D-p drive probe and cas12a cascade amplification method based on the probe

Abstract

The application discloses a D-p driving probe and a Cas12a cascade amplification method based on the probe, and belongs to the technical field of molecular diagnosis. The basic structure of the D-p driving probe is a hairpin type DNA with a breathing site, which is composed of a double-stranded stem and a single-stranded loop. The single-stranded loop is a trans-cleavage region. The double-stranded stem includes an activator, a foothold, a blocking region and a breathing site. In the above manner, the D-p driving probe has the dual functions of Cas12a trans-cleavage substrate and target activator, can connect the target recognition and trans-cleavage activity of Cas12a in series, realizes efficient signal cascade amplification, and simultaneously introduces the breathing site in the D-p driving probe, so that only the activator foothold is exposed after cutting, without affecting the key functional region, thereby avoiding false cutting and guaranteeing effective signal transmission. In addition, the ortho-allosteric activation mechanism based on the D-p driving probe can significantly inhibit the off-target effect of Cas12a and effectively improve the recognition specificity.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics technology, specifically to a Dp-driven probe and a Cas12a cascade amplification method based on the probe. Background Technology

[0002] CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / Cas) is an adaptive immune mechanism derived from prokaryotes. This system mainly consists of a specific guide RNA (crRNA) and the Cas protease. When the crRNA binds to the target DNA sequence through complementary pairing, it precisely guides the Cas protein to perform cis-cutting of the target site, thereby achieving efficient gene editing. Due to its ease of operation, high reliability, strong specificity, and excellent sensitivity, the CRISPR / Cas system has become an important tool in the field of biotechnology. In particular, the CRISPR / Cas12a system, since its discovery of highly efficient non-specific trans-cutting activity, has been widely used in the field of molecular diagnostics and is hailed as a "next-generation molecular diagnostic tool."

[0003] However, traditional CRISPR / Cas12a systems follow a "one target activates one Cas12a" model, with linear signal amplification, which typically limits detection sensitivity to the pM level, making it difficult to meet the clinical need for detecting low-abundance targets. Therefore, many early detection systems (such as DETECTR) relied on target pre-amplification steps to improve sensitivity. However, the introduction of pre-amplification makes the detection system complex and cumbersome to operate.

[0004] Furthermore, existing Cas12a detection systems are primarily suitable for routine nucleic acid targets, such as the identification of fusion gene subtypes and homogeneous detection of protein biomarkers, but they still have significant limitations. The main reason is that Cas12a protein not only exhibits strict activation specificity but also displays helicase activity, requiring only target nucleic acids of 14 or more bases for effective activation, especially in the "seed region" near the PAM sequence. Therefore, Cas12a / crRNA is prone to off-target effects when recognizing fusion gene junctions; even gene fragments that have not undergone fusion may non-specifically activate the system. Similarly, in current homogeneous protein detection based on the principle of adjacent junctions, the high off-target activity of Cas12a can lead to the activation of Cas12a by unbound protein probes, causing background signal leakage.

[0005] Based on this, the present invention designs a Dp-driven probe and a Cas12a cascade amplification method based on the probe to solve the above problems. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, this invention provides a Dp-driven probe and a Cas12a cascade amplification method based on the probe.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A Dp-driven probe, the basic structure of which is a hairpin DNA with a breathing site, consisting of a double-stranded stem and a single-stranded loop; wherein the single-stranded loop is a trans-cleavage region (c).

[0009] The double-stranded stem includes an activator (a), a foothold (b), a closed area (d), and a respiratory site;

[0010] The activator (a) of the Cas12a activation function is connected in series with the trans-shear region (c) of the trans-shear function;

[0011] The activator (a) is blocked by the blocking region (d). Both the activator (a) and the blocking region (d) are single-chain. At the same time, a breathing site is introduced in the blocking region (d). The breathing site allows the Dp probe to expose the foothold (b) of the activator (a) under the drive of respiration after being cleaved by Cas12a. Then, chain replacement is performed through the foothold (b) to activate the next Cas12a molecule. This cycle is repeated to form an exponential amplification effect of one target activating N Cas12a molecules.

