CN113324956B - Modularized detection platform based on CRISPR technology and frame nucleic acid coupling, construction method and application thereof - Google Patents
Modularized detection platform based on CRISPR technology and frame nucleic acid coupling, construction method and application thereof Download PDFInfo
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Abstract
The invention provides a modularized detection platform for coupling CRISPR technology with framework nucleic acid, a construction method and application thereof. The detection platform comprises the following 2 modules: (1) Constructing an identification module by using CRISPR technology, which is used for identifying nucleic acid (new coronavirus, human papillomavirus, hepatitis B virus nucleic acid and the like) and non-nucleic acid (protein, antibiotics, metal ions and the like) targets; (2) And constructing signal modules with different valence states by adopting framework nucleic acid, and outputting the signals through controllable amplification. And finally, integrating the identification module and the signal module on the electrochemical chip or the paper chip at the same time. The invention utilizes CRISPR/Cas to identify the target with high sensitivity and high specificity, combines the signal amplification of the frame nucleic acid signal group, realizes the sensitive, rapid, simple and convenient and timely detection of the nucleic acid and the non-nucleic acid targets in the complex matrix, and has good medical diagnosis and food safety monitoring application prospects.
Description
Technical Field
The invention belongs to the technical field of biotechnology and detection, and particularly relates to a modular detection platform construction based on CRISPR technology and frame nucleic acid coupling and detection application of the modular detection platform construction to nucleic acid and non-nucleic acid targets.
Background
CRISPR (Clustered regularly interspaced short palindromic repeats) -Cas (CRISPR-associated) system was originally derived from the prokaryotic adaptive immune system to recognize and degrade invading nucleic acids, an important invention in the 21 st century gene editing field. Because of its superior specificity and sensitivity, CRISPR/Cas systems are of great interest for applications in the biosensing field in addition to gene editing. Compared with CRISPR/Cas9, CRISPR/Cas12a, CRISPR/Cas13, CRISPR/Cas14 have both targeted specific DNA/RNA cleavage activity and paralytic non-specific DNA/RNA cleavage activity under crRNA mediation, and when Cas-crRNA complex is activated in combination with target DNA/RNA, specific cleavage of target DNA/RNA and non-specific cleavage of any adjacent DNA/RNA will occur. Due to the existence of the attribute, the protein such as CRISPR/Cas12a, CRISPR/Cas13 and CRISPR/Cas14 can be applied to construct a detection platform by marking a signal molecule on a single chain as a report chain, so that the detection and the capture of nucleic acid, metal ions, protein and even cells can be realized.
For the CRISPR technique, achieving amplification of signals in complex actual samples is a breakthrough in the technique. The number of single-stranded DNA/RNA modified signal molecules is limited and there is no capacity to resist non-specific adsorption on complex matrices. Therefore, the sensitivity and specificity of target detection in complex matrices are limited by using single-stranded DNA/RNA modified signal molecules as reporter chains, which is difficult to put into practical use. The DNA nano material has the characteristics of editability, addressability, rigidity and the like, so that the DNA nano material can be applied to solving the technical problem. In order to realize amplification and transmission of signals in a practical complex sample, framework nucleic acid (such as DNA tetrahedron and TDF) is adopted as a signal group, arm chains with different numbers are extended at the vertex positions of the framework nucleic acid, and signal molecules are modified, so that controllable amplification output of complex matrix detection signals is realized. By coupling the CRISPR technology with the framework nucleic acid, high sensitivity and high specificity detection of nucleic acids and non-nucleic acid targets is finally achieved.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to solve the signal output problem in the actual complex sample detection of the CRISPR technology, and the controllable amplification output of the signal can be realized by using rigid frame nucleic acid as a signal group. The output of multiple signals is achieved by modifying the redox indicator or fluorophore-quencher groups at the arm chain of the DNA tetrahedron, and multiplexing can be achieved. The modularized detection platform has good medical diagnosis and food safety monitoring application prospects.
