"RESET" Effect: Random Extending Sequences Enhance the Trans-Cleavage Activity of CRISPR/Cas12a.

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing applications. However, the lack of exploration on the fundamental properties of CRISPR/Cas12a not only discourages further in-depth studies of the CRISPR/Cas12a system but also limits the design space of CRISPR/Cas12a-based applications. Herein, a "RESET" effect (random extending sequences enhance trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity.

[Regeneration characteristics of three natural forests in the Three-River Headwater Region of Qinghai Province, China].

The aims of this study were to clarify the regeneration characteristics and dominant factors affecting the regeneration of three natural forests in the Three-River Headwater Region of Qinghai Province, and thus to provide a reference for the protection and management of natural forests. We evaluated the natural regeneration levels of forests, and the effects of stand factors and soil factors on natural regeneration. The results showed that three natural forests were poorly regenerated, with insufficient regeneration potential.

Signal amplification and output of CRISPR/Cas-based biosensing systems: A review.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) proteins are powerful gene-editing tools because of their ability to accurately recognize and manipulate nucleic acids. Besides gene-editing function, they also show great promise in biosensing applications due to the superiority of easy design and precise targeting. To improve the performance of CRISPR/Cas-based biosensing systems, various nucleic acid-based signal amplification techniques are elaborately incorporated.

DNA nanostructure-based nucleic acid probes: construction and biological applications.

In recent years, DNA has been widely noted as a kind of material that can be used to construct building blocks for biosensing, imaging, drug development, and disease therapy because of its advantages of good biocompatibility and programmable properties. However, traditional DNA-based sensing processes are mostly achieved by random diffusion of free DNA probes, which were restricted by limited dynamics and relatively low efficiency. Moreover, in the application of biosystems, single-stranded DNA probes face challenges such as being difficult to internalize into cells and being easily decomposed in the cellular microenvironment.

DNA nanolantern-based split aptamer probes for ATP imaging in living cells and lighting up mitochondria.

Accurate and specific analysis of adenosine triphosphate (ATP) expression levels in living cells can provide valuable information for understanding cell metabolism, physiological activities and pathologic mechanisms. Herein, DNA nanolantern-based split aptamer nanoprobes are prepared and demonstrated to work well for in situ analysis of ATP expression in living cells. The nanoprobes, which carry multiple split aptamer units on the surface, are easily and inexpensively prepared by a "one-pot" assembly reaction of four short oligonucleotide strands.

Terminal deoxynucleotidyl transferase combined CRISPR-Cas12a amplification strategy for ultrasensitive detection of uracil-DNA glycosylase with zero background.

A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design.

CRISPR/Cas12a-based dual amplified biosensing system for sensitive and rapid detection of polynucleotide kinase/phosphatase.

We reported a CRISPR/Cas-based dual amplified sensing strategy for rapid, sensitive and selective detection of polynucleotide kinase/phosphatase (PNKP), a DNA damage repair-related biological enzyme. In this strategy, a PNKP-triggered nicking enzyme-mediated strand displacement amplification reaction was introduced to enrich the activator DNA strands for CRISPR/Cas. Such an isothermal DNA amplification step, together with subsequent activated CRISPR/Cas-catalyzed cleavage of fluorescent-labeled short-stranded DNA probes, enable synergetic signal amplification for sensitive PNKP detection.

Isothermal cross-boosting extension-nicking reaction mediated exponential signal amplification for ultrasensitive detection of polynucleotide kinase.

A novel nucleic acid-based isothermal signal amplification strategy, named cross-boosting extension-nicking reaction (CBENR) is developed and successfully used for rapid and ultrasensitive detection of polynucleotide kinase (PNK) activity. Only two simple oligonucleotides (recognition substrate (RS) and TaqMan probe) are applied to construct the PNK-sensing platform. In the presence of PNK, the 3'-phosphate end of RS will be converted to the 3'-hydroxyl one, and then extended to a long poly-adenine (poly-A) sequence under the catalysis of terminal deoxynucleotidyl transferase (TdT).

Terminal deoxynucleotidyl transferase-activated nicking enzyme amplification reaction for specific and sensitive detection of DNA methyltransferase and polynucleotide kinase.

