Structural probe and use thereof

Abstract

Provided are a structural probe and use thereof. A nucleic acid probe that is a single-stranded oligonucleotide. The single-stranded oligonucleotide has a stem-loop structure, and the number of stem-loops is greater than 2. The nucleic acid probe, as an RNase reaction substrate, has ultra-high sensitivity higher than that of common single-stranded fluorescent probes, and can detect RNase A at concentrations as low as 0.1 pg / mL. The nucleic acid probe has a broad-spectrum capacity; the probe has the ability to detect a variety of ribonucleases.

Description

Structural probes and uses thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a structural probe and uses thereof. BACKGROUND

[0002] Ribonuclease (RNase) is a class of enzymes that can catalyze the degradation of RNA into small fragments, including dozens of different types. Common RNases include RNase A, RNase B, RNase C, RNase T, RNase P, etc. Among them, RNase A is the most widely used, which can specifically degrade the C and U residues of single-stranded RNA, catalyze the cleavage of the phosphodiester bond between the 5'-ribose of nucleotide and the 3'-ribose of adjacent pyrimidine nucleotide, and form oligonucleotides with 2', 3'-cyclic phosphate derivatives.

[0003] RNase exists widely, mainly produced by bacteria, microorganisms and even experimental personnel themselves, which can directly enter the experimental system through gloves, pipettes or droplets, etc., and affect the results of the experiment. Some experimental materials also have endogenous RNase contamination, which is difficult to detect and avoid, and has a great impact. Extremely small amount of RNase (pg level or above) can cause a large amount of RNA in the sample to be degraded in a short time. Up to now, its high heat resistance and activity still lead to that there is no effective means to avoid its generation and no suitable method to safely and reliably remove RNase existing in the contaminated raw materials in the conventional laboratory. Therefore, for those skilled in the art, the pre-detection of RNase in the experimental sample has become one of the key factors to judge whether the experiment can be successful.

[0004] The detection methods of RNase mainly include (1) radioactive isotope method (Egly, J. M., and J. Kempf. Detection and estimation of very low ribonuclease activities in biological fluids. Febs Letters 1976, 63(2): 250-254.), which has a sensitivity of 0.01 pg / ml, but requires the use of raw materials that may cause personal injury, so it is not suitable for use as a routine method; (2) electrochemical method (JC Ni and H Lin. Homogeneous Electrochemiluminescence Biosensor for the Detection of RNase A Activity and Its Inhibitor. Anal. Chem., 2019, 91: 14751-14756), which has extremely high detection sensitivity, but has high requirements for raw material synthesis, experimental operation, and test equipment, and is not easy to implement quality control in ordinary laboratories; (3) spectrophotometric method (Witmer, M. R, et al. U-3'-BCIP: a chromogenic substrate for the detection of RNase A in recombinant DNA expression systems. Nucleic Acids Research 1991, 19(1): 1-4.), which is simple to operate but has poor detection sensitivity; (4) fluorescence method (JY Du, YZ Dong, H Liu, LL Gong, S Lu, CY Yang, Y Li. Thioflavin T as luminescence biosensors for nucleic acid study and RNase A activity detection. Microchemical Journal, 2019, 147: 842-847), which has high specificity and selectivity, but requires that the measured component does not contain metal ions, greatly limiting its universal application.

[0005] In summary, RNase detection is a key link in quality control, and the existing technologies still have defects. In order to overcome the shortcomings of the existing RNase detection methods, such as complex experiments, low sensitivity, high instrument requirements, etc., to help technicians in the field easily, quickly and accurately master the RNase contamination in the sample to be tested and the environment, it is necessary to propose a new RNase detection method. SUMMARY

[0006] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide a structure probe and its application for solving the problems in the prior art.

[0007] To achieve the above-mentioned purpose and other related purposes, the first aspect of the present application provides a nucleic acid probe, which is a single-stranded oligonucleotide, and the single-stranded oligonucleotide has a stem-loop structure; the number of stem-loop structures is ≥2.

[0008] In any embodiment of the present application, the stem-loop structure comprises a stem and a loop connected, and the single-stranded oligonucleotide further comprises a single-stranded fragment, and the stems of adjacent stem-loop structures are directly connected by a single strand or connected by a single-stranded fragment. Preferably, the length of the single-stranded fragment is 1-10 nt, the sequence length of the loop is 3-10 nt, and the sequence length of the stem is ≥2 bp. Optionally, the stem contains mismatched base pairs, degenerate or non-matching nucleotides. The sequence of the single-stranded fragment contains C bases, U bases or G bases.

[0009] In any embodiment of the present application, the length of the single-stranded fragment is 2-10 nt, and the sequence length of the stem is 2-10 bp. The sequence of the single-stranded fragment contains C bases or U bases.

[0010] In any embodiment of the present application, the number of stem-loop structures is 2-3.

[0011] In any embodiment of the present application, any base of the nucleic acid probe is a natural, modified or unnatural nucleoside base.

[0012] In any embodiment of the present application, optionally, the nucleic acid probe contains modified ribose, and the modification mode comprises one or more of the following: 2’-O-methylation modification, 2’-methoxyethyl modification, 2’-F modification, 2’-deoxy modification, and 2’-amino modification.

