Deoxyribonucleases and their uses

Abstract

This invention relates to a deoxyribonuclease, a kit containing the deoxyribonuclease, and a composition containing the deoxyribonuclease. The deoxyribonuclease can be used as a candidate drug for gene therapy, or to prepare biosensors or chips for detecting metal ions or nucleic acids.

Description

Technical Field

[0001] This invention relates to deoxyribonucleases, their applications in the preparation of gene therapy drugs and biosensors, kits containing the deoxyribonucleases, and pharmaceutical compositions containing the deoxyribonucleases. Background Technology

[0002] Deoxyribonucleases are a class of DNA molecules with catalytic activity, and their substrates are generally ribonucleic acid (RNA). Existing deoxyribonucleases are all metal-dependent enzymes. Deoxyribonucleases bind to their substrate nucleic acids through complementary base pairing. With the assistance of metal ions, they form specific higher-order structures, undergo a catalytic reaction, and cleave the substrate nucleic acid at specific sites, breaking it into two parts and rendering it non-functional. Therefore, deoxyribonucleases can be considered a gene therapy option. Simultaneously, since metal ions are essential for the catalytic reaction of deoxyribonucleases, this reaction can be used to detect metal ions, thus deoxyribonucleases can also serve as biosensors for metal ion detection.

[0003] As gene therapy drugs, deoxyribonucleases can catalyze the cleavage of pathogenic RNA (overexpressed RNA from diseases, viral RNA, bacterial RNA, etc.), thereby inhibiting the translation of these pathogenic RNAs and achieving therapeutic goals.

[0004] The auxiliary metal ions required by these deoxyribonucleases are closely related to life activities. Mg 2+ Ca 2+ Zn 2+ Mn 2+ Cu 2+ These are essential trace elements for the human body; both excess and deficiency can cause a series of pathological reactions. Pb... 2+ Cd 2+ Cr 3+ These are metal ions that are harmful to human physiological activities. If ingested from the environment at harmful concentrations, they can lead to various pathological reactions. Therefore, monitoring these metal ions in the human body and the environment is crucial, and it is necessary to develop rapid, accurate, and sensitive detection methods. Utilizing the reaction between deoxyribonucleases and substrate nucleic acids, and with the aid of analytical methods, deoxyribonucleases have been used to develop biosensors for these metal ions.

[0005] One method for detecting metal ions and substrate nucleic acids using deoxyribozymes is based on changes in the fluorescence intensity of fluorescent signal molecules. For example... Figure 1As shown, a fluorescent group, such as 6-carboxyfluorescein (FAM), is attached to the 5' end of the substrate nucleic acid of the deoxyribonuclease; a fluorescence quenching group, such as BHQ1, is attached to the 3' end. When the substrate nucleic acid binds to the deoxyribonuclease through base pairing, the fluorescent molecule and the quenching group approach each other. In the presence of catalytic metal ions, the substrate is cleaved into two parts, dissociating from the deoxyribonuclease. The fluorescent molecule moves away from the quenching group, and upon introduction of excitation light, it gains energy and produces fluorescence. The intensity of the fluorescence signal is correlated with the number of dissociated nucleic acid fragments, that is, it is related to the reaction rate, the amount of metal ions, and the presence of the substrate nucleic acid. These correlations with the fluorescence signal can be used to detect the concentration of metal ions or substrate nucleic acid.

[0006] Several deoxyribozymes with catalytic cleavage function have been reported, such as 8-17, Mg5, 17E, GR5 and its variant GR5M4, etc., and their structures are as follows: Figure 2 As shown, they exhibit similar catalytic domain morphologies. They were obtained under different screening conditions (as shown in Table 1). They all require divalent metal ions to assist the catalytic reaction, but their dependence on metal ions differs.

[0007] Table 1 Examples of deoxyribonuclease screening conditions

[0008]

[0009] These deoxyribozymes exhibit slow catalytic reaction rates and are highly dependent on metal ions, requiring relatively high concentrations of metal ions for the catalytic reaction to proceed smoothly. Furthermore, each deoxyribozyme is only active in the presence of specific metal ions.

