Application of phi29 DNAP in dNaM-dTPT3 rolling circle amplification

Abstract

The invention discloses an application of phi29DNAP (deoxyribonucleic acid) in rolling circle amplification of dNaM-dTPT3 (deoxyribonucleic acid / deoxyribonucleic acid). According to the invention, the amplification capability of phi29DNAP on a representative non-natural base pair dNaM-dTPT3 and an analogue thereof is found, and two rolling circle amplification methods of ExRCA and ExMRCA are constructed. The ExRCA and ExMRCA methods provided by the invention can be used for preparing DNA nano-molecules with functional markers, preparing products for detecting human alpha-thrombin, carrying out enzyme immobilization, preparing cell marking and imaging products, preparing glucose detection products and preparing hydrogel with transcriptional activity. Wide application prospects are shown in the fields of expanding genetic alphabets, constructing function-enhanced oligonucleotides, DNA catalysts, drugs, sensors, materials and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to application of phi29 DNAP in dNaM-dTPT3 rolling circle amplification. BACKGROUND

[0002] DNA plays a central role in storing and transmitting genetic information in nature, and has been widely used in modern biotechnology and nanotechnology. The function and application of DNA are highly dependent on the specific base pairing between nucleotides. In the past few decades, a series of unnatural base pairs (UBPs) have been developed to expand the genetic alphabet, some of which have shown efficiencies similar to that of natural base pairs replication. The pairing of these UBPs is based on hydrogen bonds, such as dZ-dP and dS-dB developed by the Benner team; or based on hydrophobicity and packing force, such as dDs-dPx and dDs-dPa developed by the Hirao team, and dNaM-d5SICS and dNaM-dTPT3 developed by the Romesberg team. These UBPs have shown their value in a variety of applications, including the preparation of high-performance aptamers, site-specific labeling of nucleic acids, and the production of proteins containing unnatural amino acids. It is worth mentioning that, with dNaM-dTPT3 and its analogues, semi-synthetic organisms (SSOs) capable of storing genetic information have been successfully constructed.

[0003] High-efficiency amplification of DNA containing UBPs is essential for many applications of UBPs, and DNA amplification containing dNaM-dTPT3 has been verified in polymerase chain reaction (PCR). However, isothermal amplification of DNA containing dNaM-dTPT3, including the widely used rolling circle amplification (RCA), has not been explored. The amplification efficiency of RCA is significantly affected by the compatibility of polymerase with UBPs, and phi29 DNA polymerase (phi29 DNAP) with high elongation, strand displacement ability and strong proofreading activity is a commonly used enzyme in RCA, which is widely used in single molecule sequencing, in situ detection and whole genome amplification, etc. However, the replication ability of phi29 DNAP on dNaM-dTPT3 and other extended base pairs (UBPs) has not been determined.

[0004] RCA can generate tandem repeats of target DNA from a circular template under isothermal conditions. With proper design, RCA products can be embedded with functional modules such as aptamer, G-quadruplex, ribozyme, probe, etc. and are widely used in biological detection, imaging and therapy. UBPs have good orthogonality with natural bases. If introduced into RCA system, the complexity and functional programmability of product sequences can be improved without interfering with the sequence function, which can expand its application potential.

[0005] In terms of DNA material construction, RCA can also be used to prepare programmable nanostructures and DNA hydrogels. In recent years, through the introduction of modified nucleotides and enzymatic amplification methods, RCA products can be further functionalized, such as coupling of fluorophores, cholesterols or enzyme molecules, etc. However, site-specific molecular coupling of DNA materials is still a challenge. The introduction of UBP with a functional connecting arm provides a new strategy for precise modification.

[0006] The present invention systematically studies the replication ability of phi29 DNAP on dNaM-dTPT3 and its analogues, establishes and optimizes an expanded genetic alphabet-based rolling circle amplification method (ExRCA), and develops a circular template preparation strategy suitable for this system. This method combines with restriction enzymes to achieve efficient preparation of single-stranded and double-stranded DNA containing UBP. The products have been successfully used in DNA tetrahedral site labeling, high-sensitivity detection of biomolecules, multivalent aptamer construction, and enzyme function regulation, etc. At the same time, the present invention also develops a multiply-primed rolling circle amplification method with an expanded genetic alphabet (ExMRCA) for constructing cross-linked six-base DNA hydrogels with specific site coupling ability and demonstrates its application prospects in the field of RNA synthesis. SUMMARY

[0007] The first object of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide the application of phi29 DNAP in the rolling circle amplification of DNA containing dNaM-dTPT3.

[0008] The second object of the present invention is to provide an expanded genetic alphabet-based rolling circle amplification method (ExRCA) and its application.

[0009] The third object of the present invention is to provide a multiply-primed rolling circle amplification method (ExMRCA) and its application.

[0010] The application achieves the above-mentioned purpose by means of the following technical solutions.

[0011] Application of phi29 DNAP in rolling circle amplification of DNA containing dNaM-dTPT3; the rolling circle amplification includes extended genetic alphabet-based rolling circle amplification (ExRCA) and multi-primer extended rolling circle amplification (ExMRCA).

[0012] An extended genetic alphabet-based rolling circle amplification method (ExRCA) comprises the following steps:

[0013] dATP, dTTP, dGTP, dCTP, BSA, phi29 DNAP, deoxyribonucleotide triphosphates containing unnatural bases and a circular template-primer complex containing dNaM are added to 1x phi29 DNAP reaction buffer, mixed and incubated.

[0014] Further, the 1x phi29 DNAP reaction buffer comprises 50mM Tris-HCl, 10mM (NH4)2SO4, 4mM DTT and 10mM MgCl2, and the pH of the 1x phi29 DNAP reaction buffer is 7.4.

[0015] Further, the concentration of dATP, dTTP, dGTP and dCTP in the mixture is 1mM, the concentration of BSA in the mixture is 0.4mg / mL, the concentration of phi29 DNAP in the mixture is 1μM, the concentration of deoxyribonucleotide triphosphates containing unnatural bases in the mixture is 0.1mM, and the concentration of the circular template-primer complex containing dNaM in the mixture is 75nM.

[0016] Further, the deoxyribonucleotide triphosphates containing unnatural bases are dTPT3TP, dTPT3 PA TP, dTPT3 Am TP or dTPT3 Al TP.

[0017] Further, the preparation of the circular template-primer complex containing dNaM comprises the following steps:

[0018] A. 5'-phosphorylated dNaM-containing oligonucleotides are mixed with a clamp primer DNA oligonucleotide to circularize the phosphorylated product, and T4 DNA ligase is added to connect the two ends of the circularized sequence together; or

[0019] B. mixing the 5'-terminal phosphorylated dNaM-containing oligonucleotide, the 5'-terminal phosphorylated oligonucleotide containing the reverse complementary sequence of the functional sequence, and the two clamp primers DNA oligonucleotides, combining the two ends of the dNaM-containing oligonucleotide and the oligonucleotide containing the reverse complementary sequence of the functional sequence with the assistance of the two clamp primers, adding T4 DNA ligase to connect the dNaM-containing oligonucleotide and the oligonucleotide containing the reverse complementary sequence of the functional sequence at the two places of the two clamp primers; or

[0020] C. mixing the 5'-phosphorylated natural template oligonucleotide and the clamp primer, circularizing the natural template oligonucleotide, adding dNaMTP and the Klenow fragment of E. coli DNA polymerase I to integrate the dNaMTP into the 3'-end of the natural template oligonucleotide, and adding T4 DNA ligase to connect the two ends of the circularized sequence together;

[0021] Further, the 5'-terminal phosphorylation is achieved by the following steps:

[0022] adding the oligonucleotide, ATP and T4 polynucleotide kinase in the T4 polynucleotide kinase reaction buffer, mixing, and incubating at 37℃ for 1h to phosphorylate the 5'-end of the oligonucleotide;

[0023] Still further, the concentration of the oligonucleotide in the mixture is 0.6μM, the concentration of ATP in the mixture is 1mM, and the concentration of T4 polynucleotide kinase in the mixture is 0.2U / μL.

[0024] Further, the incubation is incubation at 30℃ for 20h.

[0025] The application of the above-mentioned rolling circle amplification method based on an extended genetic alphabet, the application comprising the following applications:

[0026] (1) application in the preparation of DNA nanomolecules with functionalized labels;

[0027] (2) application in the preparation of detection of human alpha-thrombin products;

[0028] (3) application in enzyme immobilization;

[0029] (4) application in the preparation of cell labeling and imaging products;

[0030] (5) application in the preparation of glucose detection products.