[0013] In any embodiment of the present application, optionally, the nucleic acid probe contains a phosphodiester bond modification of linked nucleotides, and the phosphodiester bond modification comprises a sulfur modification or a boron modification of a non-bridge oxygen atom in the phosphate skeleton.

[0014] In any embodiment of the present application, the total length of the nucleic acid probe is 10-100 nt. The 5’ end and the 3’ end of the nucleic acid probe have 0-10 nt of asymmetric bases.

[0015] In any embodiment of the present application, the single-stranded oligonucleotide is RNA.

[0016] In any embodiment of the present application, the sequence of the nucleic acid probe is shown in SEQ ID NO:3-17 or SEQ ID NO:21.

[0017] In any embodiment of the present application, the nucleic acid probe has a fluorescent group and a quencher group at its two ends, the fluorescent group is selected from FAM, HEX, TET, ROX, TAMRA, JOE, VIC, NED, Quasar, Taxas Red or Cy; preferably, the fluorescent group is FAM; the quencher group is selected from BHQ, TRAMA, FAM, MGB, Eclipse or Dabcyl; preferably, the quencher group is BHQ.

[0018] The second aspect of the present application provides the use of the aforementioned nucleic acid probe as a substrate of RNase.

[0019] In any embodiment of the present application, the RNase includes a combination of one or more of RNase A, RNase H and RNase I.

[0020] The third aspect of the present application provides the use of the aforementioned nucleic acid probe in the preparation of a product for detecting ribonuclease.

[0021] The fourth aspect of the present application provides a method for detecting ribonuclease, wherein the aforementioned nucleic acid probe is added to a sample to be tested to react, and the ribonuclease in the sample to be tested is qualitatively or quantitatively detected according to the fluorescence value in the reaction product.

[0022] In any embodiment of the present application, the ribonuclease includes a combination of one or more of RNase A, RNase T, RNase I and RNase H.

[0023] In any embodiment of the present application, the working concentration of the nucleic acid probe is 0.01-10 pmol / μL.

[0024] In any embodiment of the present application, the reaction further includes a reaction buffer and enzyme-free water; preferably, the reaction buffer includes 10-100 mM Tris and 0-0.1 M MgCl2.

[0025] In any embodiment of the present application, when the fluorescence value of the sample to be tested after the reaction changes by more than 1.1 times compared with the fluorescence value before the reaction, it is determined that the sample to be tested contains ribonuclease, otherwise the sample to be tested does not contain ribonuclease.

[0026] In any embodiment of the present application, in quantitative detection, a standard curve is further constructed, the fluorescence value of the sample to be tested is compared with the standard curve to obtain the content of ribonuclease in the sample to be tested; preferably, the standard curve is obtained according to the concentration of ribonuclease standard product with different concentrations and the corresponding fluorescence value.

[0027] In any embodiment of the present application, the method for determining the fluorescence value comprises detection by a fluorescence spectrophotometer, PCR, isothermal amplification, electrophoresis, HPLC, electrochemistry, or naked eye observation under specific light.

[0028] The fifth aspect of the present application provides a detection kit for a ribonuclease, comprising the nucleic acid probe, the reaction buffer, and the enzyme-free water as described above; preferably, the detection kit further comprises an RNase standard.

[0029] In any embodiment of the present application, the working concentration of the nucleic acid probe is 0.01-10 pmol / μL.

[0030] In any embodiment of the present application, the reaction buffer comprises 10-100 mM Tris and 0-0.1 M MgCl2.

[0031] In any embodiment of the present application, the RNase of the RNase standard is selected from RNase A, RNase T, RNase I, or RNase H.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] 1. The detection method of the present application has high sensitivity, and as a substrate for RNase reaction, it has higher sensitivity than ordinary single-stranded fluorescent probes, and can detect as low as 0.1 pg / ml of RNase A.

[0034] 2. The detection method of the present application has fast reaction speed, and the result can be obtained within one hour, which can be observed in real time and has lower requirements for equipment, and is more suitable for routine laboratory quality control.

[0035] 3. The probe designed in the present application contains more stem-loop structures, and has stronger stability than ordinary single-stranded probes.

[0036] 4. The probe of the present application has broad spectrum, and has detection ability for various ribonucleases.

[0037] 5. The present application has wide application scenarios, and can detect experimental raw materials, environmental samples, etc.

[0038] 6. The present application is safe, and the materials used do not contain harmful ingredients. BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1 is a schematic diagram of the structure of the probe.

[0040] FIG. 2 is a comparison of the response of single-stranded probes and structural probes to RNase.

[0041] FIG. 3 is a standard curve for detecting low-concentration RNase A by SEQ ID NO: 3.

[0042] Figure 4 is the response of different sequences of structural probes to RNase A.

[0043] Figure 5 is the response of different lengths of structural probes to RNase A.

[0044] Figure 6 is the response of structural probes with different stem lengths to RNase A.

[0045] Figure 7 is the response of structural probes with different single strand lengths to RNase A.

[0046] Figure 8 is the response of structural probes with different single strand sequences to RNase A.

[0047] Figure 9 is the response of structural probes with different stem loop numbers to RNase A.

[0048] Figure 10 is the response of structural probes with different asymmetric base lengths to RNase A.

[0049] Figure 11 is a commercial enzyme and environmental pollution detection example.

[0050] Figure 12 is the amplification curve of RNase detection PCR method.

[0051] Figure 13 is the standard curve of RNase detection PCR method.