[0010] On the one hand, due to intracellular metal ions (Mg 2+ Ca 2+ Zn 2+ Mn 2+ Low concentrations of certain metal ions (such as nitrogen, phosphorus, and hydrogen) are insufficient to support the efficient cleavage of pathogenic RNA by deoxyribozymes, thus affecting their application as gene therapy drugs. On the other hand, the high concentration requirements of metal ions result in low sensitivity of deoxyribozymes as biosensors for detecting metal ions.

[0011] The catalytic reaction rate of deoxyribozymes, and their dependence on metal ion concentration, have become key factors restricting their development into gene therapy drugs and biosensors. Improving the catalytic reaction rate of deoxyribozymes and reducing their dependence on metal ion concentration are urgent problems to be solved. Summary of the Invention

[0012] This invention relates to the deoxyribozyme represented by formula 17NE or a derivative thereof.

[0013] 5'-d(N) m -dT 2.1 -d(CTCAGCGAGACGXA)-d(N) m’ -3'

[0014] 17NE

[0015] Where: d(N) m and d(N) m’ These represent the substrate nucleic acid recognition arms at the 5' and 3' ends of the deoxyribonuclease, respectively. Their base sequences are complementary to the substrate nucleic acid sequences. The composition and length of their base sequences are determined according to the substrate nucleic acid sequences. Each N is an independent nucleotide residue, and m and m' represent the number of N, each being a natural number greater than or equal to 6.

[0016] dT 2.1 Represents a deoxythymidine residue, which pairs with the cleavage site rG or dG of the substrate nucleic acid sequence;

[0017] X is a nucleotide residue represented by formula I or I', where R is -(CH2). n R' is selected from amino, mercapto, hydroxy, imidazolyl, phenyl, or pyrene, and n is a natural number greater than or equal to 1 and less than or equal to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8).

[0018]

[0019] The derivative differs from the deoxyribonuclease only in that its 5' or 3' end is labeled with a marker, and it retains the function of the deoxyribonuclease, namely, the ability to catalyze the cleavage of substrate nucleic acids.

[0020] In some embodiments, in the deoxyribozyme or its derivative represented by Formula 17NE of the present invention, R is -(CH2). n R', where R' is an amino, thiol, hydroxyl, or imidazole group, and n is defined as described in this invention.

[0021] In some embodiments, in the deoxyribozyme or its derivative represented by Formula 17NE of the present invention, R is -(CH2). n R', where R' is an amino group, and n is defined as described in this invention.

[0022] In some embodiments, in the deoxyribozyme or its derivative represented by Formula 17NE of the present invention, R is -(CH2). n R', where R' is defined as described in this invention, and n is a natural number greater than or equal to 2 and less than or equal to 6, such as 2, 3, 4, 5, or 6.

[0023] In some embodiments, in the deoxyribozyme or its derivative represented by Formula 17NE of the present invention, R is -(CH2). n R', where R' is defined as described in this invention, and n is 2, 3, or 4.

[0024] In some embodiments, in the deoxyribozyme or its derivative represented by Formula 17NE of the present invention, R is -(CH2). n R', where R' is defined as described in this invention, and n is 3.

[0025] In some embodiments, in the deoxyribozyme or its derivative represented by Formula 17NE of the present invention, R is -(CH2)3NH2.

[0026] In some embodiments, the deoxyribozyme or its derivative represented by Formula 17NE of the present invention has a catalytic domain of 5'-d(CTCAGCGAGACGXA)-3', and the base positions in its sequence are labeled as 5'-d(C 3 T 4 C 5 A 6 G 7 C 8 G 9 A 10 G 11 A 12 C 13 G 14 X 15 A 15.0 The catalytic domain is divided into three parts: 1) consisting of three pairs of complementary bases, namely C 3 T 4 C 5 With G 11 A 10 G 9 The stem formed between them; 2) Terminal ring structure A 6 G 7 C 8 ;3) Large ring structural domain A 12 C 13 G 14 X 15 A 15.0 .

[0027] In some embodiments, in the deoxyribozyme or its derivative represented by Formula 17NE of the present invention, m and m' are each independently a natural number greater than or equal to 6 and less than or equal to 12, for example 6, 7, 8, 9, 10, 11, 12.