[0031] A multi-primer extended version of rolling circle amplification method based on an extended genetic alphabet, comprising the following steps:

[0032] adding random hexamer primers and dNaM-containing circular template-primer complex to 1× phi29 DNAP reaction buffer, mixing to obtain mixture A, annealing at 95℃, adding dATP, dTTP, dGTP, dCTP, dNaMTP, BSA, phi29 DNAP, and unnatural base-containing deoxyribonucleoside triphosphates, mixing to obtain mixture B, incubating.

[0033] Further, the 1× phi29 DNAP reaction buffer comprises 50mM Tris-HCl, 10mM (NH4)2SO4, 4mM DTT, and 10mM MgCl2, and the pH of the 1× phi29 DNAP reaction buffer is 7.4.

[0034] Further, the concentration of the dNaM-containing circular template-primer complex in mixture A is 75nM; and the concentration of the random hexamer primers in mixture A is 5μM.

[0035] Further, the preparation of the dNaM-containing circular template-primer complex comprises the following steps:

[0036] A. mixing 5'-terminal phosphorylated dNaM-containing oligonucleotides with clamp primer DNA oligonucleotides to circularize the phosphorylated product, and adding T4 DNA ligase to ligate the two ends of the circularized sequence together; or

[0037] B. mixing 5'-terminal phosphorylated dNaM-containing oligonucleotides, 5'-terminal phosphorylated oligonucleotides containing the reverse complement of a functional sequence, and two clamp primer DNA oligonucleotides to combine the two ends of the dNaM-containing oligonucleotides and the oligonucleotides containing the reverse complement of a functional sequence with each other with the aid of the two clamp primers and circularize them, and adding T4 DNA ligase to ligate the dNaM-containing oligonucleotides and the oligonucleotides containing the reverse complement of a functional sequence at the two places of the two clamp primers; or

[0038] C. mixing 5'-phosphorylated natural template oligonucleotides with clamp primers to circularize the natural template oligonucleotides, adding dNaMTP and Klenow fragment of E. coli DNA polymerase I to integrate dNaMTP into the 3'-end of the natural template oligonucleotides, and adding T4 DNA ligase to ligate the two ends of the circularized sequence together;

[0039] Further, the 5'-terminal phosphorylation is achieved by the following steps:

[0040] adding oligonucleotides, ATP, and T4 polynucleotide kinase to T4 polynucleotide kinase reaction buffer, mixing, and incubating at 37℃ for 1h to phosphorylate the 5'-end of the oligonucleotides;

[0041] Still further, the concentration of the oligonucleotide in the mixture is 0.6 μM, the concentration of ATP in the mixture is 1 mM, and the concentration of T4 polynucleotide kinase in the mixture is 0.2 U / μL;

[0042] Further, the concentration of dATP, dTTP, dGTP and dCTP in the mixture B is 1 mM, the concentration of dNaMTP in the mixture B is 0.1 mM, the concentration of BSA in the mixture B is 0.4 mg / mL, the concentration of DNA polymerase in the mixture is 1 μM, and the concentration of the deoxyribonucleotide triphosphate containing unnatural base in the mixture is 0.1 mM;

[0043] Further, the deoxyribonucleotide triphosphate containing unnatural base is dTPT3TP, dTPT3 PA TP, dTPT3 Am TP or dTPT3 Al TP.

[0044] Further, the incubation is 30℃ incubation for 20 h.

[0045] The above multi-primer extension version of rolling circle amplification method is applied to the preparation of hydrogel with transcription activity.

[0046] The present application has the following advantages and effects relative to the prior art:

[0047] 1. By introducing unnatural base pairs (UBPs), the genetic alphabet is significantly expanded, combined with rolling circle amplification (RCA) technology, to establish an extended genetic alphabet-based rolling circle amplification (ExRCA) and its multi-primer amplification (ExMRCA) method, promoting the development of biotechnology, biosensing and DNA material science.

[0048] 2. The phi29 DNAP was found to have the ability to amplify representative UBP: dNaM-dTPT3 and its analogues, a method for generating periodic UBP-containing super-long single-stranded and branched double-stranded DNA was developed, and three circular template preparation strategies were designed to meet the needs of diversified applications.

[0049] 3. Combined with restriction endonuclease sequence preparation template, the large-scale preparation of ssDNA and dsDNA containing unnatural bases is realized, which provides an effective technical means for the preparation of UBP-containing aptamers, DNA enzymes, functional oligonucleotides, etc.

[0050] 4. The ExRCA technology was successfully applied to develop a super-sensitive molecular detection method for human alpha-thrombin.

[0051] 5. The specific site labeling and functionalization of DNA materials, including fluorescent labeling and enzymatic labeling, are achieved by using analogs containing functionalized unnatural base dTPT3TP.

[0052] 6. The multivalent aptamer sensor with ratiometric fluorescent labeling is constructed by using the characteristics of the unnatural base pair having little effect on the natural DNA components, and the cell labeling and imaging are achieved.

[0053] 7. The DNA cascade catalyst with fine regulation characteristics is constructed based on the ExRCA technology, and the application value in glucose detection is demonstrated.

[0054] 8. The functional DNA hydrogel with specific site cross-linking is prepared by ExMRCA technology, and its excellent performance in functional RNA transcription is verified.

[0055] 9. In summary, the ExRCA and ExMRCA technologies show wide application prospects in the fields of expanding genetic alphabet, constructing functional enhanced oligonucleotides, DNA catalysts, drugs, sensors and materials, etc. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 For the recognition of phi29 DNAP to unnatural nucleoside triphosphates and the analysis of functional products. A shows the chemical structures of dNaM-dTPT3 and its analogs dTPT3 PA , dTPT3 Am , dTPT3 Al and dTPT3 Bio . B investigates the primer extension ability of phi29 DNAP to dTPT3TP and its analogs based on DNA templates containing dNaM, and the products are analyzed by denaturing PAGE. C uses dTPT3 Am TP, dTPT3 Al TP or dTPT3 Bio TP to perform primer extension reaction, and the products are reacted with NHS-FAM, N3-FAM or streptavidin, respectively, and then analyzed by denaturing PAGE or biotin gel migration experiment to analyze the functionalization efficiency and specificity. SA: streptavidin band; P: band of primer extension product containing dNaM-dTPT3 Bio ; S: band of primer extension product containing dNaM-dTPT3 Bio incubated with SA.

[0057] Figure 2Characterization of extended genetic alphabet-based rolling circle amplification (ExRCA) and its products. A is the schematic of two ExRCA strategies: (a) single primer ExRCA to generate super-long ssDNA containing UBPs; (b) multiple primer ExRCA (ExMRCA) to generate branched dsDNA containing UBPs. B is the product gel image of ExRCA reaction with dTPT3TP and its analogues. Lanes 1-6: under single primer condition, dNTPs, dTPT3TP, dTPT3 PA TP, dTPT3 Am TP, dTPT3 Al TP, dTPT3 Bio TP; Lanes 7-12: under hexamer primer condition, the same nucleotide combinations. The products were analyzed by 1% agarose gel, and the band intensity was calculated by ImageJ (n=3). C is the scanning electron microscope (SEM) images of ExRCA products obtained under 10 mM Mg 2+ and 0.1 mM dTPT3TP or its analogues. Scale bar: 1 μm. D is the high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of nanoflowers generated under 10 mM Mg 2+ and 0.1 mM dTPT3TP condition and the corresponding elemental mapping analysis. Scale bar: 1 μm. E is the SEM characterization of nanoflowers produced using 10 mM Mg 2+ and different concentrations of dTPT3TP. Scale bar: 1 μm.