In order to solve the technical problems, one of the technical schemes adopted by the invention is as follows:
providing a modular detection platform based on CRISPR technology coupled to a framework nucleic acid, the platform comprising:
(1) For nucleic acid targets, containing mediated crRNA aiming at the nucleic acid targets, incubating with Cas protein in advance to obtain Cas-crRNA complex as an identification module, and additionally containing frame nucleic acid signal modules with different valence states; the different valence state framework nucleic acid signal module is a DNA Tetrahedron (TDF) signal group with vertex extended by 1, 2, 3 or 4 arm chains and modifying electric signals or fluorescent signal molecules at the tail ends of the arm chains; or alternatively
(2) For a non-nucleic acid target, an aptamer containing non-nucleic acid target specific binding is paired with complementary strand aDNA in advance to form a double-chain aptamer-aDNA complex; the aDNA and crRNA are complementary strand aDNA and mediating crRNA designed according to the specific aptamer of the non-nucleic acid target. The mediated crRNA is pre-incubated with Cas protein to obtain Cas-crRNA complex as recognition module, and the mediated crRNA further comprises frame nucleic acid signal modules with different valence states; the different valence state framework nucleic acid signal modules are DNA Tetrahedron (TDF) signal groups with peaks extending 1, 2, 3 or 4 arm chains and modifying electric signals or fluorescent signal molecules at the tail ends of the arm chains.
The platform is used for capturing nucleic acid targets by a recognition module Cas-crRNA complex to form a Cas-crRNA-target complex, activating Cas protein side cleavage activity, and cleaving a TDF signal group to generate a changed current signal or a fluorescence signal; or alternatively
For non-nucleic acid targets, the target competes with the aptamer-aDNA duplex to form an aptamer-target complex, releasing aDNA. The released aDNA is captured by the recognition module Cas-crRNA complex to form the Cas-crRNA-aDNA complex, and simultaneously, the side cleavage activity of the Cas protein is activated to cleave the TDF signal group, so as to generate a changed current signal or a fluorescence signal.
Wherein the nucleic acid target can be a novel coronavirus, a human papillomavirus or a hepatitis B virus nucleic acid;
the non-nucleic acid targets can be proteins, antibiotics or metal ions, etc.
The Cas-crRNA complex is typically present in a buffer solution, which is typically 1×nebuffer 2.1 (containing 10U ribonuclease inhibitor).
The concentration of the Cas-crRNA complex is 50 nM-1 mu M.
The framework nucleic acid in the present invention is a DNA Tetrahedron (TDF).
The DNA tetrahedral buffer solution is TM buffer solution (50mM MgCl2, 20mM Tris,pH 8.0).
The DNA tetrahedron has a side length of 7bp, 13bp, 17bp, 26bp or 37bp.
The DNA tetrahedron arm chain is an oligonucleotide sequence extending from the vertex of the DNA tetrahedron, and the length is 10 nt-50 nt.
The redox indicator is methylene blue MB, ferrocene Fc, biotin, or a fluorescent group FAM-quenching group DABCYL.
The invention also provides a construction method of the modularized detection platform based on the coupling of the CRISPR technology and the framework nucleic acid, which comprises the following steps:
(1) Constructing a recognition module Cas-crRNA complex by using a CRISPR/Cas technology, and realizing high-sensitivity and high-specificity capturing of nucleic acid and non-nucleic acid targets;
(2) Constructing different valence state frame nucleic acid signal modules by using a DNA nanotechnology, and realizing controllable amplification output of detection signals;
(3) And (3) integrating the identification module obtained in the step (1) and the signal module obtained in the step (2) on an electrochemical chip or a paper chip to realize high-sensitivity and high-specificity detection of the target object.
Wherein the step (1) is as follows: and a CRISPR/Cas technology is utilized to construct an identification module Cas-crRNA complex, so that high-sensitivity and high-specificity capture of nucleic acid and non-nucleic acid targets is realized.
The method for constructing the recognition module Cas-crRNA complex comprises the following steps: designing crRNA hybridized with the gene sequence of the nucleic acid target; specific aptamers (aptamers) are selected for non-nucleic acid targets, and aDNA and crRNA for hybridization reaction are designed according to the aptamers. And then mixing the Cas protein and the crRNA in a concentration ratio of 1:1 in a 1 XNEBbuffer 2.1 and 10U RNase inhibitor solution, and incubating for 10 minutes at room temperature to obtain the final product.
Wherein the Cas protein is Cas12a, cas13, cas14.
Wherein the nucleic acid target is a novel coronavirus, human papillomavirus or hepatitis B virus nucleic acid.
Wherein the non-nucleic acid targets are proteins, antibiotics or metal ions, etc.
Wherein the concentration ratio of Cas protein to crRNA is 1:1. The concentration of the Cas-crRNA complex is 50 nM-1 mu M. The Cas-crRNA captures the target with a reaction time of 5-200 minutes. The concentration ratio of the aptamer to aDNA is 1:1, and the aptamer and aDNA are heated and annealed to form double chains.