[0052] Figure 14 is the sensitivity result of RNase detection isothermal amplification method. DETAILED DESCRIPTION

[0053] In order to make the purposes, technical solutions and beneficial effects of the present application clearer, the present application will be further described below in conjunction with examples. It should be understood that the examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure.

[0054] The inventors of the present application have found a structural probe and its application after a large amount of exploration and research, and on this basis, the present application is completed.

[0055] The first aspect of the present application provides a nucleic acid probe, which has a connected stem loop structure; the number of stem loops is ≥2. The probe designed in the present application contains more stem loop structures, and has stronger stability than ordinary single-stranded probes. The present application research has found that probes containing only single-stranded fragments and probes with one stem loop structure have poor quantitative detection effect on RNase with a concentration of less than 10 pg / mL, and probes containing two or more stem loops can effectively improve the detection sensitivity of the probe. In some preferred embodiments, the number of stem loops is 2-3.

[0056] Each base of the nucleic acid probe is a natural, modified or unnatural nucleobase. Alternatively, the nucleic acid probe contains modified ribose, and the modification includes one or more of the following: 2'-O-methylation, 2'-methoxyethylation, 2'-F modification, 2'-deoxy modification, and 2'-amino modification. The nucleic acid probe contains a modification of the phosphodiester bond connecting the nucleotides, and the modification includes a sulfur modification or a boranation modification of a non-bridge oxygen atom in the phosphate backbone. The probe provided in the present application has high design flexibility and can be designed and adjusted to achieve different purposes. For example, a DNA chimeric fragment can be added to improve the detection sensitivity of a specific ribonuclease, a modified group such as a dioxymethyl modification can be used to further improve the stability of the probe, and the stability of the secondary structure can be adjusted by mismatch design of the sequence, so that the fluorescent or quenching group is released faster when reacting with the ribonuclease, thereby improving the reaction speed of the experiment.

[0057] In the nucleic acid probe provided in the present application, the stem-loop structure includes a stem and a loop connected thereto, and the single-stranded oligonucleotide further includes a single-stranded fragment. The stems of adjacent stem-loop structures are directly connected by a single strand or connected by a single-stranded fragment. The stem is directly connected by a single strand means that the stems are directly connected by any one of the strands in the stem double strand.

[0058] In the nucleic acid probe provided in the present application, the length of the single-stranded fragment is 1-10 nt; preferably, the length of the single-stranded fragment is 2-10 nt; specifically, it can be 2-5 nt, 5-7 nt or 7-10 nt, etc. The present application finds that the detection effect of the 2-5 base single-stranded fragment part of the probe is the best. In some embodiments, the sequence of the single-stranded fragment contains C base, U base or G base; preferably, the sequence of the single-stranded fragment contains C base or U base. The present application finds that the effect of the sequence of the single-stranded fragment containing C base or U base is better than that containing A base and better than that containing G base.

[0059] The nucleic acid probe provided in the present application has a loop with a sequence length of 3-10 nt; specifically, it can be 3-5 nt, 5-7 nt, or 7-10 nt, etc. The stem has a sequence length of 2 bp or more. Preferably, the stem has a sequence length of 2-10 bp; specifically, it can be 2-5 bp, 5-8 bp, or 8-10 bp, etc. Optionally, the stem contains mismatched base pairs, degenerate, or mismatched nucleotides. Among them, the mismatched base pairs refer to the paired bases that do not comply with the Watson / Crick base pairing principle, for example, the sequence "5'-A-C-G-U-3'" and "5'-G-C-G-U-3'" are complementary; the degenerate nucleotides refer to universal bases that can form pairs with each natural RNA or DNA, for example, hypoxanthine, 7-deaza-2-deoxyadenosine; the mismatched nucleotides refer to bases that do not pair, such as "5'-G-G-G-A-C-G-G-G-3'" and "5'-C-C-C-G-A-C-C-C-3'" when the two sequences are complementary, the "A-A" bases do not pair, or the number of any single strand in the stem double strand is not equal to the other strand, such as the complementary sequences "5'-A-C-G-G-C-U-3'" and "5'-A-C-C-G-U-3'". The present application found that when the number of paired bases in the stem is greater than 10, the sensitivity of the probe decreases with the increase of the sequence in the stem.

[0060] The nucleic acid probe provided in the present application has a total length of 10-100 nt; specifically, it can be 10-20 nt, 20-50 nt, or 50-100 nt, etc.

[0061] The nucleic acid probe provided in the present application has 0-10 nt asymmetric bases at the 5' end and the 3' end of the nucleic acid probe. As shown in FIG. 1, the stem of the present application is a paired base, the loop is a structure in which the same set of paired bases are connected at both ends, and the single-stranded fragment is a structure in which different paired bases are connected at both ends.

[0062] The nucleic acid probe provided in the present application has a single-stranded oligonucleotide that is RNA.

[0063] In some embodiments, the RNA sequence of the nucleic acid probe is as shown in SEQ ID NO: 3-17 or SEQ ID NO: 21.

[0064] The nucleic acid probe provided in the present application has a fluorescent group and a quencher group at both ends of the nucleic acid probe, and the fluorescent group is selected from FAM, HEX, TET, ROX, TAMRA, JOE, VIC, NED, Quasar, Taxas Red, or Cy; preferably, the fluorescent group is FAM.