[0058] Figure 3 Schematic and validation of various ExRCA circular template preparation strategies and their applications in ssDNA and dsDNA amplification production and functionalization. A is the schematic of three circular template preparation strategies: (a) direct circularization of dNaM-containing oligonucleotide strands; (b) assembly of dNaM-containing oligonucleotide and native oligonucleotide strands (with the reverse complementary sequence of desired function); (c) preparation by native oligonucleotide strands and dNaMTP. B is the denaturing PAGE analysis of circular templates obtained from strategies a and b. M: low molecular weight ssRNA marker; Lanes 1 and 3: linear dNaM-containing oligonucleotide; Lanes 2, 5: circularized or assembled templates; Lane 4: native oligonucleotide. C is the template preparation analysis of strategy c. Lane 1: native oligonucleotide; Lane 2: product after Klenow fragment of E. coli DNA polymerase I [Kf(exo-) incorporation of dNaM; Lane 3: circularized template. D is the agarose gel analysis of products after ExRCA reaction with circular templates prepared by different strategies. Lanes 1-3 correspond to strategies a-c in A. E is the schematic of large-scale production of UBPs-containing ssDNA oligonucleotides based on ExRCA. F is the biotin gel shift assay to detect dTPT3Bio Incorporation in ssDNA and ssDNA library. The meaning of the annotations is as follows: H: band of the hairpin structure cleaved off; ssP: band of ssDNA product containing dTPT3 Bio ; S: migration band of ssDNA product containing dTPT3 Bio after incubation with SA. G is the preparation of ssDNA containing fluorescently modified dTPT3 FAM and the construction of DNA tetrahedron structure with specific site labeling. H is the schematic diagram of the strategy for amplifying dsDNA oligonucleotide containing UBP based on ExMRCA. I is the analysis of the retention of UBP in the dsDNA product generated by ExRCA under different reaction times. dsP: band of dsDNA product containing dTPT3 Bio ; S: migration band of dsDNA product containing dTPT3 Bio after incubation with SA.

[0059] Figure 4 ExRCA technology to achieve specific introduction of functional sites of DNA materials and its biological applications. A is the detection of human α-thrombin using ExRCA amplification detection signal: (a) HRP catalyzes color generation to generate detection signal; (b) control group: only add human α-thrombin; (c) traditional detection: biotinylated aptamer and SA-HRP detection; (d) ExRCA detection: after the binding of aptamer and target, a large number of dTPT3 Bio is introduced at the 3' end to achieve signal amplification; (e) absorbance signals generated by different treatment groups, error bars are standard deviations (n = 3), *** indicates P < 0.001, t test. B is the schematic diagram of the preparation of DNA hydrogel with site-specific function by ExRCA and its fluorescence characterization. (a) Incorporation of dTPT3 Am and labeling with NHS-FAM; (b) incorporation of dTPT3 Al and labeling with N3-FAM through click reaction; (c) incorporation of dTPT3 Bio and binding with SA-YF488 fluorescent label. Scale bar: 250 μm. C is the construction and verification of DNA hydrogel that can specifically fix enzymes (HRP) based on ExRCA. (a) Schematic diagram of HRP fixation and color development reaction; (b) absorbance change of substrate solution supernatant after 20 minutes, error bars are standard deviations (n = 3), *** indicates P < 0.001, t test.

[0060] Figure 5A, Schematic diagram of the preparation of ExRCA-based multivalent aptamer sensor. ExRCA was used to introduce multiple copies of aptamer sequences and functionalization sites for efficient cell recognition and signal amplification. B, MCF-7 and SK-BR-3 cancer cells were used as models to label the multivalent aptamer sensor prepared by ExRCA, and the cell imaging effect was observed by fluorescence microscopy. Scale bar: 50 μm.

[0061] Figure 6 Cascading catalytic DNA structure prepared based on ExRCA. A, Schematic diagram of the construction of DNA structure containing glucose oxidase (GOx) and G-quadruplex / heme complex based on ExRCA to achieve cascading catalytic reaction. B, The cascading structure was resuspended in a solution containing 1 mM glucose and 0.8 mM ABTS, and incubated at room temperature for 40 min for colorimetric detection. C, Time course monitoring of the cascading reaction. Error bars represent standard deviation of the mean (n = 3).

[0062] Figure 7 DNA hydrogel with site-specific crosslinking function prepared based on ExMRCA. A, Schematic diagram of the construction of site-specific crosslinking DNA hydrogel for RNA transcription using ExMRCA. B, Formation process of site-specific crosslinking DNA hydrogel. C, Scanning electron microscope (SEM) images of DNA hydrogel before and after crosslinking. D, Transcription and activity detection of Broccoli RNA aptamer in crosslinked hydrogel. The DNA hydrogel was suspended in a buffer containing T7 RNA polymerase, NTPs, DTT and RNase inhibitor, and incubated at 37 °C for 4 h. The RNA product in the supernatant was analyzed by 8 M urea denaturation 20% PAGE gel. M: low molecular weight ssRNA marker; RP: RNA band transcribed from the hydrogel. After incubation of the transcription product with 50 μM DFHBI, the fluorescence was read using a microplate reader. Error bars are standard deviations (n = 3), *** indicates P < 0.001, t test. DETAILED DESCRIPTION

[0063] The present application is further described in conjunction with the following examples and accompanying drawings, of which the implementation of the present application is not limited. It should be noted that if the following is not specifically described in detail, it can be realized or understood by those skilled in the art with reference to the prior art. If the reagent or instrument used is not marked with the manufacturer, it is considered to be a conventional product that can be purchased on the market.

[0064] The materials or reagents involved in the following examples are as follows:

[0065] dNTPs kit purchased from: New England Biolabs, model N0446S

[0066] rNTPs kit was purchased from New England Biolabs, Cat# N0450S

[0067] T4 polynucleotide kinase and reaction buffer were purchased from New England Biolabs, Cat# M0201S

[0068] T4 DNA ligase and buffer were purchased from New England Biolabs, Cat# M0202S

[0069] T7 RNA polymerase was purchased from New England Biolabs, Cat# M0251S

[0070] BsaI-HFv2 was purchased from New England Biolabs, Cat# R3733S

[0071] HindIII-HF was purchased from New England Biolabs, Cat# R3104S

[0072] ATP was purchased from New England Biolabs, Cat# P0756S

[0073] 1 kb DNA ladder was purchased from New England Biolabs, Cat# N3232S

[0074] DHFBI was purchased from Glpbio, Cat# GC30098

[0075] NHS-FAM was purchased from Shanghai Tuo Yang Biological Technology Co., Ltd., Cat# HY-15938

[0076] Cyber Gold was purchased from Biotang, Cat# TJ702

[0077] Streptavidin was purchased from New England Biolabs, Cat# N7021S

[0078] Human α-thrombin was purchased from Sigma-Aldrich, Cat# 605190

[0079] OPD (o-phenylenediamine dihydrochloride) tablets were purchased from Sigma-Aldrich, Cat# P9187

[0080] DBCO-PEG4-NHS ester was purchased from Shaanxi Xinyanbome Biological Technology Co., Ltd., Cat# Y-PE-2145

[0081] N3-PEG5-NHS ester was purchased from Shaanxi Xinyanbome Biotechnology Co., Ltd., model Y-PE-2063

[0082] FAM-N3 was purchased from Shaanxi Xinyanbome Biotechnology Co., Ltd., model Y-R-3280

[0083] NHS-PEG6-NHS was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., model B383205

[0084] Streptavidin-YF488 was purchased from Shanghai Baysi Biological Technology Co., Ltd., model YS0078S

[0085] Horseradish Peroxidase labeled Streptavidin (SA-HRP) was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd., model A0305

[0086] DAPI dye was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd., model C1002

[0087] RNase inhibitors were purchased from Shanghai Biyun Tian Biological Technology Co., Ltd., model R0102

[0088] Klenow fragment (exo - ) was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd., model D7041M

[0089] Cyber Gold nucleic acid gel electrophoresis dye was purchased from Xi'an Baihuo Biological Technology Co., Ltd., model TJ1702

[0090] 2xTBE-urea loading buffer was purchased from Shanghai Sunny Biotech Co., Ltd., model C506046

[0091] Streptavidin (SA) was purchased from Shanghai Sunny Biotech Co., Ltd., model A100497

[0092] Dithiothreitol (DTT) was purchased from Shanghai Sunny Biotech Co., Ltd., model A620058

[0093] Glucose oxidase (GOx) was purchased from Shanghai Sunny Biotech Co., Ltd., model A620244

[0094] Hemin was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., model H811002

[0095] MCF-7 (human breast cancer cells) purchased from: Wuhan Pons Life Science Co., Ltd., model number CL-0149

[0096] SK-BR-3 (human adenocarcinoma cells) purchased from: Wuhan Pons Life Science Co., Ltd., model number CL-0211

[0097] DNA Clean & Concentrator-5 kit purchased from: Zymo Research, model number D4014

[0098] Zymo ssDNA / RNA Clean & Concentrator kit purchased from: Zymo Research, model number D7011

[0099] The following examples use deoxyribonucleotide triphosphates containing unnatural base pairs dNaMTP, dTPT3TP and analog dTPT3 PA TP, dTPT3 Am TP, dTPT3 Al TP purchased from WuXi AppTec.