Wherein the step (2) is as follows: and constructing nucleic acid signal modules with different valence frameworks by using a DNA nanotechnology, and realizing controllable amplification output of detection signals.
The method for constructing the signal module of the framework nucleic acid (TDF signal group) with different valence states comprises the following steps: TDF signal groups with different valence states are as described in example 3, and TDF-1 (one arm chain) consists of TDF ID NO. 1-TDF ID NO. 8; TDF-2 (two arm chains) consists of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 6-TDF ID NO 9; TDF-3 (three arm chains) is composed of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 7-TDF ID NO 10; TDF-4 (four arm chains) is composed of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 8-TDF ID NO 11. Mixing the 8 DNA strands constituting each TDF in TM buffer (pH 8.0) at equal ratio, heating to 95deg.C for 10 min, rapidly cooling to 4deg.C, and maintaining at 4deg.C for 5 min.
The TDF signal bolus may be 7bp, 13bp, 17bp, 26bp and 37bp in side length.
The DNA tetrahedron arm chain is an oligonucleotide sequence extending from the vertex of the DNA tetrahedron, and the length is 10 nt-50 nt.
The TDF signaling group may modify the redox indicator Methylene Blue (MB), ferrocene (Fc) or biotin (biotin), the fluorescent group FAM-quenching group DABCYL, and the like.
The incubation concentration of the TDF signal group at the interface is 25 nM-500 nM. Wherein, the signal change rate is maximum at 50 nM.
Wherein the step (3) is as follows: the recognition module and the signal module are integrated on an electrochemical chip or a paper chip, and after the recognition module is specifically combined with a target, the activated Cas protein cuts the signal module, so that a changed current signal or a fluorescence signal is generated, and high-sensitivity and high-specificity detection of the target object is realized.
Wherein the nucleic acid targets, after being recognized and captured by the recognition module Cas-crRNA complex, form Cas-crRNA-target complex while activating the paralytic cleavage activity of Cas protein. The non-nucleic acid targets compete with aptamer-aDNA for binding, releasing aDNA. After the released aDNA is recognized and captured by the recognition module Cas-crRNA complex, a Cas-crRNA-aDNA complex is formed, and simultaneously, the Cas protein side cleavage activity is activated. The activated Cas protein cleaves the signaling module TDF signaling bolus, producing a varying current signal or fluorescent signal. The cutting time is 5-200 minutes. The signal may be a current signal or a fluorescent signal.
The modular detection platform based on the coupling of the CRISPR technology and the framework nucleic acid is obtained, an identification module is constructed based on the CRISPR technology, the target is not required to be amplified in advance, and the signal controllable amplification output is realized by utilizing the signal module constructed by the framework nucleic acid, so that the detection with high sensitivity and high specificity is realized, and the false positive/false negative problem caused by the target amplification is effectively avoided; based on the frame nucleic acid as a signal module, sequence design and modification do not need to be carried out according to a target, the method has universality, not only has the effect of resisting nonspecific adsorption, but also can controllably amplify signals, and further realizes accurate and rapid detection of complex actual samples. The advantages of the two modules are combined, and the detection platform has good medical diagnosis and food safety monitoring application prospects.
Drawings
FIG. 1 is a representation of polyacrylamide gel electrophoresis of TDF signal groups of different valencies after purification;
FIG. 2 is a graph showing the morphology of an atomic force microscope of a purified TDF-4 signal group (containing 4 arm chains);
FIG. 3 is a representation of agarose gel electrophoresis of cleavage of TDF signal groups of different valencies with and without target capture by a recognition module;
FIG. 4 is a graph of the current of alternating volts before and after the addition of 1nM and 100nM HPV viral nucleic acid, wherein HPV is human papillomavirus;
FIG. 5 is a graph of the rate of change of signals from recognition modules incubated on different valency signal clusters.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1 preparation of Cas12a-crRNA Complex
crRNA was designed against human papillomavirus HPV16 nucleic acid sequences. The crRNA can be incubated with Cas12a at room temperature to form a Cas12a-crRNA complex. The HPV16 nucleic acid sequence and crRNA sequence are shown in HPV ID NO. 1-3, respectively.
Table 1 DNA and RNA sequences involved in the examples
Example 2 preparation of recognition module Cas12a-crRNA complexes
Cas12a protein is purchased from us New England Biolabs (NEB) technologies limited. crRNA and Cas12a protein were mixed in a concentration ratio of 1:1 in 1 x NEBbuffer 2.1, 10U rnase inhibitor solution and incubated for 10 minutes at room temperature.