[0028] In some embodiments, in the deoxyribozyme or its derivative represented by Formula 17NE of the present invention, m and m' are each independently a natural number greater than or equal to 6 and less than or equal to 9, for example 6, 7, 8, 9.

[0029] In some embodiments, the substrate nucleic acid described in this invention is 3'-(N'). x -G-rA-(N') y The -5' symbol represents an RNA or a DNA-RNA-DNA chimeric structure containing at least one RNA unit rA, where G represents rG or dG, 3'-G-rA-5' is the cleavage site of the deoxyribonuclease, rA does not participate in base pairing, and rG or dG interacts with the T in the deoxyribonuclease. 2.1 Pairing, (N') x It is the substrate nucleic acid recognition arm (N) that can interact with the 5' end of the deoxyribozyme. m This forms a DNA or RNA sequence with complementary base pairings, (N') y It is the substrate nucleic acid recognition arm (N) that can interact with the 3' end of the deoxyribozyme. m’ A DNA or RNA sequence is formed with complementary base pairings, where each N' is an independent nucleotide residue, x and y represent the number of N's, and x and y are each independent natural numbers greater than or equal to 6.

[0030] The deoxyribozyme or its derivative represented by Formula 17NE described in this invention has the ability to catalyze the cleavage of substrate nucleic acids.

[0031] In some embodiments, the substrate nucleic acid described in this invention is synthetic RNA or DNA-RNA-DNA.

[0032] In some embodiments, the substrate nucleic acid described in this invention is RNA derived from plasmid expression or RNA derived from a living organism.

[0033] In some embodiments, in the substrate nucleic acid of the present invention, x and y are each independently natural numbers greater than or equal to 6 and less than or equal to 12, such as 6, 7, 8, 9, 10, 11, 12.

[0034] In some embodiments, in the substrate nucleic acid of the present invention, x and y are each independently a natural number greater than or equal to 6 and less than or equal to 9, such as 6, 7, 8, 9.

[0035] In some embodiments, in the substrate nucleic acid described in this invention, x and m are equal, y and m' are equal, and (N') x With the substrate nucleic acid recognition arm (N) in deoxyribozymes. m The bases are completely complementary, (N') y With the substrate nucleic acid recognition arm (N) in deoxyribozymes.m’ The bases are completely complementary.

[0036] In some embodiments, in the substrate nucleic acid described in this invention, x>m, y>m', (N') x A partial base sequence adjacent to G-rA in the middle of the deoxyribonuclease interacts with the substrate nucleic acid recognition arm (N). m Complementary, (N') y A partial base sequence adjacent to G-rA in the middle of the deoxyribonuclease interacts with the substrate nucleic acid recognition arm (N). m’ Complementary.

[0037] In some embodiments, the sequence of the deoxyribozyme represented by Formula 17NE of the present invention is: d(AGG ATCTAT CTC AGC GAG ACG XA GGC TCC AT), where X is a nucleotide residue represented by Formula 1 or 1'.

[0038]

[0039] In some embodiments, the sequence of the deoxyribozyme derivative of Formula 17NE described in this invention is: d(AGG ATC TAT CTC AGC GAG ACG XA GGC TCC AT)-BHQ1, where X is a nucleotide residue represented by Formula 1 or 1'.

[0040]

[0041] The present invention also relates to the use of the deoxyribonuclease or its derivatives represented by Formula 17NE as described herein in the preparation of sensors or chips for detecting metal ions.

[0042] The present invention also relates to a chip or sensor comprising:

[0043] a) The deoxyribozyme or its derivative represented by Formula 17NE as described in this invention, and

[0044] b) The substrate nucleic acid described in this invention or the substrate nucleic acid capable of being catalytically cleaved by the deoxyribozyme or its derivative represented by Formula 17NE described in this invention, or

[0045] The chip or sensor contains:

[0046] a) The deoxyribozyme or its derivative represented by Formula 17NE as described in this invention, and

[0047] c) Metal ions.

[0048] In some embodiments, in the chip or sensor of the present invention, the 5' or 3' end of the substrate nucleic acid described in b) may be labeled with a marker.