[0100] All oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. The sequence is shown in Table 1

[0101] Table 1. Oligonucleotide sequences used

[0102]

[0103]

[0104]

[0105] X = NaM

[0106] N = A, T, G or C

[0107] Example 1 Recognition of unnatural base pair dNaM-dTPT3 and its analogs by Phi29 DNAP

[0108] 1) Primer extension experiment mediated by Phi29 DNAP to pair dTPT3TP and its analogs with dNaM on the template

[0109] The 1×phi29 DNAP reaction buffer contains 50 mM Tris-HCl, 10 mM (NH4)2SO4, 4 mM DTT, and 10 mM MgCl2, with the balance being ddH2O. The pH of the 1×phi29 DNAP reaction buffer is 7.4. 0.02 μM primer FAM-T1-p (as shown in Table 1), 0.4 μM template T1 (as shown in Table 1), 0.1 mM dATP, 0.1 mM dTTP, 0.1 mM dGTP, 0.1 mM dCTP, and 0.1 mM dTPT3TP or its analogues (dTPT3) are added. PA TP, dTPT3 Am TP, dTPT3 Al TP and dTPT3 Bio TP)(dTPT3、dTPT3 PA dTPT3 Am dTPT3 Al and dTPT3 Bio Chemical formula such as Figure 1 (As shown in A) was mixed in 1×phi29 DNAP reaction buffer (each concentration is the final concentration of each component in the mixture after adding all components), incubated at 95°C for 10 minutes, and then slowly cooled to room temperature for annealing. 1 μM phi29 DNAP and 0.2 mg / mL BSA were added (each concentration is the final concentration of each component in the mixture after adding DNAP and BSA), and the mixture was reacted at 30°C for 15 minutes. After the reaction was terminated, the products were analyzed using 20% ​​denaturing PAGE containing 8M urea. The control group lacked dTPT3TP or its analogues. Results are as follows: Figure 1 As shown in B, when no non-natural nucleotides are present, primer extension stops at the dNaM position; after adding dTPT3TP or its analogues, full-length extension products are obtained, indicating that phi29DNAP can efficiently and accurately incorporate dTPT3TP and functionalized analogues.

[0110] 2) Labeling assay and biotinylated gel migration assay of UBP-containing DNA oligonucleotides prepared by phi29 DNAP-mediated primer extension.

[0111] The 1×phi29 DNAP reaction buffer contained 50 mM Tris-HCl, 10 mM (NH4)2SO4, 4 mM DTT, and 10 mM MgCl2, with the balance being ddH2O. The pH of the 1×phi29 DNAP reaction buffer was 7.4. To verify the incorporation of phi29 DNAP into functionalized dTPT3TP analogs and to demonstrate the labeling of non-natural nucleotides, primer extension experiments were performed by mixing 50 nM primers FAM-T1-p (for dTPT3... Bio (TP extension) or primer T1-p (for dTPT3) AmTP or dTPT3 Al TP (with extension of the TP primer), 100 nM template Tl, 200 μΜ dATP, 200 μΜ dTTP, 200 μΜ dGTP, 200 μΜ dCTP, 10 μΜ dTPT3 TP analog (dTPT3 Bio TP, dTPT3 Am TP or dTPT3 Al TP) in lx phi29 DNAP reaction buffer. The mixture was incubated at 95 °C for 10 min, then slowly cooled to room temperature to anneal the primers to the template, then 1 μΜ phi29 DNAP and 0.2 mg / mL BSA were added (each concentration is the final concentration of each component in the mixture after the DNAP and BSA were added). The mixture was incubated at 30 °C for 30 min. After purification using Zymo ssDNA / RNA Clean & Concentrator kit, dTPT3 Bio TP, dTPT3 Am TP or dTPT3 Al TP.

[0112] After purification, dTPT3 Bio TP primer extension product was co-incubated with 0.5 mg / mL streptavidin, PAGE analysis showed that the product almost 100% shifted (as shown in C in Figure 1 , indicating high incorporation and labeling efficiency.

[0113] After purification, dTPT3 Am TP extension product was mixed with 10 μΜ NHS-FAM in lx PBS buffer containing 150 mM NaCl, 7.6 mM NaH2P04, and 2.4 mM Na2HP04(pH 8.5) and incubated at 37 °C for 2 h. The product was analyzed using 20% denaturing PAGE gel with 8 M urea and visualized with Cyber gold staining before and after staining and gel imager.

[0114] After purification, dTPT3 Al TP primer extension product was mixed with 10 μΜ N3-FAM, 0.1 mM CuS04, 0.5 mM tris(hydroxypropyl) triazolylmethyl amine (THPTA), 2.5 mM amino guanidine hydrochloride, and 12.5 mM sodium ascorbate (NaAsc) (each concentration is the final concentration of each component in the mixture after all components were added) in lx PBS buffer containing 150 mM NaCl, 7.6 mM NaH2P04, and 2.4 mM Na2HP04(pH 7.4) and incubated at 37 °C for 2 h. The product was analyzed using 20% denaturing PAGE gel with 8 M urea and visualized with Cyber gold staining before and after staining and gel imager.

[0115] Mixing in solution, incubate at 37°C for 2 hours. Analyze the products using 20% denaturing PAGE gel with 8M urea, and visualize the gel before and after staining with Cyber gold staining and gel imager.

[0116] As shown in C of Figure 1 , both of the coupling products showed fluorescent bands before the gel was stained with Cyber gold, indicating that dTPT3 Am and dTPT3 Al were successfully incorporated into the extension products and labeled efficiently by NHS-FAM and N3-FAM.

[0117] Example 2 Establishment of ExRCA method

[0118] Based on the high efficiency and high fidelity of phi29 DNAP in replicating dNaM-dTPT3 and its analogs, we further tried to establish an ExRCA method based on extended genetic alphabet, by which we can generate long repetitive single-stranded DNA containing unnatural base dTPT3 or its analogs (schematic diagram of the method is shown in (a) of A of Figure 2 ).

[0119] The ExRCA method includes the following steps: mix 75 nM of circular template-primer complex containing dNaM with 1 mM dATP, 1 mM dTTP, 1 mM dGTP, 1 mM dCTP, 0.1 mM dTPT3TP or its analogs, 0.4 mg / mL BSA and 1 μM phi29 DNAP in 1 × phi29 DNAP reaction buffer (each concentration is the final concentration of each component in the mixture after all components are added). The mixture is incubated at 30°C for 20 hours. The construction of the circular template-primer complex is constructed according to step 1) of Example 5.

[0120] The ExRCA product was analyzed using 1% agarose gel containing ethidium bromide (EB), and the results showed that only when unnatural nucleotide triphosphates were added, high molecular weight DNA was produced, and no obvious product was produced for natural dNTPs. Different unnatural nucleotides have different yields, among which dTPT3, dTPT3 PA and dTPT3 Al have higher yields (as shown in B of Figure 2 ).

[0121] Example 3 Establishment of ExMRCA method

[0122] At the same time, we used random hexamer primers to perform ExMRCA reaction on circular template-primer complex containing dNaM (schematic diagram of the method is shown in (b) of A of Figure 2 ).

[0123] The ExMRCA method comprises the following steps:

[0124] 75 nM of the dNaM-containing circular template-primer complex was mixed with 5 mM of the random hexamer primer (as shown in Table 1) in 1x phi29 DNAP reaction buffer (each concentration is the final concentration of each component in the mixture after all components are added). After annealing at 95 °C, 1 mM dATP, 1 mM dTTP, 1 mM dGTP, 1 mM dCTP, 0.1 mM dNaMTP, 0.1 mM dTPT3TP or the like, 0.4 mg / mL BSA and 1 mM phi29 DNAP were added (each concentration is the final concentration of each component in the mixture after all components are added), and the mixture was incubated at 30 °C for 20 hours. The construction of the circular template-primer complex was constructed according to the steps of Example 5, 1).