[0065] The nucleic acid probe provided in the present application has a quencher selected from the group consisting of BHQ, TRAMA, FAM, MGB, Eclipse and Dabcyl; preferably, the quencher is BHQ. When the nucleic acid probe is intact, the fluorescent group and the quencher are close to each other, and the fluorescent background value is low. When there is a ribonuclease, the probe is rapidly degraded, the fluorescent group and the quencher are separated, the fluorescent value is greatly increased, and the ribonuclease in the sample can be qualitatively or quantitatively determined.

[0066] In the present application, the fluorescent group and the quencher can adopt the technical solutions commonly used in the art, and are not particularly limited herein.

[0067] The second aspect of the present application provides the use of the aforementioned nucleic acid probe as a substrate for RNase. In the use provided in the present application, the RNase includes one or a combination of RNase A, RNase H and RNase I. The nucleic acid probe provided in the present application has a higher sensitivity than ordinary single-stranded fluorescent probes, and can detect RNase as low as 0.1 pg / ml.

[0068] The third aspect of the present application provides the use of the aforementioned nucleic acid probe in the preparation of a product for detecting ribonuclease.

[0069] The fourth aspect of the present application provides a method for detecting ribonuclease. The aforementioned nucleic acid probe is added to a sample to be tested to react, and the ribonuclease in the sample to be tested is qualitatively or quantitatively detected according to the fluorescent value in the reaction product. The detection method uses the nucleic acid probe provided in the present application as a substrate. If there is RNase in the sample to be tested, the substrate will be degraded, the fluorescent group will be released to emit fluorescence, and the content of RNase can be obtained according to the fluorescent signal value. The method is simple, sensitive and accurate because it uses the RNase substrate specially designed in the present application, and the detection can be completed in about 1 hour, and is suitable for high-throughput detection.

[0070] In the detection method provided in the present application, the ribonuclease includes one or a combination of RNase A, RNase T, RNase I and RNase H.

[0071] In the detection method provided in the present application, the working concentration of the nucleic acid probe is 0.01-10 pmol / μL; specifically, it can be 0.01-0.5 pmol / μL, 0.5-11 pmol / μL or 1-10 pmol / μL, etc.

[0072] In the detection method provided in the application, the reaction also includes a reaction buffer and enzyme-free water. The reaction buffer includes 10-100 mM Tris and 0-0.1 M MgCl2. The reaction buffer herein is a 10x formula, and needs to be diluted to a working concentration of 1x before actual use.

[0073] In the detection method provided in the application, when the change in the fluorescence value of the sample to be tested after the reaction and the fluorescence value before the reaction exceeds 1.1 times, it is determined that the sample to be tested contains ribonuclease, otherwise the sample to be tested does not contain ribonuclease.

[0074] In the detection method provided in the application, when the change in the fluorescence value of the sample to be tested after the reaction and the fluorescence value before the reaction exceeds 1.1 times, it is determined that the sample to be tested contains ribonuclease, otherwise the sample to be tested does not contain ribonuclease.

[0075] In the detection method provided in the application, the method for measuring the fluorescence value includes detection by a fluorescence spectrophotometer, real-time quantitative PCR, electrophoresis, HPLC, electrochemistry or naked eye observation under specific light.

[0076] In some embodiments, when the fluorescence value is detected by a fluorescence spectrophotometer, the reaction temperature is 20-30℃, and the reaction time is 0.5-1.5 h.

[0077] In some embodiments, when real-time quantitative PCR is used, DNA polymerase and reverse transcriptase also need to be added for reaction, and the PCR program is specifically 94℃ for 10 s and 55℃ for 25 s for one cycle, and a total of 45 cycles.

[0078] The fifth aspect of the application provides a detection kit for ribonuclease, which includes the nucleic acid probe, the reaction buffer and the enzyme-free water described above; preferably, the detection kit also includes an RNase standard.

[0079] In the detection kit provided in the application, the working concentration of the nucleic acid probe is 0.01-10 pmol / μL; specifically, it can be 0.01-0.5 pmol / μL, 0.5-1 pmol / μL or 1-10 pmol / μL, etc.

[0080] In the detection kit provided in the application, the reaction buffer includes 10-100 mM Tris and 0-0.1 M MgCl2.

[0081] In the detection kit provided in the application, the RNase of the RNase standard is selected from RNase A, RNase T, RNase I or RNase H.

[0082] In some embodiments, the aforementioned detection method or the aforementioned detection kit can be used for detecting the content or relative content of RNase in a sample to be detected, or for comparing the content or relative content of RNase in two or more samples to be detected. The detection method of the present application has high sensitivity, fast reaction speed, and can be observed in real time, is suitable for routine laboratory quality control, has the ability to detect various RNases, and can easily, quickly and accurately grasp the RNase contamination in the sample to be detected and the environment.

[0083] The present application will be further described by examples, but not limited to the scope of the present application.

[0084] The materials, reagents and the like used in the examples are obtained by commercial means unless otherwise specified. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer.

[0085] Unless otherwise specified, the professional and scientific terms used in this text have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present application.