[0012] The length of the foothold is 7-9 nt;

[0013] The length of the reverse shear zone is 10-15 nt;

[0014] The trans-cleavage region of the Dp-driven probe is located at the 3' end of the crRNA;

[0015] The sequence of crRNA is SEQ ID NO.6;

[0016] The Dp-driven probe is prepared by hybridization of H and L chains. The sequence of the H chain is SEQ ID NO.1, and the sequence of the L chain is SEQ ID NO.2.

[0017] To better achieve the objectives of this invention, the present invention also provides an application of a Dp-driven probe in the Cas12a exponential amplification reaction.

[0018] To better achieve the objectives of this invention, this invention also provides a method for preparing a Dp-driven probe, which is prepared by hybridization of H chain and L chain, wherein the sequence of H chain is SEQ ID NO.1 and the sequence of L chain is SEQ ID NO.2.

[0019] Furthermore, the specific preparation process is as follows:

[0020] The Dp-driven probe was prepared by hybridization of 1 μM H chain and 1.5 μM L chain under the following conditions: denaturation at 95℃ for 5 minutes and incubation at 37℃ for 60 minutes. After the reaction, it was stored at -20℃ for later use. The reaction buffer was NEB 1×buffer 2.1.

[0021] Compared with the prior art, the beneficial effects of this invention are as follows: 1. Dp has the dual functions of Cas12a trans-cleavage substrate and target activator, which can link the target recognition and trans-cleavage activity of Cas12a in series to achieve efficient signal cascade amplification.

[0022] 2. The respiratory sites introduced in Dp ensure that only the activator's foothold is exposed after cutting, without affecting key functional areas, thereby avoiding accidental cutting and ensuring effective signal transmission.

[0023] 3. The Dp-based ortho-allision activation mechanism can significantly inhibit the off-target effect of Cas12a. Under the blocking effect of the stem, the loop region is structurally stable and conventional 14-base DNA probes are difficult to open, effectively improving the recognition specificity. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0025] Figure 1 This is a schematic diagram of the structure of the Dp-driven probe of the present invention.

[0026] Where c represents the trans-shearing zone, a represents the activator, b represents the foothold, d represents the closed zone, and ▲ represents the breathing site;

[0027] Figure 2 This is a diagram showing the results of polyacrylamide gel electrophoresis performed after the Dp-driven probe was prepared according to the present invention.

[0028] Figure 3 The image shows the results of fluorescence spectroscopy analysis performed after the Dp-driven probe was prepared according to this invention.

[0029] Figure 4 This is a result of the feasibility verification of the Dp-driven probe-induced Cas12a cascade amplification of the present invention. Figure 1 ;

[0030] Figure 5 This is a result of the feasibility verification of the Dp-driven probe-induced Cas12a cascade amplification of the present invention. Figure 2 ;

[0031] Figure 6 This is a result of the feasibility verification of the Dp-driven probe-induced Cas12a cascade amplification of the present invention. Figure 3 ;

[0032] Figure 7 This is a schematic diagram of the Cas12a cascade amplification of the present invention;

[0033] Figure 8 The effect of the position of the trans-shearing region on the reaction in order to optimize the Dp-driven probe of this invention;

[0034] Figure 9 The influence of the length of the foothold of the Dp-driven probe of this invention on the reaction;

[0035] Figure 10 The influence of the length of the trans-shearing region on the reaction in order to optimize the Dp-driven probe of this invention;

[0036] Figure 11 This is a graph showing the sensitivity results of Cas12a cascade amplification induced by the Dp-driven probe of this invention.

[0037] Figure 12 This is a schematic diagram illustrating the principle of Dp-driven probe ortho-alteral metamorphism-induced Cas12a activation in this invention.