[0125] The ExRCA product was analyzed using 1% agarose gel containing EB. The ExRCA product was super high molecular weight and almost completely blocked in the gel well (as shown in B of Figure 2 As a control, only dNTPs (dATP, dTTP, dGTP and dCTP) were added without dNaMTP, dTPT3TP and the like, and almost no product was detected after 20 hours of reaction using only natural dNTPs. This again indicates that phi29 DNAP can copy dNaM-dTPT3 and the like with high fidelity, and can generate UBP-containing multi-branch double-stranded DNA through ExMRCA. ExMRCA has potential for sensitive detection of unnatural nucleotide triphosphates, and dTPT3 and the like have high yield.

[0126] Example 4. Characterization of ExRCA product by scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

[0127] According to the steps described in Example 2, a 20 mL ExRCA reaction was performed using dTPT3TP or the like, and the product was resuspended after multiple water washes. The precipitate was placed on a glass slide, dried, and a layer of gold was sprayed on its surface, and then imaged using SEM. For TEM, the precipitate was placed on a carbon-coated copper grid, dried, and imaged.

[0128] As shown in C of Figure 2 Under the test conditions, nanoflowers with an average diameter of about 1.5 pm were observed in the ExRCA product generated using dTPT3TP, dTPT3 PA TP and dTPT3 Al TP. Using dTPT3 BioSmaller nanoflowers with an average diameter of about 500 nm and more small petals were observed in the ExRCA product generated by TP. Interestingly, the use of dTPT3 Am No obvious nanoflower structure was observed in the ExRCA product generated by TP.

[0129] The formed nanoflowers were further characterized by transmission electron microscopy (TEM), high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), and energy dispersive X-ray spectroscopy (EDS). The TEM image of the dTPT3-ExRCA product showed a porous flower-like structure, consistent with the observation in the SEM image. As shown in D of Figure 2 The elemental distribution in the nanoflowers was investigated by HAADF-STEM and STEM-EDS elemental mapping analysis. C, N, O, P, and Mg are the representative elements of the mineralized organic template DNA and inorganic component Mg2PPi. Meanwhile, the representative element S of dTPT3 was also observed to be uniformly distributed in the nanoflowers, which further indicates that dTPT3 was successfully incorporated into the ExRCA product.

[0130] The concentration of dTPT3 TP was also explored to affect the microstructure of the ExRCA product. Based on the experimental protocol of Example 2, 0.1 mM dTPT3 TP was adjusted to 0.05 mM, 0.025 mM, or 0.0125 mM, and the results are shown in E of Figure 2 As shown in E of, the SEM imaging experiment indicates that within the range of 12.5 μΜ to 100 μΜ, the higher the concentration of dTPT3 TP usually leads to the generation of larger and more petal-dense nanoflowers in the ExRCA product. This, together with the above results, indicates that dTPT3 TP has a high degree of participation in the ExRCA reaction.

[0131] Example 5. Preparation of a circular template containing a non-natural base for ExRCA

[0132] 1) Preparation of a circular template containing a non-natural base using a DNA oligonucleotide chain containing a non-natural base

[0133] The first strategy is to use a bridging oligonucleotide to circularize the 5'-phosphorylated dNaM-containing oligonucleotide, and then use T4 DNA ligase to connect the two ends together (a schematic diagram of which is shown in (a) of A of Figure 3 includes the following steps:

[0134] A 0.6 mM dNaM-containing DNA oligonucleotide T4 (sequence shown in Table 1) was mixed with 1 mM ATP and 0.2 U / µL T4 polynucleotide kinase in lx T4 polynucleotide kinase reaction buffer (each concentration is the final concentration of each component in the mixture after all components are added) at 37 °C for 1 hour to achieve 5'-end phosphorylation. After the reaction was terminated by incubation at 65 °C for 20 minutes, the product was purified using a Zymo ssDNA / RNA Clean & Concentrator kit. The 1 µM purified product was mixed with 2 µM splint primer DNA oligonucleotide S4 in lx T4 DNA ligase buffer (each concentration is the final concentration of each component in the mixture after all components are added), denatured at 95 °C for 5 minutes, slowly cooled to room temperature, and incubated on ice for 5 minutes to allow annealing. Subsequently, 20 U / µL T4 DNA ligase was added, and the mixture was incubated at 16 °C overnight to complete circularization.

[0135] The product was analyzed by denaturing PAGE with 8 M urea and stained with Cyber gold, and detected using a gel imager. The results showed that the circular product was successfully obtained, and its band was higher than that of the linear oligonucleotide in the electrophoretogram, indicating successful ligation (results shown in Figure 3 B of FIG. 1).

[0136] 2) Preparation of a non-natural base circular template containing the reverse complementary sequence of a functional sequence

[0137] In the second strategy, a 5'-phosphorylated dNaM-containing oligonucleotide and another 5'-phosphorylated oligonucleotide containing the reverse complementary sequence of a functional sequence were co-circularized with the aid of two oligonucleotide splint primers, and then ligated together using T4 DNA ligase (a schematic diagram thereof is shown in (b) in A of FIG. 1). The following steps were included: Figure 3

[0138] The 1 µM 5'-phosphorylated dNaM-containing DNA oligonucleotide T4 was mixed with 1 µM 5'-phosphorylated DNA oligonucleotide T5 (containing the reverse complementary sequence of a functional sequence) and 2 µM of each of the two splint primers (S1-T5 and S2-T5) in lx T4 DNA ligase buffer, denatured at 95 °C for 5 minutes, slowly cooled to room temperature, and incubated on ice for 5 minutes to allow annealing. 20 U / µL T4 DNA ligase was added, and the mixture was incubated at 16 °C overnight to complete circularization.

[0139] The product was analyzed by denaturing PAGE and imaged after staining, and the results showed that the band of the obtained circular product was obviously moved up, indicating that the desired template was successfully prepared (results shown in B of FIG. 1). Figure 3

[0140] ​​3) Preparation of circular templates containing unnatural bases using natural DNA oligonucleotide strands

[0141] The third strategy does not require the use of dNaM-containing oligonucleotides, but only relies on natural oligonucleotides and dNaMTPs to complete the introduction of unnatural bases and template circularization. (The schematic diagram is shown in (c) in A in Figure 3

[0142] 1 μΜ 5'-phosphorylated natural template oligonucleotide strand T6 was mixed with 2 μΜ splint primer S6 in 1x CutSmart buffer, incubated at 95 °C for 5 min, then slowly cooled to room temperature, and incubated on ice for 5 min to anneal the oligonucleotides. 50 μΜ dNaMTPs and 0.2 U / μL Klenow fragment of E. coli DNA polymerase I [Kf(exo-)] were added, and the mixture was incubated at 37 °C for 30 min to integrate dNaMTPs to the 3' end of T6. After heating at 95 °C for 10 min to terminate the reaction, 20 U / μL T4 DNA ligase and buffer were directly added, and incubated at 16 °C overnight to achieve circularization.

[0143] The resulting product was analyzed by denaturing PAGE and imaging, and the results showed that Kf(exo-) could efficiently incorporate dNaMTPs and form stable circular products.

[0144] (The results are shown in C in Figure 3 ).

[0145] To test the quality and usability of the circular templates prepared by different strategies, we used these templates to perform ExRCA experiments, and analyzed the products by agarose gel. As shown in D in Figure 3 , for the ExRCA reactions performed using circular templates prepared by the three strategies, a large amount of product was generated in the presence of dTPT3TP, while little product was generated in the absence of dTPT3TP, indicating that these circular templates have good quality and usability. Obviously, the application range of ExRCA has been further expanded through these circular template preparation strategies.

[0146] Example 6. Large-scale preparation of DNA oligonucleotides or oligonucleotide libraries containing unnatural bases using ExRCA

[0147] Taking advantage of the property that ExRCA can efficiently generate super-long single-stranded DNA of repetitive sequences, this example establishes a method combining ExRCA and IIs-type restriction endonuclease cleavage for batch preparation of DNA oligonucleotides or libraries containing any unnatural bases. (The schematic diagram is shown in E in Figure 3 ).