[0049] This invention also relates to a method for detecting metal ions, comprising:

[0050] (1) Provide the sample to be tested;

[0051] (2) Contact the sample to be tested with the deoxyribozyme or its derivative as shown in Formula 17NE of the present invention and the substrate nucleic acid or substrate nucleic acid that can be catalytically cleaved by the deoxyribozyme or its derivative as shown in Formula 17NE of the present invention.

[0052] (3) Measure the lysis of the substrate nucleic acid.

[0053] In some embodiments, in the method for detecting metal ions according to the present invention, the 5' or 3' end of the substrate nucleic acid described in (2) can be labeled with a marker.

[0054] This invention also relates to a method for detecting nucleic acids, comprising:

[0055] (1) Provide the sample to be tested;

[0056] (2) Contact the sample to be tested with the deoxyribozyme or its derivatives as shown in Formula 17NE of the present invention and metal ions;

[0057] (3) Measure the lysis of nucleic acid in the sample to be tested.

[0058] The present invention also relates to a kit comprising the deoxyribonuclease or a derivative thereof represented by Formula 17NE as described in the present invention.

[0059] In some embodiments, the kit of the present invention further comprises: the substrate nucleic acid of the present invention or the substrate nucleic acid capable of being catalytically cleaved by the deoxyribozyme or its derivative represented by Formula 17NE of the present invention.

[0060] In some embodiments, the 5' or 3' end of the substrate nucleic acid in the kit of the present invention may be labeled with a marker.

[0061] In some embodiments, the kit of the present invention further comprises: metal ions.

[0062] In some embodiments, the metal ion of the present invention is selected from Mg. 2+ Ca 2+ Zn 2+ Pb 2+ Mn 2+ Cd 2+ Ba 2+ Co 2+ Ni 2+ Fe 2+ Fe 3+Cr 3+ Hg 2+ Cu 2+ Tl + Ag + and any combination thereof.

[0063] In some embodiments, the kit described in this invention further comprises a buffer substance.

[0064] In some embodiments, the buffering substance of the present invention is selected from 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), morpholine ethanesulfonic acid (MES), and tris(hydroxymethyl)aminomethane.

[0065] The present invention also relates to the use of the deoxyribonuclease or its derivatives represented by Formula 17NE as described herein in the preparation of gene therapy drugs.

[0066] The present invention also relates to a composition comprising the deoxyribonuclease or a derivative thereof represented by formula 17NE as described in the present invention, and a pharmaceutically acceptable carrier or excipient.

[0067] In some embodiments, the marker described in this invention is a substance capable of generating a detection signal, such as a substance capable of generating fluorescence (e.g., 6-carboxyfluorescein), a substance capable of quenching a light signal (e.g., fluorescence quenching molecule (BHQ1)), or a substance capable of generating a radioactive signal (e.g., a radioactive isotope, such as a radioactive...). 32 P) or substances that can generate electrical signals.

[0068] In some embodiments, for detection purposes, a label is attached to the 5' or 3' end of the deoxyribozyme or substrate nucleic acid described in this invention. The labeled deoxyribozyme and the labeled substrate nucleic acid undergo a catalytic cleavage reaction under the action of metal ions, generating a change in signal. The metal ions or substrate nucleic acid can be detected by detecting the change in signal.

[0069] In some embodiments, the deoxyribozyme represented by Formula 17NE of the present invention is a DNA enzyme capable of catalytically cleaving substrate nucleic acids. Its catalytic domain is 5'-CTCAGCGAGACGXA-3', divided into three parts: (1) consisting of three pairs of complementary bases, namely C... 3 T 4 C 5 With G 11 A 10 G 9 (2) End ring structure A 6 G 7 C 8 (3) Macrocyclic structural domain A 12 C 13 G 14 X15 A 15.0 (N) m and (N) m’ It consists of recognition arms at the 5' and 3' ends, respectively, and N is a nucleotide unit complementary to the cleaved nucleic acid. The number of N, m and m', in each recognition arm may be the same or different, ranging from 6 to 12.

