D-p driving probe and Cas12a cascade amplification method based on same

Abstract

The invention discloses a D-p driving probe and a Cas12a cascade amplification method based on the D-p driving probe, and belongs to the technical field of molecular diagnosis, the basic structure of the D-p driving probe is hairpin type DNA with respiration sites, the D-p driving probe is composed of a double-stranded stem part and a single-stranded loop part, and the single-stranded loop part is a trans-shear region; the double-stranded stem includes an activator, a foothold, a closure region, and a breathing site. Through the mode, the D-p driving probe has dual functions of a Cas12a trans-cleavage substrate and a target activator, and target recognition and trans-cleavage activity of Cas12a can be connected in series, so that efficient signal cascade amplification is realized; meanwhile, a breathing site introduced into the D-p driving probe enables only an activator footing point to be exposed after cutting, and a key functional area is not affected, so that mistaken cutting is avoided, and effective transmission of signals is guaranteed; in addition, an ortho-position allosteric activation mechanism based on the D-p driving probe can significantly inhibit the off-target effect of Cas12a, and the recognition specificity is effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular diagnostics, in particular to a D-p driven probe and a Cas12a cascade amplification method based on the probe. BACKGROUND

[0002] CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / Cas) is an adaptive immune mechanism derived from prokaryotes. The system is mainly composed of a specific guide RNA (crRNA) and a Cas proteinase; when the crRNA is combined with the target DNA sequence through complementary pairing, it can accurately guide the Cas protein to perform "cis cleavage" on the target, thereby realizing efficient gene editing. With its characteristics of simple 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 it was found to have high non-specific "trans cleavage" activity, it has been widely used in the field of molecular diagnostics and has been hailed as a "new generation of molecular diagnostic tool".

[0003] However, the traditional CRISPR / Cas12a system follows the mode of "one target activates one Cas12a", and its signal amplification process is linear amplification, which limits the detection sensitivity to usually pM level, making it difficult to meet the detection needs of low-abundance targets in clinical practice. Therefore, the early established detection system (such as DETECTR) needs to rely on the pre-amplification step of the target to improve the sensitivity. However, the introduction of pre-amplification makes the detection system components complex and the operation cumbersome.

[0004] In addition, the existing Cas12a detection system is mainly suitable for conventional nucleic acid targets, such as the identification of fusion gene subtypes and the homogeneous detection of protein markers, and still has obvious deficiencies, the main reason for which is that the Cas12a protein not only has strict activation specificity, but also shows helicase activity, and only a target nucleic acid of 14 or more bases can be effectively activated, especially in the "seed region" near the PAM sequence, which is more sensitive. Therefore, Cas12a / crRNA is prone to off-target effects when recognizing the junction of fusion genes, and even gene fragments that have not been fused may also non-specifically activate the system. Similarly, in the current homogeneous protein detection based on the principle of adjacent ligation, the high off-target activity of Cas12a can cause the free probe of the unbound protein to also activate Cas12a, causing background signal leakage.

[0005] Based on this, the present application designs a D-p driven probe and a Cas12a cascade amplification method based on the probe to solve the above problems. SUMMARY

[0006] In view of the above-mentioned shortcomings of the prior art, the present application provides a D-p driven probe and a Cas12a cascade amplification method based on the probe.

[0007] To achieve the above object, the present application is realized by the following technical solutions: A D-p driven probe, the basic structure of the D-p driven probe is a hairpin type DNA with a breathing site, which is composed of a double-stranded stem and a single-stranded loop; wherein the single-stranded loop is a transcleavage region (c); The double-stranded stem includes an activator (a), a foothold (b), a blocking region (d) and a breathing site; The activator (a) of the activation function of Cas12a is connected in series with the transcleavage region (c) of the transcleavage function; The activator (a) is blocked by the blocking region (d), and the activator (a) and the blocking region (d) are both single-stranded, and a breathing site is introduced in the blocking region (d), the breathing site enables the D-p probe to expose the foothold (b) of the activator (a) under the driving of respiration after being cut by Cas12a, and then performs strand displacement through the foothold (b), activates the next Cas12a molecule, and so on, forming an exponential amplification effect of activating N Cas12a target.

[0008] Further, the length of the foothold is within 7-9 nt, the strand displacement ability is strong, and the activation efficiency of Cas12a is high.

[0009] Further, the length of the transcleavage region is within 10-15 nt, the strand displacement ability is strong, and the activation efficiency of Cas12a is high.