[0038] Figure 13 This is the result of the Dp-driven probe ortho-alteral metathesis-induced Cas12a activation in this invention. Figure 1 ;

[0039] Figure 14 This is the result of the Dp-driven probe ortho-alteral metathesis-induced Cas12a activation in this invention. Figure 2 . Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0041] Definitions of abbreviations and key terms:

[0042] Dp drives the probe: Dp.

[0043] CRISPR / Cas12a: The acquired immune system in prokaryotes, now widely used in gene editing, nucleic acid diagnostics and other fields.

[0044] Shear substrate: ss-FQ.

[0045] Guide RNA: crRNA, an RNA molecule in the CRISPR / Cas system, mainly guides Cas proteins to locate and recognize target DNA, and to pair complementary DNA with the target DNA.

[0046] Single-chain activator: Target.

[0047] Example 1: Design, fabrication and optimization of Dp-driven probes

[0048] 1. Design of Dp-driven probe

[0049] The basic structure of the Dp-driven probe is a hairpin DNA with a breathing site, consisting of a double-stranded stem and a single-stranded loop.

[0050] like Figure 1 As shown, the Dp probe is a specially designed DNA target probe whose main function is to break the traditional linear amplification mode of "one target activates one Cas12a" and construct an exponential amplification mode of "one target activates N Cas12a". This mode overcomes the challenge of low-background detection of fusion genes and protein biomarkers in existing CRISPR / Cas12a systems. To meet these practical needs, we have created an original Dp probe:

[0051] First, to construct the exponential amplification mode of "one target activating N Cas12a", it is necessary to cascade the dual functional modules of "activation" and "trans-splicing" of Cas12a. Therefore, the Dp probe needs to include the "activation" sequence (i.e., activator a) and the "trans-splicing" sequence (i.e., trans-splicing region c).

[0052] Meanwhile, when the Cas12a detection system is in a quiescent state (without a detection target), in order to ensure that Cas12a is not non-specifically "activated" by the "sequence activator" of the Dp probe, and at the same time that the "sequence activator" is not non-specifically "trans-cleaved" by the activated Cas12a, the sequence activator needs to be designed in a single-stranded state and needs to be blocked by another DNA single strand, i.e., the blocking region d;

[0053] When the Cas12a detection system is activated, to ensure that the Dp probe releases the blocked activator a after Cas12a trans-shearing, we introduced a breathing site ▲ in the blocking region d. This site allows the Dp probe to expose the site b of activator a through respiration after shearing; then, chain displacement occurs through this site b, activating the next Cas12a molecule. This cycle repeats, forming an exponential amplification effect of "one target activating N Cas12a molecules".

[0054] In addition, the Dp probe needs to drive the exponential amplification of "one target activating N Cas12a", and it also needs to be activated by recognition probes of fusion genes and protein markers to initiate the amplification process.

[0055] 2. Preparation of Dp-driven probes

[0056] The Dp-driven probe was prepared by hybridization of 1 μM H chain and 1.5 μM L chain under the following conditions: denaturation at 95℃ for 5 minutes and incubation at 37℃ for 60 minutes. After the reaction, it was stored at -20℃ for later use. The reaction buffer was NEB 1×buffer 2.1.

[0057] like Figure 2 As shown, the results of polyacrylamide gel electrophoresis (PAGE) indicate that the H chain and L chain successfully hybridized to form a double-stranded product.

[0058] To verify that the obtained product has a hairpin conformation, the fluorescent group FAM (HF) was labeled at the 5' end of the H chain, and the quencher group BHQ1 (LB) was labeled at the 3' end of the L chain, as follows: Figure 3 As shown, fluorescence spectroscopy analysis revealed that the fluorescence signal of HF was significantly quenched, indicating that the Dp-driven probe had successfully formed the expected structure.

[0059] 3. Optimization of Dp-driven probes

[0060] To improve the effect of the Dp-driven probe in driving the Cas12a cascade reaction, this study further optimized the key parameters of the Dp-driven probe.