[0070] The substrate nucleic acid of the deoxyribozyme represented by Formula 17NE in this invention is: 3'-(N'). x -G-rA-(N') y -5', where G represents rG or dG, 3'-G-rA-5' is the cleavage site of the deoxyribonuclease, rA does not participate in base pairing, and rG or dG interacts with the dT of the deoxyribonuclease recognition arm. 2.1 In the pairing process, the N-shaped sequence of the recognition arms at both ends of the deoxyribozyme specifically binds to the N'-shaped sequence of the substrate nucleic acid through base pairing. A pairs with T, G pairs with C, and vice versa.

[0071] The deoxyribozyme represented by Formula 17NE of this invention can undergo catalytic cleavage of substrate nucleic acids in the presence of the following ion: Mg 2+ Ca 2+ Zn 2+ Pb 2+ Mn 2+ Cd 2+ Ba 2+ Co 2+ Ni 2+ Fe 2+ Fe 3+ Cr 3+ Hg 2+ Cu 2+ Tl + Ag + .

[0072] The deoxyribozyme represented by formula 17NE described in this invention, when X is X1, that is, when X is a nucleotide residue represented by formula 1 or 1', the deoxyribozyme is deoxyribozyme 17NEM, and its structural schematic diagram is shown below. Figure 3 As shown.

[0073] In some implementation schemes, multiple methods can be used to detect nucleic acid lysis. Two commonly used methods are: The first is using radioactivity... 32 The 5' end of the P-labeled nucleic acid substrate was separated by denaturing gel electrophoresis, and the radiolabeled substrate and product were separated. The radioactivity levels of both were quantitatively analyzed to calculate reaction kinetic parameters or to quantify nucleic acid cleavage and metal ion concentration.

[0074] The second method is fluorescence spectroscopy, such as... Figure 2 As shown. A fluorescent molecule is used to label the 5' or 3' end of the nucleic acid substrate, while a quencher molecule is labeled at the other end to reduce background interference. A quencher molecule is introduced at the complementary end of the deoxyribozyme. For example, if the fluorescent molecule FAM is labeled at the 5' end of the nucleic acid substrate, the quencher molecule BHQ1 is labeled at its 3' end, and the quencher molecule BHQ1 is also labeled at the 3' end of the deoxyribozyme. A stable complex structure is obtained when the recognition arms at both ends of the deoxyribozyme can form complete base pairing with the substrate nucleic acid. The 5'-FAM of the substrate is close to the 3'-BHQ1 of the deoxyribozyme, resulting in fluorescence quenching. When a catalytic metal ion is present, it drives the catalytic cleavage reaction of the deoxyribozyme, cleaving the substrate nucleic acid into two smaller fragments that dissociate from the complex. The product fragment containing 5'-FAM is far from the 3'-BHQ1 of the deoxyribozyme and emits specific fluorescence under the excitation wavelength. The intensity of the fluorescence reflects the number of dissociated fragments or the extent of the reaction. Therefore, fluorescence changes can be used to calculate the catalytic reaction parameters of deoxyribozymes, or to quantify the cleavage of nucleic acids and the concentration of metal ions.

[0075] In some embodiments, the deoxyribozyme represented by Formula 17NE, such as deoxyribozyme 17NEM, is labeled with BHQ1 at its 3' end, the substrate nucleic acid is labeled with the fluorescent molecule FAM at its 5' end, and the quencher molecule BHQ1 is labeled at its 3' end. Therefore, the excitation and emission wavelengths of FAM can be used to monitor the catalytic cleavage reaction and obtain the catalytic reaction kinetic parameters of the deoxyribozyme.

[0076] The two analytical methods described above are merely illustrative examples, and the analytical methods for deoxyribozymes based on Formula 17NE of this invention are not limited to the examples provided.

[0077] The deoxyribozymes represented by formula 17NE described in this invention, such as deoxyribozyme 17NEM, can exert catalytic activity in a variety of buffer systems, including (1) 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (50 mM HEPES, pH 7.0), which can be used in Ca 2+ and Mg 2+ (2) Morpholine ethanesulfonic acid (MES) system (50 mM MES, pH 6.0), which can be used for Zn 2+ (3) 50mM HEPES-100mM NaNO3-pH 7.0, which can be used for Pb catalytic reactions; 2+ (4) Tris-hydroxymethylaminomethane (Tris) system (50mM Tris-HCl, pH 7.5).