EXAMPLE 3 Synthesis and purification of TDF Signal Condition
The oligonucleotide sequences and extended arm strands required for TDF signal cluster synthesis were all synthesized by Shanghai Ind technologies Inc. The sequences of the oligonucleotides are shown in TDF ID No. 1 to TDF ID No. 11, respectively. TDF-1 (an arm chain) is composed of TDF ID NO 1-TDF ID NO 8; TDF-2 (two arm chains) consists of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 6-TDF ID NO 9; TDF-3 (three arm chains) is composed of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 7-TDF ID NO 10; TDF-4 (four arm chains) is composed of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 8-TDF ID NO 11. The concentration of 8 DNA strands constituting each TDF was mixed in equal proportion in a TM buffer (pH 8.0), heated to 95℃for 10 minutes, then rapidly lowered to 4℃and maintained at 4℃for 5 minutes.
TDF signal clusters were purified using an Agilent 1260HPLC system, equipped with a size exclusion chromatography column (Phenomenex BioSec-SEC-4000,300mm-7.8 mm), and the chromatogram recorded at 260 nm. The buffer was replaced with a suitable structurally stable TM buffer by centrifugation using an Amicon Ultra-0.5mL ultrafiltration tube (MWCO 100 kDa) at 3000g for 10 minutes, followed by replenishment of the ultrafiltration tube with TM buffer to a total volume of 500uL and further centrifugation at 3000g for 10 minutes. The concentration of TDF signal clusters was then determined with an ultraviolet-visible absorption spectrophotometer. Agarose Gel Electrophoresis (AGE) confirmed the TDF synthesis results and polyacrylamide gel electrophoresis (PAGE) confirmed the modified methylene blue TDF synthesis results.
For comparison with the experimental group TDF signal bolus, TDF-0 without arm chain was synthesized as a control group.
The experimental results are shown in fig. 1, and the mobility of the band is reduced along with the increase of valence state, so that the successful synthesis of TDF signal groups with different valence states is proved, and the TDF signal groups can be used for further experimental study.
The morphology of TDF-4 (4-valent TDF signal group, containing 4 arm chains) was imaged using an atomic force microscope, as shown in FIG. 2, demonstrating that the morphology of the TDF-4 signal group is tetrahedral.
TABLE 2 DNA sequences involved in TDF in examples
TABLE 3 DNA sequences involved in TDF of different valences in the examples
Example 4 preparation of Cas12a-crRNA-target Complex
2. Mu.L of sample containing target is added into 98. Mu.L of Cas12a-crRNA complex, and the reaction is carried out for 10 minutes at room temperature, so as to obtain the Cas12a-crRNA-target complex. At this point, cas12a protein paralytic cleavage activity is activated.
As shown in fig. 3, after the recognition module Cas12a-crRNA complex captures the target and forms the Cas12a-crRNA-target complex, the activity of Cas12a protein is successfully activated, the TDF signal group with different valence states is cleaved, and the mobility of the TDF signal group strip after cleavage becomes fast.
Example 5 integration of identification Module and Signal Module on electrochemical chip
To achieve low cost detection, the identification module and the signal module are integrated on an electrochemical chip. The electrode chip was cleaned and purged with nitrogen (N) 2 ) Blow-dry, incubate TDF signal clusters on the working electrode surface, and then place the chip in a wet box overnight. The electrode chip was rinsed with 1 XPBS and N 2 And (5) blow-drying. The recognition module (containing target DNA) was incubated in the experimental group, the control group (without target DNA) was set, the electrode chip was rinsed with 1×pbs after 30 minutes, and finally 7 μl of PBS buffer solution was added dropwise for electrochemical measurement. Square wave voltammetry and alternating current voltammetry are used to measure the electrical signals: square wave voltammetry frequency is 10Hz; the ac voltammetry frequency was 50Hz.
As shown in fig. 4, the current signal gradually decreased after incubation of the control group (without target DNA), the recognition module (with 10nM target DNA), and the recognition module (with 100nM target DNA) on the signal module, respectively, and the signal change rate increased, indicating that the recognition module successfully cleaved the signal bolus.