DNA methyltransferase (MTase) and polynucleotide kinase (PNK) are both DNA-dependent enzymes that play important roles in DNA methylation and DNA repair processes, respectively. Dysregulation of their activities is associated with various human diseases. Herein, we present a specific and sensitive biosensing strategy, named terminal deoxynucleotidyl transferase (TdT)-activated nicking enzyme amplification reaction (TdT-NEAR), for their activity detection.

A modified exponential amplification reaction (EXPAR) with an improved signal-to-noise ratio for ultrasensitive detection of polynucleotide kinase.

We applied a modified exponential amplification reaction (EXPAR) strategy to design a label-free and "one-pot" biosensor for ultrasensitive detection of polynucleotide kinase (PNK). This method was also successfully applied in the screening of PNK inhibitors and analysis of the endogenous PNK activity at the single-cell level.

Label-Free Telomerase Detection in Single Cell Using a Five-Base Telomerase Product-Triggered Exponential Rolling Circle Amplification Strategy.

Telomerase is a universal biomarker of malignant tumors. Sensitive and reliable analysis for telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, a novel fluorescent strategy was proposed for sensitive and label-free detection of telomerase activity.

Terminal Deoxynucleotidyl Transferase-Catalyzed Preparation of pH-Responsive DNA Nanocarriers for Tumor-Targeted Drug Delivery and Therapy.

Developing a highly efficient carrier for tumor-targeted delivery and site-specific release of anticancer drugs is a good way to overcome the side effects of traditional cancer chemotherapy. Benefiting from the nontoxic and biocompatible characteristics, DNA-based drug carriers have attracted increasing attention. Herein, we reported a novel and readily manipulated strategy to construct spherical DNA nanocarriers.

Dual enzyme-assisted one-step isothermal real-time amplification assay for ultrasensitive detection of polynucleotide kinase activity.

A novel, simple, one-step and one-tube detection method was developed for ultrasensitive detection of polynucleotide kinase (PNK) activity on the basis of dual enzyme-synergistic signal amplification. This method was also demonstrated to work well for PNK inhibitor screening and endogenous PNK detection in cell lysates at a single-cell level.

Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase.

As one of the key initiators of the base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis.

Amplified detection of genome-containing biological targets using terminal deoxynucleotidyl transferase-assisted rolling circle amplification.

We proposed a terminal deoxynucleotidyl transferase (TdT)-assisted rolling circle amplification (RCA) strategy for the amplified detection of genome-containing biological targets. The synergetic signal amplification of DNase I-mediated genomic DNA fragmentation and subsequent RCA allow the sensing platform to have extraordinarily high sensitivity.

Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA.

An exonucleolytic digestion-assisted exponential rolling circle amplification (RCA) strategy was developed for sensitive and sequence-specific detection of target DNA embedded in long-stranded genomic DNA. Herein, Phi29 DNA polymerase plays two important roles as exonuclease and polymerase. Long-stranded genomic DNAs can be broken into small DNA fragments after ultrasonication.

Sensitive fluorescent detection of DNA methyltransferase using nicking endonuclease-mediated multiple primers-like rolling circle amplification.

Sensitive and reliable detection of DNA methyltransferase (MTase) is of great significance for both early tumor diagnosis and therapy. In this study, a simple, label-free and sensitive DNA MTase-sensing method was developed on the basis of a nicking endonuclease-mediated multiple primers-like rolling circle amplification (RCA) strategy. In this method, a dumbbell RCA template was prepared by blunt-end ligation of two molecules of hairpin DNA.

Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification.

Optimization of strand displacement amplification-sensitized G-quadruplex DNAzyme-based sensing system and its application in activity detection of uracil-DNA glycosylase.

As an isothermal nucleic acid amplification technique, strand displacement amplification (SDA) reaction has been introduced in G-quadruplex DNAzyme-based sensing system to improve the sensing performance. To further provide useful information for the design of SDA-amplified G-quadruplex DNAzyme-based sensors, the effects of nicking site number in SDA template DNA were investigated. With the increase of the nicking site number from 1 to 2, enrichment of G-quadruplex DNAzyme by SDA is changed from a linear amplification to an exponential amplification, thus greatly increasing the amplification efficiency and subsequently improving the sensing performance of corresponding sensing system.