[0078] In some embodiments, the deoxyribozymes represented by Formula 17NE, such as deoxyribozyme 17NEM, are preferably present in a reaction system at a concentration of 10 nM to 2 μM. Furthermore, in the reaction system, the concentration of the substrate nucleic acid is preferably 10-200 nM, and the concentration of the polyvalent metal ions is preferably 0.1 nM to 10 mM.

[0079] In some embodiments, in the catalytic reaction of the deoxyribonuclease represented by Formula 17NE (e.g., deoxyribonuclease 17NEM) of the present invention, the deoxyribonuclease, substrate nucleic acid, and metal ions are mixed in a buffer solution and incubated at 37°C. Measurements can be taken using a Tecan 1000-pro microplate reader with an excitation wavelength of 496 nm (slit width 5.0 nm) and an emission wavelength of 524 nm (slit width 10.0 nm). The catalytic reaction kinetics are calculated using the fluorescence emission intensity of the FAM.

[0080] The carriers described in this invention include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycerol, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax, and lanolin.

[0081] The excipients described in this invention refer to additives in pharmaceutical preparations other than the active pharmaceutical ingredient (API). They are stable in nature, have no incompatibilities with the API, do not produce side effects, do not affect efficacy, are not easily deformed, cracked, moldy, or infested by insects at room temperature, are harmless to the human body, have no physiological effects, do not produce chemical or physical reactions with the API, and do not affect the content determination of the API. Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; and preservatives, antioxidants, flavoring agents, flavoring agents, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in oral liquid preparations.

[0082] The compositions described in this invention can be prepared into various dosage forms, including but not limited to tablets, capsules, solutions, suspensions, granules, or injections, using conventional methods in the art, for administration via routes such as oral or parenteral.

[0083] In this invention, the letter 'r' preceding a nucleic acid sequence or nucleotide indicates that the sequence or nucleotide is a ribonucleic acid sequence or ribonucleotide. For example, rG represents guanine ribonucleotide, and r(CTCAGCGAGACGXA) represents that the sequence is an RNA sequence. The letter 'd' preceding a nucleic acid sequence or nucleotide indicates that the sequence or nucleotide is a deoxyribonucleic acid sequence or deoxyribonucleotide. For example, dG represents guanine deoxyribonucleotide, and d(CTCAGCGAGACGXA) represents that the sequence is a DNA sequence.

[0084] It should also be noted that the dosage and method of administration of the deoxyribonuclease described in this invention, as a gene therapy drug, depend on many factors, including the patient's age, weight, sex, natural health condition, nutritional status, the activity intensity of the compound, the duration of administration, metabolic rate, severity of the disease, and the subjective judgment of the treating physician. The preferred dosage is between 0.0001 and 1000 mg / kg body weight / day. This daily dose can be administered once or in multiple divided doses, depending on the situation.

[0085] Beneficial effects of the present invention

[0086] The deoxyribozyme represented by formula 17NE, such as deoxyribozyme 17NEM, as described in this invention has one or more of the following advantages:

[0087] 1) The catalytic rate is significantly improved.

[0088] 2) It exhibits broad metal ion dependence and is active for a variety of metal ions.

[0089] 3) It reduces dependence on metal ion concentration and can utilize physiological concentrations of metal ions as cofactors to obtain highly efficient catalytic cleavage of nucleic acids.

[0090] Compared to 8-17, Mg5, 17E, and GR5M4, the deoxyribozyme represented by formula 17NE described in this invention, such as deoxyribozyme 17NEM, is active against a variety of metal ions, including Mg. 2+ Ca 2+ Zn 2+ Pb 2+ Mn 2+ Cd 2+ Ba 2+ Co 2+ Ni 2+ Fe 2 + Fe 3+ Cr 3+ Hg 2+ Cu 2+ Tl + Ag +In a solution with a pH of 6 to 7, the deoxyribonuclease represented by Formula 17NE, such as deoxyribonuclease 17NEM, can specifically recognize and bind to the substrate nucleic acid, and undergo a catalytic cleavage reaction with the assistance of polyvalent metal ions, breaking down the nucleic acid substrate into two fragments.