[0010] Further, the transcleavage region of the D-p driven probe is located at the 3' end of the crRNA.

[0011] Further, the sequence of the foothold is SEQ ID NO. 16.

[0012] Further, the sequence of the transcleavage region is SEQ ID NO. 26.

[0013] Further, the sequence of the crRNA is SEQ ID NO. 6.

[0014] In order to better achieve the object of the present application, the present application further provides an application of the D-p driven probe in a Cas12a exponential amplification reaction.

[0015] In order to better achieve the purpose of the present application, the present application also provides a preparation method of the D-p driving probe, which is prepared by hybridization of an H chain and an L chain, the sequence of the H chain is SEQ ID NO. 1, and the sequence of the L chain is SEQ ID NO. 2.

[0016] Further, the specific process for preparation is as follows: The D-p driving probe is prepared by hybridization of 1 μM H chain and 1.5 μM L chain, the hybridization condition is denaturation at 95℃ for 5 minutes and incubation at 37℃ for 60 minutes, and after the reaction is completed, the product is stored at -20℃ for standby; and the reaction buffer is NEB 1×buffer 2.1.

[0017] Compared with the prior art, the present application has the following beneficial effects: 1. The D-p has the dual functions of Cas12a trans-cleavage substrate and target activator, can combine the target recognition and trans-cleavage activity of Cas12a in series, and realizes efficient signal cascade amplification; 2. The introduction of the breathing site in the D-p exposes only the activator foothold after cleavage, without affecting the key functional region, thereby avoiding mis-cleavage and protecting the effective transmission of the signal; 3. The ortho-allosteric activation mechanism based on the D-p can significantly inhibit the off-target effect of Cas12a, under the blocking effect of the stem part, the structure of the loop part region is stable, and the conventional 14-base DNA probe is difficult to open it, thereby effectively improving the recognition specificity. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0019] Figure 1 is a structural schematic diagram of the D-p driving probe of the present application, wherein c represents the trans-cleavage region, a represents the activator, b represents the foothold, d represents the blocking region, and▲represents the breathing site; Figure 2 is a result diagram of polyacrylamide gel electrophoresis after the D-p driving probe of the present application is prepared; Figure 3 is a result diagram of fluorescence spectrum analysis after the D-p driving probe of the present application is prepared; Figure 4 is a result of feasibility verification of the D-p driving probe of the present application inducing Cas12a cascade amplification Figure 1 ; Figure 5This is a result of the feasibility verification of the Dp-driven probe-induced Cas12a cascade amplification of the present invention. Figure 2 ; 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 ; Figure 7 This is a schematic diagram of the Cas12a cascade amplification of the present invention; 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; Figure 9 The influence of the length of the foothold of the Dp-driven probe of this invention on the reaction; 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; Figure 11 This is a graph showing the sensitivity results of Cas12a cascade amplification induced by the Dp-driven probe of this invention. Figure 12 This is a schematic diagram illustrating the principle of Dp-driven probe ortho-alteral transformation inducing Cas12a activation in this invention. Figure 13 This is the result of the Dp-driven probe ortho-alteral metathesis-induced Cas12a activation in this invention. Figure 1 ; Figure 14 This is the result of the Dp-driven probe ortho-alteral metathesis-induced Cas12a activation in this invention. Figure 2 . Detailed Implementation

[0020] 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.

[0021] Definitions of abbreviations and key terms: Dp drives the probe: Dp.

[0022] CRISPR / Cas12a: The acquired immune system in prokaryotes, now widely used in gene editing, nucleic acid diagnostics and other fields.

[0023] Shear substrate: ss-FQ.

[0024] Guide RNA: crRNA, an RNA molecule in the CRISPR / Cas system, mainly guides the Cas protein to locate and recognize the target DNA, and is complementary to the target DNA.

[0025] Single-stranded activator: Target.

[0026] Example 1: Design, preparation and optimization of D-p driving probe 1. Design of D-p driving probe The basic structure of D-p driving probe is a hairpin DNA with a breathing site, which is composed of a double-stranded stem and a single-stranded loop.