[0148] ​The circular template-primer complex of ExRCA was prepared according to the method in Example 5-2) using 5'-phosphorylated template oligonucleotide Tre and T3 and splint primer S1-Tre and S2-Tre (see Table 1). Then dTPT3 Bio was used for ExRCA reaction and incubated for 8 hours according to the ExRCA procedure described in 2.4. The solution of ExRCA product was incubated at 95 °C for 5 min and then slowly cooled to room temperature to fold into a hairpin structure containing a restriction site. Then 1 U / μL Bsal-HFv2 and 1x CutSmart buffer were directly added into the ExRCA product and the mixture was incubated at 37 °C for 12 hours for enzyme digestion. To verify the retention rate of dTPT3 Bio in the ssDNA product, a portion of the purified product was mixed with 0.5 mg / mL streptavidin and incubated at 37 °C for 2 hours, and then analyzed using a 9% PAGE gel.

[0149] The results show that the product band almost completely migrates after binding with streptavidin (as shown by F in Figure 3 , indicating that the unnatural base (such as dTPT3 Bio ) is well retained. If the DNA oligonucleotide library is the target product, only the sequence L1 (see Table 1) is used to replace T3 when constructing the circular template, and the rest of the steps are the same. Figure 3 The results show that the product band almost completely migrates after binding with streptavidin (as shown by F in

[0150] Example 7. Preparation of site-specifically labeled DNA nanostructure based on ExRCA

[0151] To demonstrate the application value of the method of the present application in the construction of DNA nanostructure containing unnatural base, this example uses an oligonucleotide containing dTPT3 Am to construct a site-specifically labeled DNA tetrahedron structure (the schematic diagram of which is shown by G in Figure 3 ).

[0152] First, according to the method in 5-1), the circular template-primer complex of ExRCA was prepared using 5'-phosphorylated template oligonucleotide T-A13 and splint primer S-A13 (see Table 1). Then, dTPT3 Am was used for ExRCA reaction and 1 U / μL Bsal-HFv2 was added after 2 hours of incubation for enzyme digestion for 12 hours to obtain A13(dTPT3 Am ). Am-A13), and purified with Zymo ssDNA / RNA Clean & Concentrator kit. 2 μΜ dTPT3 Am -A13 was mixed with 1 mM NHS-FAM in 1x PBS buffer (pH 8.5) (each concentration is the final concentration of each component in the mixture after all components are added), and incubated at 37 °C for 2 hours to allow the fluorescent labeling reaction at the amino position. After the reaction, free NHS-FAM was removed by purification again to obtain the FAM-labeled product dTPT3 FAM -A13.

[0153] To assemble site-specifically labeled DNA tetrahedron, oligonucleotide dTPT3 FAM -A13, B13, C13 and D13 (see Table 1) were mixed in 1x Tris-Acetic-EDTA-Mg 2+ (TAE / Mg 2+ ) buffer (40 mM Tris base, 20 mM acetic acid, 2 mM EDTA, 12.5 mM Mg acetate, pH 8.0) at equimolar ratio. The final concentration of each oligonucleotide was 1 μΜ. The oligonucleotide mixture was incubated at 90 °C for 5 min, and then placed on ice for 1 hour. The product was analyzed using 10% PAGE gel, which was run in 1x TBE running buffer containing 12.5 mM Mg 2+ at 4 °C. As shown in G in Figure 3 , the stained bands showed good assembly efficiency. In the FAM-labeled group, the migration bands detected by fluorescence imaging were consistent with the target structure, and the migration rate was consistent with that of the tetrahedral structure without unnatural base, indicating that the incorporation of dTPT3 Am did not affect the structure assembly. In summary, the unnatural base oligonucleotide prepared by this method can be used to construct DNA nanostructures with functionalized labels, and has good universality and application prospect.

[0154] Example 8. Mass production of dsDNA containing UBP based on ExMRCA

[0155] This example establishes a method combining ExMRCA and restriction enzyme digestion to achieve large-scale isothermal synthesis of double-stranded DNA containing unnatural base pairs (UBP), which is suitable for constructing DNA materials with extended genetic alphabet and semi-synthetic organisms (such as Figure 3 as shown in H in BioTP and dNaMTP were subjected to ExMRCA reaction for 12 hours. Then 7 U / μL HindIII and 1x CutSmart buffer (each concentration is the final concentration of each component in the mixture after all components are added) were directly added into the reaction product and incubated at 37°C for 4 hours to cleave the ExMRCA product. To verify the retention of UBP, the purified product was subjected to PAGE analysis after incubation with 0.5 mg / mL streptavidin at 37°C for 2 hours. Figure 4 As shown in lane I of FIG. 6, more than 95% of the bands in each time point product obtained at 2, 4, 8 and 12 hours of reaction can bind to streptavidin and migrate, proving that the unnatural base pairs are well retained in dsDNA.

[0156] Example 9. Ultra-sensitive detection of human α-thrombin using ExRCA

[0157] RCA technology has been widely used in signal amplification in molecular detection and imaging, and its principle usually relies on hybridization of single-stranded DNA probes complementary to the amplification product. In contrast, the super-long single-stranded DNA generated by ExRCA periodically contains unnatural bases, which can be used to introduce specific functional groups at a high density in the product by introducing functional unnatural nucleotide triphosphates, to achieve covalent or non-covalent coupling with fluorescent molecules or enzymes. Compared with traditional RCA methods, ExRCA does not require probe hybridization, and only a single base site is required for the binding of labeled molecules, which is suitable for systems where probes are difficult to access or have low hybridization efficiency, and the labeling density can be flexibly controlled by adjusting the template length. To demonstrate the application of ExRCA in ultra-sensitive biomolecular detection, we used human α-thrombin as a model target to be detected (as shown in lane A of FIG. 6). Figure 4

[0158] First, according to the method in 5-1), ExRCA circular template-primer complex was prepared using 5'-phosphorylated template oligonucleotide T4 and splint primer S-T4a containing thrombin aptamer sequence (31TBA). In a 20 μL reaction system, dTPT3 Bio ​TP for ExRCA, incubated at 37°C for 30 min. As a control, 2 μΜ of biotin-labeled 31TBA (31TBA-biotin) was used. The reaction products were mixed with 80 μΐ, of 1 x binding buffer (50 mM NaCl, 2 mM CaCl2, 20 mM HEPES, pH 7.3) and allowed to fold by slowly cooling to room temperature after heating at 95°C for 10 min. Next, 100 μΐ, of 0.05 mg / mL human α-thrombin solution was added to a high-adsorption 96-well plate, incubated at 4°C overnight, washed 6 times with PBST buffer containing 0.05% (v / v) Tween-20, and blocked with 5% BSA for 2 h. Subsequently, ExRCA products or control samples were added, respectively, and incubated at 37°C for 2 h, followed by the addition of 200 μΐ, of 1 μg / mL streptavidin-HRP and incubation for another 30 min and washing. The color was developed by adding HRP substrate OPD / H2O2 for 10 min, and the reaction was stopped by adding 3 M H2SO4. The absorbance was read at 490 nm.

[0159] As shown in (e) of A in FIG. 6, the signal intensity of ExRCA products was about 6 times that of biotinylated 31TBA, significantly enhancing the detection sensitivity. This verified that ExRCA can be used for efficient and ultra-sensitive biomolecular detection without the need for a probe and has broad application potential. Figure 4

[0160] Example 10. Preparation of specifically labeled or functionalized DNA materials by ExRCA and characterization thereof

[0161] Controllable labeling and functionalization of biomaterials, including DNA materials, are essential for expanding their application range. Due to its ultra-high molecular weight product, RCA has been widely used in the manufacture of various DNA materials. Inspired by these works and influenced by the efficiency of ExRCA, we further explored the method of preparing DNA materials with specific site labeling or functionalization by ExRCA combined with modified non-natural nucleotide triphosphates and coupling with functional molecules. We used dTPT3 Am TP, dTPT3 Al TP and dTPT3 Bio ExRCA reactions of 30 h were performed with TP, dTPT3 Am , dTPT3 Al and dTPT3 Bio to prepare DNA hydrogels containing dTPT3 Figure 4 , dTPT3

[0162] 1) dTPT3 Am modified ExRCA products were labeled with N-hydroxysuccinimide (NHS) ester

[0163] ​First, follow the method in Example 2 using dTPT3 Am After the TP was subjected to the ExRCA reaction, it was washed with ddH2O. The resulting product was incubated with 1 mM NHS-FAM in 1×PBS buffer (pH 8.5) at 37°C for 2 hours (1 mM is the final concentration of NHS-FAM after being added to the buffer). After purification, it was purified with ddH2O, stained with 4',6-diamino-2-phenylindole (DAPI), and subjected to fluorescence microscopy imaging.