[0086] Example 1, comparison of sensitivity with existing RNase A detection technology

[0087] 1. Material preparation

[0088] In the existing RNase detection technology, the probe method is the most recognized by those skilled in the art because of its simplicity and low cost. The action substrate used in this method is mainly a short single-stranded RNA probe without special secondary structure. The length of this probe is 5-20 nt, one end of which is labeled with a fluorescent group and the other end is labeled with a quencher group, which can be released from the fluorescence by the action of RNase. However, its detection sensitivity for RNase is relatively low, and the detection line is only 10 pg / ml, which cannot effectively meet the quantitative detection needs of users for low concentration RNase. The substrate sequence of the probe is designed and optimized in the present application. The optimized structure design is represented by a double stem loop structure, which is a collection of two sequences of any length of stem loop structure connected by a single-stranded fragment of 2-10 nt.

[0089] The following uses the probe designed by the application and short single-stranded probe (SEQ ID NO: 1, SEQ ID NO: 2) for control test to show the superiority of the application. The nucleic acid probe sequence is shown in Table 1, SEQ ID NO: 1 and SEQ ID NO: 2 are both single-stranded probes without special structure (hereinafter referred to as single-stranded probes), and SEQ ID NO: 3 is a special probe with double stem-loop structure designed by the patent. For easy viewing, the secondary structure of each probe is given in the form of an illustration in the corresponding sensitivity diagram, and the legend shows the total amount of RNase added in the system, i.e. a total of 10, 1, 0.5, 0.1, 0.01 pg of RNase is added in the system. In the following table, the solid line and the wavy line respectively represent the single-stranded fragment sequence and the loop structure sequence of the structural probe of the application. If not otherwise specified, the 5' end of all probes is a FAM fluorescent group, and the 3' end is a BHQ fluorescent quenching group. All RNases used are RNase A.

[0090] Table 1. Single-stranded probe and double stem-loop probe sequence

[0091] 2. System preparation

[0092] The system is prepared according to the following table. Among them, RNase A (Thermo Scientific; #EN0531; 10 mg / ml) is sequentially gradient diluted with enzyme-free water to 1 pg / μL, 0.1 pg / μL, 0.05 pg / μL, 0.01 pg / μL, 0.001 pg / μL, to obtain different concentration gradient standard positive reference; enzyme-free water is used instead of RNase A as negative control.

[0093] Each probe is configured with 2 groups as a duplicate hole.

[0094] 3. Reaction detection

[0095] SpectraMax iD5 multifunctional enzyme marker is used for fluorescence value detection. In fact, any instrument that can observe fluorescence changes can be used for this step. According to the fluorescence group of the probe, the excitation wavelength is set to 485 nm, and the emission wavelength is set to 520 nm. The reaction is carried out at 25℃ for 1 hour, and the data reading is carried out once every 1 minute.

[0096] 4. Data analysis

[0097] The fluorescence detection data of each probe were averaged, and then a line graph was plotted with fluorescence value (RFU) as the ordinate and time as the abscissa, as shown in Figure 2. The values ​​of SEQ ID NO:1 to SEQ ID NO:2 sequentially show the detection sensitivity of SEQ ID NO:1 to SEQ ID NO:3 at different concentrations of RNase A. Under the same reaction conditions, concentrations, and times, SEQ ID NO:1 and SEQ ID NO:2 could not distinguish RNase standards in the concentration range of 1 pg / mL to 10 pg / mL, i.e., they lacked the ability to quantify RNase within this range; SEQ ID NO:3 showed a more significant change in fluorescence value. A standard curve for the detection of low concentrations of RNase A by SEQ ID NO:3 was obtained by plotting RNase A concentration as the ordinate and fluorescence data (RFU) at the fifth minute of reaction time as the abscissa, as shown in Figure 3. The quantification range is 1–100 pg / mL. The standard curve formula is shown in the lower right corner of Figure 3. 2 The value of 0.998 indicates that the quantitative relationship of SEQ ID NO:3 is accurate, meaning that compared with commonly used single-chain probes, the probe designed in this invention has higher quantitative detection sensitivity for low concentrations of RNase A.

[0098] Example 2: Structure dependence of the detection of RNase A by the structural probe of the present invention

[0099] The interaction between enzymes and nucleic acids is often affected by the nucleic acid sequence and length. To verify the effectiveness of the structural probes designed in this invention, that is, the sensitivity improvement is related to the probe structure rather than caused by random factors caused by sequence and length, a series of double stem-loop structure probes with different lengths and sequences were designed. Experiments have shown that the detection sensitivity of probes with this structure is always higher than that of ordinary single-stranded probes.

[0100] 1. Structural probes with different sequences

[0101] The probes shown in Table 2 were synthesized. SEQ ID NO:1 is a single-stranded probe used as a control, and the remaining probe sequences are all double stem-loop structures with the same length but different sequences.

[0102] Table 2. Structural probes with different sequences

[0103] In Example 2, the concentrations of RNase A used in the system preparation for each probe were 10 pg / μL, 1 pg / μL, 0.5 pg / μL, and 0.1 pg / μL. Apart from these differences, the system preparation method and detection method were the same as in Example 1.

[0104] The RFU of the probes in Table 2 against time was plotted as a line graph, as shown in Figure 4. Although there is a certain difference in sensitivity due to different sequences, the sensitivity of all double stem-loop probes is still significantly higher than that of single-stranded probes.

[0105] 2. Structural probes of different lengths

[0106] The probes shown in Table 3 were synthesized. SEQ ID NO: 1 is a single-stranded probe for control, and the remaining probe sequences are double stem-loop structural probes of different lengths.