[0027] As shown in Figure 1 , D-p probe is a specially designed DNA target probe, which mainly functions to break the traditional linear amplification mode of “one target activating one Cas12a”, and construct an exponential amplification mode of “one target activating N Cas12a”, and this mode can overcome the difficulty of low background detection of fusion genes and protein markers that cannot be achieved by existing CRISPR / Cas12a system. In order to meet the above practical needs, we designed the D-p probe: Firstly, to construct the exponential amplification mode of “one target activating N Cas12a”, the “activation” and “transcriptional cleavage” dual functional modules of Cas12a need to be linked in series, therefore, D-p probe needs to include the sequence of “activation” (i.e. activator a) and the sequence of “transcriptional cleavage” (i.e. transcriptional cleavage region c); At the same time, when the Cas12a detection system is in a silent state (no detection target), in order to ensure that Cas12a is not activated by the “sequence activator” of D-p probe, and the “sequence activator” is not transcribed and cleaved by the activated Cas12a, the sequence activator needs to be designed as a single-stranded state, and needs to be closed by another DNA single strand, i.e. closing region d; When the Cas12a detection system is activated, in order to ensure that the D-p probe can release the closed “activator a” after being “transcriptional cleaved” by Cas12a, we introduce a breathing site▲ in the closing region d. This site makes the D-p probe cut and expose the foothold b of “activator a” under the driving of respiration; then through the foothold b, the next Cas12a molecule is activated. In this way, the exponential amplification effect of “one target activating N Cas12a” is formed.

[0028] In addition, D-p probe needs to drive the exponential amplification of “one target activating N Cas12a”, and also needs to be activated by the recognition probe of fusion genes and protein markers to start the amplification process.

[0029] 2. Preparation of D-p driving probe The D-p driving probe was prepared by hybridization of 1 μM H strand and 1.5 μM L strand under the following conditions: denaturation at 95 °C for 5 min, incubation at 37 °C for 60 min, and storage at -20 °C after reaction completion; the reaction buffer was NEB 1× buffer 2.1.

[0030] As shown in the polyacrylamide gel electrophoresis (PAGE) results, the H strand was successfully hybridized with the L strand to form a double-stranded product. Figure 2

[0031] To verify that the product has a hairpin conformation, a fluorescent group FAM was labeled at the 5' end of the H strand (H-F), and a quencher group BHQ1 was labeled at the 3' end of the L strand (L-B), as shown in Figure 3 Fluorescence spectrum analysis showed that the fluorescence signal of H-F was significantly quenched, indicating that the D-p driving probe had successfully formed the expected structure.

[0032] 3. Optimization of D-p driving probe To improve the effect of D-p driving probe in driving Cas12a cascade reaction, the key parameters of D-p driving probe were further optimized.

[0033] First, based on the 5'-3' direction of crRNA, the position of the trans-cleavage region was investigated for its effect on the reaction, as it directly determines the exposure direction of the activator foothold.

[0034] In theory, when the trans-cleavage region is located at the 5' end of crRNA, the foothold activates Cas12a through strand displacement, which should be more effective; however, the experimental results showed that the effect was more significant when the trans-cleavage region was located at the 3' end of crRNA, as shown in Figure 8

[0035] Second, the length of the foothold has a key impact on the efficiency of strand displacement: generally, a longer toehold can enhance the ability of strand displacement, thereby improving the activation efficiency of Cas12a; however, too long can easily lead to an increase in background signal and may be non-specifically cleaved by Cas12a.

[0036] Therefore, the toehold lengths of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, and 10 nt were tested, as shown in Figure 9 Finally, it was found that H-8 nt had the best effect, as it ensured strong strand displacement ability while having the lowest background signal.

[0037] ​​In addition, the length of the trans-splicing region not only affects the trans-cleavage efficiency of Cas12a, but also relates to the structural stability of D-p.

[0038] Comparing 5 nt, 10 nt, 15 nt and 20 nt 4 trans-splicing region lengths, as Figure 10 The experimental results show that the reaction performance is best when the length of the trans-splicing region is 15 nt.

[0039] In summary, when the trans-splicing region of D-p is located at the 3' end of crRNA, the toehold length is 8 nt, and the length of the trans-splicing region is 15 nt, the effect of Cas12a cascade amplification can be maximally enhanced.

[0040] Example 2: Verification of the feasibility of D-p induced Cas12a cascade amplification First, 500 nM fluorescently labeled D-p driving probe was added to the pre-activated Cas12a amplification system (containing 50 nM Cas12a, 100 nM crRNA and 50 nM Target), as Figure 4 As shown, the fluorescence signal was significantly improved, indicating that the loop region of D-p can be trans-cleaved by Cas12a, thereby releasing the fluorescence signal.

