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

Abstract

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.

Scheme 1. Schematic of AMC based on nicking endonuclease Nt.BstNBI. (a) Recognition sequence and cleavage site of Nt.BstNBI. (b) Basic structure of the proposed DNA aligner and working principle of AMC. Nt.BstNBI firstly binds to the recognition sequence in the stem of DNA aligner (Loading), and is aligned with a specific locus through the hybridization of the two side arms with target DNA (Localization), then cleaves at that site (Reaction). (c) Programmable, sequence-specific cleavage of DNA via AMC. By simply varying the sequences of the aligner's side arms, a break can be made at any locus of target DNA.

Fig. 1. AMC makes a break exclusively on target DNA. (a) Representative sequence/structure of DNA aligner (Left). The denaturing PAGE image shows that only the DNA aligner with one extra base pair beyond the recognition site can induce cleavage (Right). (b) Schematics of DNA complexes formed by fluorescently labelled target DNA and aligners (Top). The denaturing PAGE image shows that aligner-mediated cleavage exclusively occurs on target DNA (Red triangle), rather than on DNA aligner (Black triangle) (Bottom). (c) Schematics of DNA complexes formed by dual-labelled (Dabcyl and FAM) aligner or target sequence (Top). Real-time fluorescence upon adding Nt.BstNBI at 55 °C also shows a break of target sequence but not DNA aligner (Bottom).

Fig. 2. Programmable, sequence-specific cleavage of DNA via AMC. (a) Schematic (Top) and denaturing PAGE image (Bottom) showing that a target strand was cut at different sites by simply varying the sequences of the DNA aligner's side arms. (b) Fluorescence study showing that the cleavage site can be tuned down to one nucleotide. The schematic (Top) displays the labelling sites of FAM and Dabcyl, as well as the positions where DNA aligners bind. In a crossover trial, temporal fluorescence changes upon adding Nt.BstNBI at 55 °C were recorded (Middle), and then the patterns of fluorescence increase for each DNA aligner were summarized (Bottom). (c) Agarose gel image showing that a plasmid was cut via AMC. SC, OC and Lin represent super-coiled, open circular and linearized plasmid, respectively.

Scheme 2. Schematic illustration of AMC-SDA. (Step 1) Target DNA, or its antisense sequence, hybridizes with DNA aligner and is cleaved by Nt.BstNBI through AMC. (Step 2) The cleaved sequence with a definite 3′-end binds a linear primer, followed by polymerase-catalyzed extension along this linear primer to generate a complete double-stranded recognition site. (Step 3) Nt.BstNBI binds to the newly formed recognition site and makes a nick four bases downstream. (Step 4) Polymerase catalyzes the extension from the nicking site, displacing the previous strand. Step 3 and Step 4 can be repeated many times.

Fig. 3. Feasibility, sensitivity and specificity of AMC-SDA. (a) Real-time fluorescence and (b) PAGE image for three target DNAs (10^–11 M) of different lengths. (c) Real-time fluorescence caused by different concentrations of target DNA (TA-1). (d) Schematic showing the positions of mismatching and the sites where the cleavage/extension occurs in forward initiation. (e) Real-time fluorescence caused by matched and single-base mismatched sequences (10^–11 M).

Fig. 4. Universality of AMC-SDA. (a and b) Real-time fluorescence for various concentrations of target sequences from HIV Gag gene and HBV S gene, respectively. (c) Schematic of a target fragment in plasmid pUC57 and the cleavage sites mediated by various forward/reverse aligners. (d) Real-time fluorescence for a 56-bp fragment from no. 1240 to 1296 base of pUC57. (e) PAGE image showing the amplification products corresponding to (d) (Left) and three fragments with different length from plasmid pUC57 when using various forward aligners/primers (Right).