple operation, high analytical sensitivity, and low requirement for instrumentation, which makes it conducive to clinical application. The proposed new parameter (MDB) could be a more direct, accurate and clinically significant indicator for evaluating the sperm DNA integrity.

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease Ⅳ-assisted exponential signal amplification / 药物分析学报

Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH,TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10-3 U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.

Endonuclease IV-Regulated DNAzyme Motor for Universal Single-nucleotide Variation Discrimination.

Single-nucleotide variation (SNV) detection plays significant roles in disease diagnosis and treatment. Generally, auxiliary probe, restricted design rules, complicated detection system, and repeated experimental parameter optimization are needed to obtain satisfactory tradeoff between sensitivity and selectivity for SNV discrimination, especially when different mutant sites need to be distinguished. To overcome these limitations, we developed a universal, straightforward, and relatively cheap SNV discrimination strategy, which simultaneously possessed high sensitivity and selectivity. The excellent performance of this strategy was ascribed to the SNV discrimination property of endonuclease IV (Endo IV) and the different hydrolysis behavior between free deoxyribozyme (DNAzyme) and the trapped DNAzyme to the substrates modified on gold nanoparticles (AuNPs). When Endo IV recognized the mutant-type target (MT), free DNAzyme was released from the probe, and the DNAzyme motor was activated with the help of cofactor Mn2+ to generate an amplified fluorescence signal. On the contrary, the wild-type target (WT) could not effectively trigger the DNAzyme motor. Moreover, for different SNV types, the corresponding probe could be designed by simply changing the sequence hybridized with the target and retaining the DNAzyme sequence. Thus, the fluorescence signal generation system does not need to change for different SNV targets. Five clinical-related SNVs were determined with the limit of detection (LOD) ranging from 0.01 to 0.05%, which exhibited competitive sensitivity over existing SNV detection methods. This strategy provided another insight into the properties of Endo IV and DNAzyme, expanded the applications of DNAzyme motor, and has great potential to be used for precision medicine.

Development of a background signal suppression probe for 8-oxoguanine DNA glycosylase detection.

8-oxoguanine DNA glycosylase (OGG), which plays a crucial role in base excision repair (BER), is an important biomarker. The existing highly sensitive fluorescent methods always need complicated amplification design. The method with high sensitivity and simple design at the same time is urgently needed. Here, we developed a highly sensitive detection method for OGG detection with lambda exonuclease and the background signal suppression probe. Through probe structure design, the steric hindrance and competitive binding effects successfully suppressed the background signal. We achieved sensitive detection of OGG with a simple design, and the limit of detection was 5.0 × 10-4 U mL-1. Moreover, the method was highly selective and successfully applied to OGG detection in biological samples, which shows the potential clinical application value.

Programmable One-Pot Enzymatic Reaction for Direct Fluorescence Detection of Ultralow-Abundance Mutations in the DNA Duplex.

Sensitive detection of low-abundance driver mutations may provide valuable information for precise clinical treatment. Compared to next-generation sequencing and droplet digital PCR methods, fluorescent probes show great flexibility in rapid detection of specific mutations with high sensitivity and easily accessible instruments. However, existing approaches with fluorescent probes need an additional step to convert duplex DNA to single-stranded DNA (ssDNA) before the detection step, which increases the time, cost, and risk of loss of low-input target strands. In this work, we attempt to integrate the ssDNA-generation step with the subsequent detection into a programable one-pot reaction by employing lambda exonuclease (λ exo), a versatile nanopore nuclease which exercises different functions on different substrates. The capability of λ exo in discrimination of mismatched bases in 5'- FAM-ended 2 nt-unpaired DNA duplexes was first demonstrated. Specific fluorescent probes were developed for EGFR exon 19 E746-A750del and PIK3CA E545K mutations with discrimination factors as high as 8470 and 884, respectively. By mixing the probes and λ exo with the PCR products of cell-free circulating DNA extracted from plasma samples, the reaction was immediately initiated, which allowed sensitive detection of the two types of mutations at an abundance as low as 0.01% within less than 2 h. Compared to existing approaches, the new method has distinct advantages in simplicity, low cost, and rapidity. It provides a convenient tool for companion diagnostic tests and other routine analysis targeting genetic mutations in clinical samples.

Research on the mechanisms of polyacrylamide nanospheres with different size distributions in enhanced oil recovery.

Crosslinked polyacrylamide microspheres are widely used as in-depth flooding agents in petroleum development due to their unique properties of thickening, salt-resistance, high-temperature resistance, low cost, etc. To solve the problem of their injections in heterogeneous reservoirs, polyacrylamide nanospheres were synthesized. However, mechanisms of polymer nanospheres in enhanced oil recovery were not investigated comprehensively. In this study, we synthesized polymer nanospheres with different size distributions and studied their mechanisms in enhancing the oil recovery. First, the effects of polyacrylamide nanospheres in enhanced oil recovery of heterogeneous sand-packed tubes was explored by sand-packed tube oil displacement experiments. Second, the rheological properties of polyacrylamide nanosphere dispersion were explored using a rheometer. Third, through the visual microchannel experiment, the mechanism of polymer nanosphere emulsion on the removal of the residual oil film was explored. Finally, through the crude oil removal experiment, it was found that polymer nanospheres with a particle size of about 54 nm can cooperate with surfactants to accelerate the removal of oil droplets.