EXAMPLE 6 investigation of the Signal Change Rate of different valence TDF Condition
TDF signal groups (modified MB) of different valences, each at 50nM, were incubated overnight on gold electrodes (diameter 2 mm), the electrodes were rinsed with 1 XPBS, and N 2 And (5) blow-drying. Recognition modules (containing 100nM target DNA) were incubated on each of the different valence signal clusters, the electrode chip was rinsed with 1 XPBS after 30 min, and finally electrochemical measurements were performed in PBS buffer. The electrical signal was measured using square wave voltammetry with a square wave voltammetry frequency of 50Hz. The rate of change of the signal clusters of different valencies is compared.
As shown in FIG. 5, the signal change rate becomes larger as the valence state increases, and the TDF-4 signal change rate becomes maximum.
The foregoing is merely a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the claims. The present invention is not described in detail in the conventional art.
Claims (8)
1. A modular detection platform based on CRISPR technology and frame nucleic acid coupling is characterized in that the platform comprises an identification module constructed by CRISPR technology and a signal module with different valence states constructed by adopting frame nucleic acid:
(1) For nucleic acid targets, containing mediated crRNA aiming at the nucleic acid targets, incubating with Cas protein in advance to obtain Cas-crRNA complex as an identification module, and additionally containing frame nucleic acid signal modules with different valence states; the different valence state framework nucleic acid signal module is a DNA Tetrahedron (TDF) signal group which extends 1, 2, 3 or 4 arm chains at the top and modifies an electric signal or fluorescent signal molecule at the tail end of the arm chain; or alternatively
(2) For a non-nucleic acid target, an aptamer containing non-nucleic acid target specific binding is paired with complementary strand aDNA in advance to form a double-chain aptamer-aDNA complex; the aDNA and crRNA are complementary strand aDNA and mediating crRNA designed according to the specific aptamer of the non-nucleic acid target; the mediated crRNA is pre-incubated with Cas protein to obtain Cas-crRNA complex as recognition module, and the mediated crRNA further comprises frame nucleic acid signal modules with different valence states; the different valence state framework nucleic acid signal module is a DNA Tetrahedron (TDF) signal group with vertex extended by 1, 2, 3 or 4 arm chains and modifying electric signals or fluorescent signal molecules at the tail ends of the arm chains; for nucleic acid targets, the targets react with the Cas-crRNA complex to form the Cas-crRNA-target complex, the Cas protein side cleavage activity is activated, and the TDF signal group is cleaved to generate a changed current signal or a fluorescence signal;
for a non-nucleic acid target, the target competes with an aptamer-aDNA double-strand to form an aptamer-target complex and release aDNA; the released aDNA reacts with the Cas-crRNA complex to form a Cas-crRNA-aDNA complex, and simultaneously, the side cleavage activity of the Cas protein is activated to cleave the TDF signal group to generate a changed current signal or a fluorescence signal;
wherein the signal molecule modified by the TDF signal group is a redox indicator methylene blue MB, ferrocene Fc, biotin or a fluorescent group FAM-quenching group DABCYL.
2. The platform of claim 1, wherein the nucleic acid is a novel coronavirus, human papillomavirus, or hepatitis b virus nucleic acid; the non-nucleic acid is protein, antibiotic or metal ion.
3. The platform of claim 1, wherein the Cas-crRNA complex concentration is 50nM to 1 μm.
4. The platform of claim 1, wherein the TDF signal clusters are DNA tetrahedrons of side lengths of 7bp, 13bp, 17bp, 26bp, or 37bp.
5. The platform of claim 1, wherein the DNA tetrahedron arm is 10nt to 50nt in length.
6. A method of constructing a modular detection platform based on CRISPR technology coupled to a framework nucleic acid according to any of claims 1 to 5, comprising the steps of:
(1) Constructing a recognition module Cas-crRNA complex by using a CRISPR/Cas technology, and realizing high-sensitivity and high-specificity capturing of nucleic acid and non-nucleic acid targets;
(2) Constructing different valence state frame nucleic acid signal modules by using a DNA nanotechnology, and realizing controllable amplification output of detection signals;
(3) And (3) integrating the identification module obtained in the step (1) and the signal module obtained in the step (2) on an electrochemical chip or a paper chip to realize high-sensitivity and high-specificity detection of the target object.
7. Use of a modular detection platform based on CRISPR technology coupled to a framework nucleic acid according to any of claims 1 to 5 for detection of a target.
8. The use of a modular detection platform based on CRISPR technology coupled to a framework nucleic acid according to claim 7 for detection of a target, wherein said use is for detection of a pathogenic nucleic acid, protein, small molecule or antibiotic.
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