[0164] 2) Labeling dTPT3 using copper-catalyzed azide-enyne cyclization reaction (CuAAC). Al Modified ExRCA products

[0165] Using dTPT3 Al After the TP was subjected to the ExRCA reaction, it was washed with ddH2O. The resulting product was mixed with 1 mM 5-carboxyfluorescein azide (N3-FAM), 0.1 mM CuSO4, 0.5 mM THPTA, 2.5 mM aminourea hydrochloride and 12.5 mM sodium ascorbate (NaAsc) in 1×PBS buffer (pH 7.4) (each concentration is the final concentration of each component in the mixture after adding all components). The mixture was incubated at 37°C for 2 hours, purified, stained with DAPI and subjected to fluorescence microscopy.

[0166] 3) Use streptavidin to label dTPT3 Bio Modified ExRCA products

[0167] Using dTPT3 Bio After the TP was subjected to the ExRCA reaction, it was washed with ddH2O. The resulting product was mixed with 1 mM SA-YF488 in 1×PBS buffer (pH 7.4), and the mixture was incubated at 37°C for 2 hours. After purification, it was stained with DAPI and subjected to fluorescence microscopy imaging. Figure 4 As shown in (a), (b), and (c) of B in the diagram, all labeled DNA hydrogels emitted bright green fluorescence, and the green fluorescent regions completely overlapped with the DAPI fluorescent regions, indicating that all DNA hydrogels were efficiently labeled using the corresponding labeling strategies. It is worth noting that dTPT3... Al With azides and dTPT3 Bio The conjugation with streptavidin is highly specific and should therefore enable the labeling or functionalization of DNA materials produced via ExRCA in complex environments (e.g., environments with high concentrations of amino groups).

[0168] Example 11. Immobilization of enzymes in DNA material generated by ExRCA

[0169] In addition to fluorophores and labeled proteins, enzymes are widely used as key functional molecules for the loading and application of biomaterials. In this example, horseradish peroxidase (HRP) was used as a model to explore the feasibility of site-specific immobilization of enzymes using ExRCA products containing modified non-natural nucleotides. First, ExRCA reactions were performed using dTPT3 Bio TP according to the method of Example 2. After washing with lx PBS buffer (pH 7.4), the product was resuspended in PBS buffer containing 100 ng / mL streptavidin-horseradish peroxidase (SA-HRP) and incubated at 4°C overnight. The unbound SA-HRP was then removed by washing again, and the DNA hydrogel was stained with Cybergold and washed with PBS to obtain the enzyme-immobilized DNA hydrogel. The resulting hydrogel was resuspended in a substrate solution containing OPD / H2O2, and the reaction was terminated after 20 minutes by adding 3M H2SO4. The absorbance of the supernatant at 490 nm was measured. As shown in Fig. 8C, the hydrogel rapidly changed color after the addition of the substrate, indicating that the immobilized HRP had good activity. As the product diffused, the overall color of the solution increased. After only 20 minutes of reaction, the absorbance of the experimental group was 117 times that of the control, indicating that the specific immobilization efficiency of HRP on the DNA hydrogel was high, and the enzyme activity was well maintained. The results show that ExRCA can be used as an efficient strategy for the preparation of DNA materials that can load enzymes or other functional proteins. Figure 5

[0170] Example 12. Preparation of a multivalent aptamer sensor with ratio fluorescent labeling by ExRCA for cell labeling and imaging

[0171] One of the advantages of ExRCA is that it has minimal impact on natural DNA sequences, enabling site-specific coupling of non-natural bases with functional molecules, and the ratio of natural functional sequences to modified sites can be precisely regulated by designing circular templates. Based on this property, a multivalent aptamer sensor with ratio fluorescent labeling was constructed in this example, and was used for the labeling and imaging of breast cancer cells MCF-7 and SK-BR-3 (schematic diagram shown in Fig. 9A). Figure 5

[0172] The circular template used for labeling MCF-7 cells was composed of T4, T-MUC1, and primers S1-MUC1 and S2-MUC1; the template used for labeling SK-BR-3 cells was composed of T4, T-HER2, and primers S1-HER2 and S2-HER2 (Table 1). According to the method of Example 2, ExRCA reactions were performed using dTPT3 Am ​​The product was denatured at 95 °C for 10 min, then slowly cooled and incubated on ice to fold into aptamer structure. Subsequently, the product was reacted with 2 mM NHS-FAM in 1x PBS (pH 8.5) at 37 °C for 2 h, and the FAM-labeled product was washed with PBS (pH 7.4) for 6 times. The product was suspended in DMEM (for MCF-7) containing 10% FBS (v / v), 1% penicillin / streptomycin (v / v) and 1% non-essential amino acids (v / v), or McCoy's 5A medium (for SK-BR-3) containing 15% FBS (v / v), respectively. MCF-7 cells were cultured in DMEM with 10% fetal bovine serum (FBS, v / v), 1% penicillin / streptomycin (v / v) and 1% non-essential amino acids (v / v), and SK-BR-3 cells were cultured in McCoy's 5A (modified) medium with 15% (heat-inactivated) FBS (v / v). Cells were incubated at 37 °C in a humidified incubator with 5% CO2. Cells were seeded in 12-well plates until the confluency of the wells reached 70-90%. The medium in each cell sample was removed, and the cells were washed with PBS twice. Then the FAM-labeled ExRCA product was added to the corresponding wells. After incubation at 37 °C for 2 h, the cells were washed with DPBS and stained with DAPI, and imaged with an inverted fluorescence microscope.

[0173] Figure 6 The results in B of FIG. 12 show that strong fluorescence signals were observed on the surface of both types of cells, indicating that the prepared aptamer sensor performed well in cell labeling and imaging.

[0174] Example 13. DNA cascade catalyst constructed based on ExRCA and its application in glucose detection

[0175] In addition to aptamers, ExRCA can also be used to construct DNA structures with catalytic functions. Among them, the natural DNA part can encode catalytic sequences, and the unnatural base site can be used to fix natural enzymes, forming a DNA cascade catalytic system with controllable ratio. This embodiment constructed a cascade catalyst containing G-quadruplex / hemin DNAzyme (with peroxidase-like activity) and glucose oxidase (GOx), with a ratio of 1:1, and demonstrated its application in glucose detection (schematic diagram as shown in A of FIG. 13). Figure 6

[0176] First, a circular template containing T4 and T-G4 (G-quadruplex reverse complementary sequence) was prepared according to Example 5-2), and ExRCA amplification was performed with primers S1-G4 and S2-G4, using dTPT3 Am ​TP as a non-natural triphosphate substrate. After purification by Amicon (100 kDa) filtration and washing with ddH2O, the product was incubated with 20 mM NHS-PEG4-DBCO in PBS buffer (pH 8.5) at 37 °C for 4 h to obtain the DBCO-modified product, which was then washed with ddH2O.

[0177] Meanwhile, 1 mM GOx was incubated with 40 mM NHS-PEG5-N3 in 1x PBS buffer (pH 8.5) at room temperature for 4 h to generate azide-modified Gox, which was then washed with PBST (pH 7.4) for 5 times. Then the DBCO-modified ExRCA product was mixed with excess azide-modified GOx in PBS buffer containing 0.05% Tween-20 and incubated at 37 °C for 3 h for click coupling. The product was washed in 1x hemin-G4 binding buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 200 mM NaCl, 20 mM KCl, 1% DMSO, 0.1% Triton X-100, pH 7.3) to remove unbound GOx.

[0178] For loading hemin, the GOx-containing ExRCA product was resuspended in 1x hemin-G4 binding buffer and supplemented with 0.2 mM hemin, and incubated at 4 °C overnight. Unbound hemin was removed by washing with cascade reaction buffer (20 mM Na2HPO4, 20 mM NaH2PO4, 200 mM NaCl, 20 mM KCl, pH 6.3) for 5 times.

[0179] Finally, the GOx- and hemin-containing ExRCA product was suspended in cascade reaction buffer, and 1 mM glucose and 0.8 mM ABTS were added, and the reaction was carried out at room temperature, and the absorbance change was monitored by a microplate reader at 420 nm. As shown in B of FIG. 14, Figure 6 As shown in B of FIG. 14, the solution of the experimental group was obviously discolored, and the absorbance rapidly increased within 40 min; the control group showed no significant change. Figure 7 As further confirmed in C of FIG. 14, the cascade catalyst reaction was highly efficient, and could be used for glucose detection.