[0061] First, using the 5'-3' orientation of crRNA as a reference, the effect of the position of the trans-cleavage region on the reaction was examined, as it directly determines the exposure direction of the activator's anchorage.

[0062] Theoretically, when the trans-splicing region is located at the 5' end of crRNA, the activation of Cas12a via strand displacement should be stronger; however, experimental results show that the effect is more significant when the trans-splicing region is located at the 3' end of crRNA. Figure 8 As shown, this is mainly because the 5' end position is prone to causing significant background signal leakage.

[0063] Secondly, the length of the toehold has a key impact on chain permutation efficiency: generally speaking, a longer toehold can enhance chain permutation ability, thereby improving the activation efficiency of Cas12a; however, an excessively long toehold can easily lead to an increase in background signal and may be non-specifically cleaved by Cas12a.

[0064] For this experiment, toehold lengths of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, and 10 nt were tested respectively. Figure 9 As shown, the best results were found when H-8 nt was used, as it had the lowest background signal while ensuring strong chain substitution capability.

[0065] Furthermore, the length of the reverse shearing zone not only affects the reverse cutting efficiency of Cas12a, but also relates to the structural stability of Dp.

[0066] Compare the lengths of four reverse shear regions: 5 nt, 10 nt, 15 nt, and 20 nt. Figure 10 As shown in the figure, the experimental results indicate that the reaction performance is optimal at 15 nt.

[0067] In summary, when the trans-splicing region of Dp is located at the 3' end of crRNA, the toehold length is 8 nt, and the trans-splicing region length is 15 nt, the effect of Cas12a cascade amplification can be maximized.

[0068] Example 2: Feasibility verification of Dp-induced Cas12a cascade amplification

[0069] First, 500 nM fluorescently labeled Dp-driven probe was added to the pre-activated Cas12a amplification system (containing 50 nM Cas12a, 100 nM crRNA, and 50 nM Target), as follows: Figure 4 As shown, its fluorescence signal is significantly enhanced, indicating that the ring region of Dp can be trans-cleaved by Cas12a, thereby releasing a fluorescence signal.

[0070] Secondly, to verify whether the cleaved Dp could effectively activate the new Cas12a protease, the cleavage product was added to a reaction system containing 50 nM Cas12a / crRNA, and a FAM / BHQ1 double-labeled single-stranded DNA (ss-FQ) was introduced as a signal probe. Figure 5 As shown, the fluorescence signal generated by this system can reach 93% of that of the Cas12a / crRNA system activated by the free single-stranded activator, indicating that the cleaved Dp-driven probe can expose the site and effectively activate the new Cas12a protein.

[0071] The results confirm that the Dp-driven probe can not only serve as a trans-cleavage substrate for Cas12a, but its cleavage product can also further activate new Cas12a proteins.

[0072] To investigate whether the Dp-driven probe could drive the cascade amplification of Cas12a, 30 nM Dp was premixed with 5 nM Target, 30 nM Cas12a / crRNA, and 500 nM ss-FQ and reacted at 37°C for 1 hour. Figure 6 As shown, the fluorescence signal intensity of this system was increased by 3 times compared with the Cas12a system activated only by a 5 nM target, indicating that the Dp-driven probe can indeed effectively drive the cascade amplification reaction of Cas12a.

[0073] See the schematic diagram of Cas12a cascade amplification. Figure 7 .

[0074] Example 3: Sensitivity of Dp-induced Cas12a cascade amplification

[0075] Based on optimal Dp conditions, the established Cas12a cascade amplification was used to detect different concentrations of the target, such as... Figure 11 As shown, the results indicate that the sensitivity can reach 10 fM.

[0076] The reaction system and conditions were 20 nM Dp, different concentrations of Target, 30 nM Cas12a / crRNA, 500 nM ss-FQ, and reaction at 37℃ for 1 hour.