[0041] Secondly, in order to verify whether the cleaved D-p can effectively activate new Cas12a protease, the cleavage product was added to the reaction system containing 50 nM Cas12a / crRNA, and a FAM / BHQ1 double-labeled single-stranded DNA (ss-FQ) was introduced as a signal probe. As Figure 5 As shown, the fluorescence signal produced by this system can reach 93% of the Cas12a / crRNA system activated by free single-stranded activator, indicating that the cleaved D-p driving probe can expose the foothold and effectively activate new Cas12a protein.

[0042] The above results confirm that the D-p driving probe not only can be used as a trans-cleavage substrate for Cas12a, but also can further activate new Cas12a protein.

[0043] In order to explore whether the D-p driving probe can drive the cascade amplification of Cas12a, 30 nM D-p was pre-mixed with 5 nM Target, 30 nM Cas12a / crRNA and 500 nM ss-FQ and reacted at 37°C for 1 hour. As Figure 6 As shown, the fluorescence signal intensity of this system is 3 times higher than that of the Cas12a system activated by only 5 nM target, indicating that the D-p driving probe can indeed effectively drive the cascade amplification reaction of Cas12a.

[0044] The schematic diagram of Cas12a cascade amplification is shown in Figure 7.

[0045] Example 3: Sensitivity of D-p induced Cas12a cascade amplification Based on the optimal D-p conditions, the established Cas12a cascade amplification was used to detect different concentrations of Target, such as Figure 11 As shown in the results, the sensitivity can reach 10 fM.

[0046] The reaction system and reaction conditions are 20 nM D-p, different concentrations of Target, 30 nM Cas12a / crRNA, 500 nM ss-FQ, and 37°C reaction for 1 hour.

[0047] Experimental example: feasibility verification of D-p driven probe para- allosteric induction of Cas12a activation Taking leukemia fusion gene BCR-ABL P210 as a fusion gene detection model, para-allosteric recognition probes BCR-p and ABL-p were designed. When the pair of probes recognize the fusion gene, para-allosteric effect occurs, which induces the allosteric change of D-p, exposes the foothold of the activator to activate Cas12a.

[0048] Because the loop region and part of the stem region of D-p need to be complementary to the para-allosteric recognition probe, according to the sequence of the para-allosteric recognition probe, under the premise of ensuring the correct structure of D-p, part of the sequence of H chain is adjusted to H*, and the corresponding crRNA sequence is adjusted to crRNA*, the reaction principle of the above reaction is shown in Figure 12 .

[0049] The system and steps of the above reaction are: First, 100 nM BCR-ABL P210, 100 nM BCR-p and 100 nM ABL-p were reacted at 37°C for 30 min, then 50 nM fluorescent D-p (formed by H*-F and L chain) was added and reacted at 37°C for 30 min. The control experiments were not added BCR-ABL P210, BCR-p, ABL-p, respectively, as shown in Figure 13 The fluorescence results show that only when BCR-ABL P210, BCR-p and ABL-p are present, the allosteric change of D-p can be started, thereby releasing the fluorescence signal.

[0050] As shown in Figure 14 The para-allosteric Cas12a was added to the mixed system of 50 nM Cas12a / crRNA complex, 40 nM D-p and 500 nM ss-FQ, and the fluorescence signal was greatly increased, indicating that the para-allosteric D-p can efficiently start the cascade amplification of Cas12a, that is, the D-p driven probe can para-allosterically induce the efficient activation of Cas12a.

[0051] The specific sequences of the nucleic acid strands involved in the above experiments are shown in Table 1: Table 1 Summary of nucleic acid sequences

[0052] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements will not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

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.

2. The Dp-driven probe according to claim 1, characterized in that, The length of the foothold is 7-9 nt.

3. The Dp-driven probe according to claim 1, characterized in that, The length of the reverse shear zone is 10-15 nt.

4. The Dp-driven probe according to claim 1, characterized in that, The trans-cleavage region of the Dp-driven probe is located at the 3' end of the crRNA.

5. The Dp-driven probe according to claim 2, characterized in that, The sequence of the foothold is SEQ ID NO.

16.

6. The Dp-driven probe according to claim 3, characterized in that, The sequence of the trans-shear region is SEQ ID NO.

26.

7. The Dp-driven probe according to claim 4, characterized in that, The sequence of crRNA is SEQ ID NO.

6.

8. The application of a Dp-driven probe according to any one of claims 1-7 in the Cas12a exponential amplification reaction.

9. A method for preparing a Dp-driven probe according to any one of claims 1-7, 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.

10. The method for preparing the Dp-driven probe according to claim 9, 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.

Images