Aligner mediated cleavage of nucleic acids for site-specific detection of single base mismatch.

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5'-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.

Aligner-mediated cleavage of nucleic acids and its application to isothermal exponential amplification.

We herein describe a simple and versatile approach to use conventional nicking endonuclease (NEase) for programmable sequence-specific cleavage of DNA, termed aligner-mediated cleavage (AMC), and its application to DNA isothermal exponential amplification (AMC-based strand displacement amplification, AMC-SDA). AMC uses a hairpin-shaped DNA aligner (DA) that contains a recognition site in its stem and two side arms complementary to target DNA. Thus, it enables the loading of an NEase on DA's stem, localization to a specific locus through hybridization of the side arms with target DNA, and cleavage thereof. By using just one NEase, it is easy to make a break at any specific locus and tune the cleavage site to the single-nucleotide scale. This capability also endows the proposed AMC-SDA with excellent universality, since the cleavage of target DNA, followed by a polymerase-catalyzed extension along a particular primer as a key step for initiating SDA, no longer relies on any special sequence. Moreover, this manner of initiation facilitates the adoption of 3'-terminated primers, thus making AMC-SDA highly sensitive and highly specific, as well as simple primer design.

Aligner-mediated cleavage-triggered exponential amplification for sensitive detection of nucleic acids.

Exponential amplification reaction (EXPAR), as a simple and high sensitive method, holds great promise in nucleic acids detection. One major challenge in EXPAR is the generation of trigger DNA with a definite 3'-end, which now relies on fingerprinting technology. However, the requirement of different endonucleases for varying target sequences and two head-to-head recognition sites in double stranded DNA, as well as the confinement of trigger DNA's 3'-end to be near/within the recognition site, usually subject EXPAR to compromised universality and/or repeated matching of reaction conditions. Herein, we report a simple and universal method for high sensitive detection of nucleic acids, termed aligner-mediated cleavage-triggered exponential amplification (AMCEA). The aligner-mediated cleavage (AMC) needs only one nicking endonuclease and can make a break at any site of choice in a programmable way. Thus, the 3'-end of target DNA can be easily redefined as required, a key step for initiating the amplification reaction. This capability endows the proposed AMCEA with excellent universality and simplicity. Moreover, it is sensitive and specific, with a detection limit at amol level, a broad dynamic range of 5～6 orders of magnitude and the ability to distinguish single nucleotide mutation. Experiments performed with human serum indicate that AMCEA is compatible with the complex biological sample, and thus has the potentials for practical applications.

Stepwise evolution of Au micro/nanocrystals from an octahedron into a truncated ditetragonal prism.

Shape transformation of Au crystals from a {111}-bounded octahedron to a {730}-bounded tetrahexahedron (THH) and then to a {310}-bounded truncated ditetragonal prism (TDP) was achieved by a one pot synthesis method. The transformation of the crystal morphology is the result of the synergistic effect of Ag+ and O2/Cl-. This work not only provides a way to prepare high-index faceted Au micro/nanocrystals, but also provides new insights into the crystal growth habit.

A novel steric effect-regulated isothermal exponential amplification technology for the one-step homogeneous sensing of proteins.

A simple and homogeneous technology, the steric effect-regulated isothermal exponential amplification reaction (SER-EXPAR), was developed to sense proteins. By using a small molecule linked DNA nanostructure, termed enzyme-binding hairpin (EBH), the protein-small molecule binding events could be readily sensed by utilizing the steric effect generated between the protein and enzyme. It set free the enzyme to be active again, thus regulating the amplification rate of EXPAR.