[0107] Table 3. Structural probes of different lengths

[0108] The system of each probe was prepared in the same way as in Example 1, and the detection method was the same.

[0109] The RFU of the probes in Table 3 against time was plotted as a line graph, as shown in Figure 5. The length of the double stem-loop probe can be arbitrarily changed, and probes of different lengths have good discrimination for low concentrations of RNase, and the effect is significantly better than that of single-stranded probes SEQ ID NO: 1.

[0110] Example 3, optimization of the design of the structural probes of the application

[0111] Obviously, while the overall structure is the same, the detection effect of the probe is still regulated by each component structure, and only when each component structure is within a certain range can the designed structural probe obtain a beneficial effect. For example, a double stem-loop structure probe with too long a stem length, although it is still a double stem-loop structure, will have very poor detection effect and will not be different from a double-stranded nucleic acid that is not conducive to RNase reaction, so it is necessary to limit the length of the stem of the structural probe. Considering the above factors, the application proposes the influence trend of the component structure of the probe on the detection effect, in order to optimize the design range of the structural probe and enable the probe in this range to obtain a detection effect better than that of the single-stranded probe.

[0112] The factors affecting the structural probe include but are not limited to those proposed in the following experiments. It should be understood that the double stem-loop structure probe is mainly used as a comparison basis in the examples, but since the design of the structural probe is not limited to the double stem-loop structure, and the overall structure design is related to each component structure, the following examples cannot be considered as limiting the scope of the claims.

[0113] In the following comparative experiments, the concentration of RNase A used in the preparation of each probe system was 1 pg / μL, 0.1 pg / μL, 0.05 pg / μL, and 0.01 pg / μL. In addition, the system preparation method and detection method were the same as in Example 1. In Figures 6-10, a is the RNase sensitivity detection graph corresponding to the single-stranded probe SEQ ID NO: 1, in order to compare the results; each probe corresponds to the letter order in the corresponding sensitivity graph from b in the order from top to bottom in the table.

[0114] 1. Effect of stem length on the detection sensitivity of structural probes

[0115] Table 4 lists a plurality of structural probe sequences with different stem lengths, and the RNase detection sensitivity graph corresponding to each probe is shown in Figure 6. The sensitivity is related to the stem length, and the sensitivity decreases with the increase of the stem length; when the single-stranded length is 2, the double-stem loop probe with a total stem length greater than 13 bp cannot be detected at an RNase addition concentration of 1 pg / μL.

[0116] Table 4. Probe sequences with different stem lengths

[0117] 2. Effect of single-stranded length on the detection sensitivity of structural probes

[0118] Table 5 lists a plurality of structural probe sequences with different single-stranded lengths, and the RNase detection sensitivity graph corresponding to each probe is shown in Figure 7. As can be seen from Figure 7, the detection ability of the probe structure SEQ ID NO: 7 without a single-stranded fragment is still stronger than that of the single-stranded probe; the addition of a single-stranded fragment can greatly improve the sensitivity of the structural probe, and the detection lower limit of the structural probe SEQ ID NO: 3 containing a 2 nt single-stranded fragment is more than 10 times higher than that of the probe SEQ ID NO: 7 without a single-stranded fragment; the single-stranded length is related to the sensitivity, and the increase of the single-stranded length (>2) leads to a relative decrease in the sensitivity, and the extension of the single-stranded length (>5 nt) leads to a stable effect of the single-stranded length on the detection.

[0119] Table 5. Probe sequences with different single-stranded lengths

[0120] 3. Effect of single-stranded sequence on the detection sensitivity of structural probes

[0121] Table 6 lists a plurality of structural probe sequences with different single-stranded sequences, and Figure 8 shows the effect of single-stranded sequence variation on the detection ability of the structural probe. As can be easily understood, RNase has sequence preference, and therefore a single-stranded sequence containing a specific base has better detection effect on the target RNase, which is related to the specific type of RNase. However, the presence or absence of a small amount of preferred base in a shorter single-stranded sequence has a relatively slight effect on the detection effect.

[0122] Table 6. Probe sequences of different single-stranded sequences

[0123] 4. Effect of stem loop number on detection sensitivity of structure probes

[0124] Table 7 lists a plurality of structure probe sequences with different stem loop numbers, and the RNase detection sensitivity diagram corresponding to each probe is shown in Figure 9. As can be seen from Figure 9, the detection effect of the probe containing one stem loop is similar to that of the single-stranded probe. Their qualitative detection range is above 10 pg / ml, and the difference between each group of data of the system concentration of 1-10 pg / ml RNase is quite small, and they have almost no quantitative detection ability. When the stem loop number is two, the detection sensitivity for low concentration RNase is improved by 100 times, the quantitative range can reach 1 pg / ml, the qualitative detection can reach 0.1 pg / ml, and the linear calibration curve is good (Figure 3). When the stem loop number is three, the qualitative detection range is similar to that of the single-stranded probe, but the quantitative ability is better than that of the single-stranded probe, and the quantitative detection limit can reach 10 pg / ml, indicating that the probe structure with three stem loops still has certain design advantages.