[0180] Example 14. Site-specific cross-linked DNA hydrogel based on ExMRCA and verification of its transcriptional activity

[0181] In addition to being used to introduce functional modifications, the unnatural bases in ExRCA / ExMRCA products can also serve as specific sites for DNA intermolecular crosslinking. This example demonstrates the construction of site-controllable crosslinked DNA hydrogel by UBP on (dNaM-dTPT3Am) and verifies its feasibility in embedded gene sequence expression. This strategy aims to enhance the mechanical properties of the material while maintaining the functional readability of the native DNA sequence (schematic shown as Figure 7 .

[0182] The circular template used for ExMRCA was prepared according to the method in Example 5-2), which contained T4 and T-trans (reverse complementary sequence containing T7 promoter, Broccoli fluorescent RNA aptamer and T7 terminator sequence) and splint primer S1-trans and S2-trans as described in Example 2. ExMRCA reaction was performed using dNaM-dTPT3 Am TP, and the reaction product was washed with ddH2O. To achieve site-specific crosslinking, the ExMRCA product was resuspended in 1x PBS buffer (pH 8.5) and 4 mM NHS-PEG6-NHS was added, and incubated at 37°C for 5 hours. After the reaction was completed, the crosslinked product was washed with ddH2O to obtain the crosslinked DNA hydrogel. Subsequently, the DNA hydrogel was resuspended in the transcription reaction solution containing 0.5 mM NTPs, 5 mM DTT, 2 U / μL T7 RNA polymerase, 1 U / μL RNase inhibitor and 1x RNA polymerase buffer, and incubated at 37°C for 4 hours for in vitro transcription. The reaction product was analyzed by 20% denaturing urea PAGE electrophoresis, and the gel was stained with Cyber Gold and imaged. To detect the functional Broccoli RNA, 50 μM 3,5-DFHBI was added to the supernatant after transcription, and incubated at room temperature for 10 minutes. The fluorescence intensity was measured using a microplate reader (excitation wavelength 454 nm, emission wavelength 506 nm).

[0183] The prepared DNA hydrogel was then resuspended in ddH2O, stained with Cyber Gold, and observed under white light and blue light. As shown in Figure 7 B, the crosslinked hydrogel exhibited high integrity. Scanning electron microscopy (SEM) images showed that obvious crosslinked regions were formed between nanostructures in the crosslinked product, which were significantly larger than the non-crosslinked ExMRCA product (as shown in Figure 7 C). ​ In addition,

[0184] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A rolling circle amplification method based on an extended genetic alphabet, characterized in that, Includes the following steps: Add dATP, dTTP, dGTP, dCTP, BSA, phi29 DNAP, deoxyribonucleoside triphosphates containing non-natural bases, and a circular template-primer complex containing dNaM to 1×phi29 DNAP reaction buffer, mix, and incubate.

2. The method according to claim 1, characterized in that, The deoxyribonucleoside triphosphates containing non-natural bases mentioned are dTPT3TP and dTPT3. PA TP, dTPT3 Am TP or dTPT3 Al TP.

3. The method according to claim 1, characterized in that, The preparation of the cyclic template-primer complex containing dNaM includes the following steps: A. Mix the 5'-terminal phosphorylated oligonucleotide containing dNaM with the clamp primer DNA oligonucleotide to circularize the phosphorylation product, and add T4 DNA ligase to ligate the two ends of the circularized sequence together; or B. Mix the 5'-terminal phosphorylated oligonucleotide containing dNaM, the 5'-terminal phosphorylated oligonucleotide containing the reverse complementary sequence of the functional sequence, and two splice primer DNA oligonucleotides. With the assistance of the two splice primers, the oligonucleotides containing dNaM and the oligonucleotides containing the reverse complementary sequence of the functional sequence combine at both ends and become circular. Add T4 DNA ligase to ligate the dNaM-containing oligonucleotides at both ends of the two splice primers to the reverse complementary sequences of the functional sequence; or C. Mix the 5'-phosphorylated natural template oligonucleotide with the splice primer to cyclize the natural template oligonucleotide. Add dNaMTP and the Klenow fragment of E. coli DNA polymerase I to integrate dNaMTP into the 3' end of the natural template oligonucleotide. Add T4 DNA ligase to ligate the two ends of the cyclized sequence together.

4. The method according to claim 1, characterized in that, The 1×phi29 DNAP reaction buffer contains 50mM Tris-HCl, 10mM (NH4)2SO4, 4mM DTT and 10mM MgCl2, and the pH of the 1×phi29 DNAP reaction buffer is 7.

4. The concentrations of dATP, dTTP, dGTP, and dCTP in the mixture were all 1 mM, the concentration of BSA in the mixture was 0.4 mg / mL, the concentration of DNA polymerase in the mixture was 1 μM, the concentration of deoxyribonucleoside triphosphates containing non-natural bases in the mixture was 0.1 mM, and the concentration of the circular template-primer complex containing dNaM in the mixture was 75 nM.

5. The application of the rolling circle amplification method based on the extended genetic alphabet as described in any one of claims 1 to 4, wherein the application includes the following: (1) Application in the preparation of DNA nanomolecules with functionalized labels; (2) Application in the preparation of products for detecting human α-thrombin; (3) Application in enzyme immobilization; (4) Applications in the preparation of cell labeling and imaging products; (5) Application in the preparation of glucose detection products.

6. A multi-primer extended version rolling circle amplification method based on an extended genetic alphabet, characterized in that, Includes the following steps: Add random hexameric primers and circular template-primer complexes containing dNaM to 1×phi29 DNAP reaction buffer, mix to obtain mixture A, anneal at 95°C, then add dATP, dTTP, dGTP, dCTP, dNaMTP, BSA, phi29 DNAP and deoxyribonucleoside triphosphates containing non-natural bases, mix to obtain mixture B, and incubate.

7. The method according to claim 6, characterized in that, The preparation of the cyclic template-primer complex containing dNaM includes the following steps: A. Mix the 5'-terminal phosphorylated oligonucleotide containing dNaM with the clamp primer DNA oligonucleotide to circularize the phosphorylation product, and add T4 DNA ligase to ligate the two ends of the circularized sequence together; or B. Mix the 5'-terminal phosphorylated oligonucleotide containing dNaM, the 5'-terminal phosphorylated oligonucleotide containing the reverse complementary sequence of the functional sequence, and two splice primer DNA oligonucleotides. With the assistance of the two splice primers, the oligonucleotides containing dNaM and the oligonucleotides containing the reverse complementary sequence of the functional sequence combine at both ends and become circular. Add T4 DNA ligase to ligate the dNaM-containing oligonucleotides at both ends of the two splice primers to the reverse complementary sequences of the functional sequence; or C. Mix the 5'-phosphorylated natural template oligonucleotide with the splice primer to cyclize the natural template oligonucleotide. Add dNaMTP and the Klenow fragment of E. coli DNA polymerase I to integrate dNaMTP into the 3' end of the natural template oligonucleotide. Add T4 DNA ligase to ligate the two ends of the cyclized sequence together.

8. The method according to claim 6, characterized in that, The deoxyribonucleoside triphosphates containing non-natural bases mentioned are dTPT3TP and dTPT3. PA TP, dTPT3 Am TP or dTPT3 Al TP.

9. The method according to claim 6, characterized in that, The 1×phi29 DNAP reaction buffer contains 50mM Tris-HCl, 10mM (NH4)2SO4, 4mM DTT and 10mM MgCl2, and the pH of the 1×phi29 DNAP reaction buffer is 7.

4. The concentration of the cyclic template-primer complex containing dNaM in mixture A was 75 nM; the concentration of the random hexameric primer in mixture A was 5 μM. The concentrations of dATP, dTTP, dGTP, and dCTP in mixture B were all 1 mM, the concentration of dNaMTP in mixture B was 0.1 mM, the concentration of BSA in mixture B was 0.4 mg / mL, the concentration of DNA polymerase in the mixture was 1 μM, and the concentration of deoxyribonucleoside triphosphates containing non-natural bases in the mixture was 0.1 mM.

10. The use of the method according to any one of claims 6 to 9 in the preparation of a hydrogel with transcriptional activity.

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