[0077] Experimental Example: Feasibility Verification of Dp-Driven Probe Ortho-Allosteric Induction of Cas12a Activation

[0078] Using the leukemia fusion gene BCR-ABL P210 as a fusion gene detection model, we designed adjacent recognition probes BCR-p and ABL-p. When these probes recognize the fusion gene, they can interact adjacently to induce an allosteric change in Dp, exposing the site of the activator to activate Cas12a.

[0079] Because the loop region and part of the stem region of Dp need to be complementary to the adjacent recognition probe, based on the sequence of the adjacent recognition probe, while ensuring the correct structure of Dp, a portion of the H chain sequence is adjusted to H*, and the corresponding crRNA sequence is adjusted to crRNA*. The reaction principle of the above reaction is described in [link to reaction details]. Figure 12 .

[0080] The system and steps of the above reaction are as follows:

[0081] First, 100 nM BCR-ABL P210, 100 nM BCR-p, and 100 nM ABL-p were reacted at 37℃ for 30 min. Then, 50 nM fluorescent Dp (formed by H*-F and L chains) was added and reacted at 37℃ for 30 min. The control experiments were conducted without BCR-ABL P210, with BCR-p and ABL-p respectively. Figure 13 As shown, the fluorescence results indicate that Dp can only undergo allosteric transformation and release a fluorescence signal when BCR-ABL P210, BCR-p, and ABL-p are all present.

[0082] like Figure 14 As shown, when ortho-altered Cas12a was added to a mixture of 50 nM Cas12a / crRNA complex, 40 nM Dp, and 500 nM ss-FQ, the fluorescence signal increased significantly, indicating that the ortho-altered Dp can efficiently initiate the cascade amplification of Cas12a, that is, the Dp-driven probe can induce efficient activation of Cas12a through ortho-alteration.

[0083] The specific sequences of the nucleic acid chains involved in the above experiments are shown in Table 1:

[0084] Table 1 Summary of Nucleic Acid Sequences

[0085]

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Dp-driven probe, characterized in that, The basic structure of the Dp-driven probe is a hairpin DNA with a breathing site, consisting of a double-stranded stem and a single-stranded loop; the single-stranded loop is the trans-cleavage region (c). The double-stranded stem includes an activator (a), a foothold (b), a closed area (d), and a respiratory site; The activator (a) of the Cas12a activation function is connected in series with the trans-shear region (c) of the trans-shear function; The activator (a) is blocked by the blocking region (d). Both the activator (a) and the blocking region (d) are single-chain. At the same time, a breathing site is introduced in the blocking region (d). The breathing site allows the Dp probe to expose the foothold (b) of the activator (a) under the drive of respiration after being cleaved by Cas12a. Then, chain replacement is performed through the foothold (b) to activate the next Cas12a molecule. This cycle is repeated to form an exponential amplification effect of one target activating N Cas12a molecules. The trans-cleavage region of the Dp-driven probe is located at the 3' end of the crRNA; The sequence of crRNA is SEQ ID NO.6; The Dp-driven probe is prepared by hybridization of H and L chains. The sequence of the H chain is SEQ ID NO.1, and the sequence of the L chain is SEQ ID NO.

2.

2. The application of the Dp-driven probe according to claim 1 in the Cas12a exponential amplification reaction.

3. A method for preparing a Dp-driven probe according to claim 1, characterized in that, It was prepared by hybridization of H chain and L chain, with the sequence of H chain being SEQ ID NO.1 and the sequence of L chain being SEQ ID NO.

2.

4. The method for preparing the Dp-driven probe according to claim 3, characterized in that, The specific preparation process is as follows: The Dp-driven probe was prepared by hybridization of 1 μM H chain and 1.5 μM L chain under the following conditions: denaturation at 95℃ for 5 minutes and incubation at 37℃ for 60 minutes. After the reaction, it was stored at -20℃ for later use. The reaction buffer was NEB 1×buffer 2.1.

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