[0125] Table 7. Probe sequences with different stem loop numbers

[0126] 5. Asymmetric base length

[0127] Table 8 lists a plurality of structure probe sequences with different asymmetric base lengths, and the RNase detection sensitivity diagram corresponding to each probe is shown in Figure 10. Prolonging the number of asymmetric bases has an adverse effect on the detection effect, and the optimal number of asymmetric bases should be less than 3.

[0128] Table 8. Probe sequences with different asymmetric base lengths

[0129] Example 4, Detection of finished enzyme, environmental pollution by RNA probe

[0130] Considering the actual application scene of the probe, the RNase pollution detection of the purchased commercial enzyme reagent and the environmental surface was carried out. The probe used was SEQ ID NO: 3.

[0131] 1. Material preparation

[0132] The information of the purchased commercial enzyme is shown in Table 9.

[0133] Table 9. Information of purchased commercial enzyme

[0134] The sample of environmental pollution was obtained by wiping the test bench table and centrifuge tube surface with enzyme-free water; the table surface before and after wiping with RNase cleaner was sampled as a control group; and the ultrapure water sample was taken as a negative control

[0135] 2. System preparation

[0136] Two kinds of commercial enzyme samples and environmental samples were set up, and each kind had two groups. When detecting DNase I, the amount of DNase I added in the system was 5 U; when detecting T7 RNA Polymerase, the amount of T7 RNA Polymerase added in the system was 100 U, and the configuration of the remaining components was the same as in Example 2.

[0137] The detection method was the same as in Example 1. The fluorescence value before the reaction was the fluorescence value of the system before adding the sample to be detected, and the fluorescence value after the reaction was the fluorescence value of the system after incubation at 37°C for 1 hour after adding the sample to be detected. The fluorescence value RFU was taken as the vertical coordinate to make a column chart, and the detection conditions of each group are shown in FIG. 11. Except that the sample of the un-wiped table produced more than 1.1 times fluorescence enhancement after the reaction, the fluorescence values of the remaining groups did not change significantly. Therefore, it can be judged that the test bench table contains RNase contamination, and the commercial enzyme and the centrifuge tube are relatively clean.

[0138] Example 5, PCR method for detecting RNase content of a sample

[0139] The probe structure designed in the patent has a faster reaction speed with RNase, and can be used as a reaction substrate without carrying a fluorescent group and a quencher to obtain higher detection sensitivity. In this embodiment, a nucleic acid sequence with a length of 86 nt is designed as an RNA template, which has 6 stem-loop structures connected by single-stranded fragments. When the template reacts with RNase, it will be rapidly degraded into fragments, and part of the intact fragments that do not react with RNase will be amplified by PCR. The reaction CT value is proportional to the RNase concentration, and the RNase content in the sample to be detected can be judged according to the CT value. It is easy to understand that the RNase content of the sample to be detected is obtained by comparing with a standard curve obtained by reacting with a series of RNase standard samples with different content gradients.

[0140] 1. Material preparation

[0141] The PCR nucleic acid sequence used in the experiment is shown below, and the information of the commercial enzyme used is shown in Table 10.

[0142] RNA template sequence:

[0143] 5'-ACCGCUAAUGCGGCGUGCUCAUUAGAGCUACCAGACUACUGGCAGAUCACUGAAAGCCUACAGGCGCAUGCACUAGCAUGUCGGUA-3'(SEQ ID NO: 17)

[0144] Upstream primer: 5'-CGTGCTCATTAGAGCTACCA-3' (SEQ ID NO: 18)

[0145] Downstream primer: 5'-CAATACCGACATGCTAGTGC-3' (SEQ ID NO: 19)

[0146] Detection probe: 5'FAM-TGGCAGATCACTGAAAGCCTACAGGCGC-3' BHQ (SEQ ID NO: 20)

[0147] 2. System preparation:

[0148] The experiment is divided into positive control (RNase A standard), negative control (enzyme-free water) and test group.

[0149] The following system is carefully prepared without enzyme to avoid mixing other contaminants.

[0150] 3. Reaction detection

[0151] The prepared PCR reaction solution is placed in a PCR instrument, and the reaction is carried out according to the following program.

[0152] 4. Data analysis

[0153] The system is placed in a real-time fluorescent PCR instrument for one-step reaction to obtain the Ct value under the action of different concentrations of RNase A standard, and the corresponding amplification curve is shown in Figure 12; the Ct value of each group of experiments is taken as the abscissa, and the RNase A concentration is taken as the ordinate to obtain the standard curve as shown in Figure 13, the quantitative range of the standard curve is 2-4 ng, R 2 0.973, which is good in linear and can be used for quantitative detection of RNase.

[0154] Example 6, application of structure probe in isothermal amplification

[0155] Compared with conventional PCR detection, isothermal amplification has lower equipment requirements. The structure probe of the present application can be used as a substrate template, which, after acting with RNase, realizes reverse transcription from RNA to DNA and amplification of DNA fragments through RT-PRA reaction. The initial RNA amount of reverse transcription is the amount of intact RNA substrate template remaining after the action of RNase, which corresponds to the amount of fluorescent group released by the fluorescent probe in the final detection step, so according to the change of fluorescence value, the content of RNase in the sample to be detected can be obtained, and the detection of RNase is realized.

[0156] 1. Material preparation

[0157] RNA template sequence:

[0158] 5'-CGUGCUCAUUAGAGCUACCAACCGCUAAUGCGGGACUACUGGCAGAUCACUGAAAGCCUACAGGCGCAUGCACUAGCAUGUCGGUAUUG-3' (SEQ ID NO: 21)

[0159] Upstream primer: 5'-CGTGCTCATTAGAGCTACCA-3' (SEQ ID NO: 19)

[0160] Downstream primer: 5'-CAATACCGACATGCTAGTGC-3' (SEQ ID NO: 20)

[0161] Detection probe: 5'-ATTACGCCCTGATGTGGC(FAM-dT)(THF)(BHQ1-dT)AGATCACTGAAAGCCTACAGGCGC-3' (phosphate) (SEQ ID NO: 22)

[0162] Wherein, SEQ ID NO: 21 is an RNA structure probe as a substrate template, which does not contain fluorescent or quenching groups at both ends; SEQ ID NO: 2

[0163] 2 FAM is a luminescent group, THF is a tetrahydrofuran site, BHQ1 is a quenching group, and phosphate is a phosphate group.

[0164] 2. System preparation

[0165] The substrate template is 100 ng; the RNase is 0, 0.1 ng, 0.15 ng, 0.3 ng, and 0.35 ng, respectively. The sample without adding RNase is the negative control. In addition, the exoRPA kit is used to prepare the system, and the RT-PRA reaction is carried out according to the instructions.

[0166] 3. Data analysis

[0167] The system is incubated at 37°C for 20 minutes, and the fluorescence value is taken as the vertical coordinate and the reaction time as the horizontal coordinate, and the graph is shown in FIG. 14. The RNase content has a correlation with the change of the fluorescence value, and the unknown sample can be compared with the results obtained from the known standard to understand the RNase content therein.

[0168] The above embodiments are only illustrative of the principles of the present application and its effects, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. A nucleic acid probe, which is a single-stranded oligonucleotide, wherein the single-stranded oligonucleotide has a linked stem-loop structure; wherein the number of stem-loops is ≥2.

2. The nucleic acid probe as described in claim 1, characterized in that, The stem-loop structure includes a connected stem and a loop, and the single-chain oligonucleotide also includes a single-chain fragment. The stems of adjacent stem-loop structures are directly connected by single chains or connected by single-chain fragments. Preferably, the single-stranded fragment is 1–10 nt in length; the loop sequence is 3–10 nt in length; the stem sequence is ≥2 bp in length; optionally, the stem contains mismatched base pairs, degenerate or unmatched nucleotides; and / or, the single-stranded fragment sequence contains C, U or G bases. More preferably, the single-stranded fragment is 2 to 10 nt in length; the stem sequence is 2 to 10 bp in length; and the single-stranded fragment sequence contains a C base or a U base.

3. The nucleic acid probe as described in claim 1, characterized in that, The number of stem rings is 2 to 3; And / or, any one base of the nucleic acid probe is a natural, modified, or non-natural nucleoside base; And / or, optionally, the nucleic acid probe contains modified ribose, the modification including one or more combinations of 2'-O-methylation, 2'-methoxyethylation, 2'-F modification, 2'-deoxy modification, and 2'-amino modification; And / or, optionally, the nucleic acid probe contains phosphodiester bond modification for linking nucleotides, the phosphodiester bond modification including thiolation or boronylation modification of non-bridging oxygen atoms in the phosphate backbone; And / or, the total length of the nucleic acid probe is 10 to 100 nt; and / or, the 5' end and 3' end of the nucleic acid probe have 0 to 10 nt of asymmetric bases; And / or, the single-stranded oligonucleotide is RNA.

4. The nucleic acid probe according to any one of claims 1 to 3, characterized in that, The RNA sequence of the nucleic acid probe is shown in SEQ ID NO:3-17 or SEQ ID NO:

21.

5. The nucleic acid probe as described in claim 1, characterized in that, The nucleic acid probe has a fluorescent group and a quenching group at both ends, respectively. The fluorescent group is selected from FAM, HEX, TET, ROX, TAMRA, JOE, VIC, NED, Quasar, Taxas Red or Cy; preferably, the fluorescent group is FAM. The quenching group is selected from BHQ, TRAMA, FAM, MGB, Eclipse, or Dabcyl; preferably, the quenching group is BHQ.

6. Use of the nucleic acid probe as an RNase substrate as described in any one of claims 1 to 5.

7. The use as described in claim 6, characterized in that, The RNase includes one or more combinations of RNase A, RNase H and RNase I.

8. Use of the nucleic acid probe according to any one of claims 1 to 5 in the preparation of products for detecting ribonuclease.

9. A method for detecting ribonuclease, comprising adding the nucleic acid probe according to any one of claims 1 to 5 to a sample to be tested for reaction, and performing qualitative or quantitative detection of ribonuclease in the sample to be tested based on the fluorescence value in the reactants.

10. The detection method as described in claim 9, characterized in that, The ribonuclease includes one or more combinations of RNase A, RNase T, RNase I, and RNase H; And / or, the working concentration of the nucleic acid probe is 0.01–10 pmol / μL; And / or, the reaction also includes a reaction buffer and enzyme-free water; preferably, the reaction buffer includes 10-100 mM Tris and 0-0.1 M MgCl2.

11. The detection method as described in claim 9, characterized in that, The detection methods include detection by fluorescence spectrophotometer, PCR, isothermal amplification, electrophoresis, HPLC, electrochemistry, or visual observation under specific light.

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