[0091] The deoxyribozyme represented by Formula 17NE, such as deoxyribozyme 17NEM, described in this invention can be used as a candidate drug for gene therapy, or as a biosensor or chip for detecting metal ions or nucleic acids. Attached Figure Description

[0092] Figure 1 This demonstrates the principle of using deoxyribozymes to determine metal ions and substrate nucleic acids by detecting changes in the fluorescence intensity of fluorescent signal molecules;

[0093] Figure 2 A schematic diagram of the structure of some deoxyribozymes is shown;

[0094] Figure 3 The diagram shows the structural schematics of the deoxyribozyme and substrate nucleic acid represented by Formulas 17NE and 17NEM of the present invention, wherein X is a nucleotide residue represented by I or I', and X1 is a nucleotide residue represented by 1 or 1'.

[0095] Figure 4 The diagram shows the structural schematics of deoxyribozyme 17NEMQ and deoxyribozyme 17EV1Q designed in the embodiments of the present invention and the substrate nucleic acid, wherein X1 is the nucleotide residue shown as 1 or 1'.

[0096] Figure 5 The phosphoramidide monomer used to synthesize the deoxyribozyme of the present invention is shown;

[0097] Figure 6 The diagram shows the structures of other deoxyribozymes used as controls;

[0098] Figure 7 The results showed that 17NEMQ and other deoxyribozymes were present at 0.156 mM Ca2+. 2+ The catalytic reaction changes over time in the presence of Ca, and the reactions of 17NEMQ and 17EV1 at different Ca concentrations. 2+ The change of catalytic reaction over time in the presence of [a specific substance];

[0099] Figure 8 Various deoxyribozymes in 1.1 mM Mg 2+ The change of catalytic reaction over time in the presence of [a specific substance];

[0100] Figure 9 Deoxyribonucleases 17NEMQ and 17EV1Q at different concentrations of Ca 2+ / Mg 2+The change of catalytic reaction over time in the presence of [a specific substance];

[0101] Figure 10 The deoxyribonucleases 17NEMQ and 17EV1Q were shown to be effective in 0.25 mM Zn 2+ The catalytic reaction changes over time in the presence of Zn, and the changes of 17NEMQ and 17EV1Q at different Zn concentrations. 2+ The change of catalytic reaction over time in the presence of [a specific substance];

[0102] Figure 11 Deoxyribonucleases 17NEMQ and 17EV1Q at different concentrations of Pb 2+ The change of catalytic reaction over time in the presence of [a specific substance];

[0103] Figure 12 The changes in the catalytic reactions of deoxyribonucleases 17NEMQ and 17EV1Q over time in the presence of 5 μM metal ions. Detailed Implementation

[0104] To make the objectives and technical solutions of this invention clearer, the invention is further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, specific experimental methods not mentioned in the following embodiments were performed according to conventional experimental methods.

[0105] All instruments, materials and reagents used in this invention are well known in the field and selected accordingly, but this does not limit the implementation of this invention. Other well known instruments and reagents in the field can also be applied to the following embodiments of this invention.

[0106] Example 1: Synthesis of Deoxyribozymes

[0107] A substrate nucleic acid sequence, 3'BHQ1-d(TCC TAG AT)-r(GA)-d(CCG AGG TA)-FAM-5', was designed and synthesized by Takara Bio Inc. It contains a cleavage site 3'-r(GA)-5', which is an RNA unit, and DNA units at both ends, 3'-d(TCC TAG AT)-5' and 3'-d(CCG AGG TA)-5', respectively. A fluorescent quencher, BHQ1, was attached to the 3' end of the substrate nucleic acid, and a fluorescent group, FAM, was attached to the 5' end. Based on this, the two recognition arms of the deoxyribonuclease 17NEM were designed to ensure complete complementary pairing between the deoxyribonuclease and the substrate nucleic acid. The rA at the cleavage site does not participate in pairing, while the rG at the cleavage site interacts with the T at the deoxyribonuclease. 2.1 pair.

[0108] Using a DNA solid-phase synthesizer, employing methods such as Figure 5The phosphoramidamide monomers of the various nucleosides shown were used to synthesize deoxyribonuclease 17NEM via the phosphoramidamide method. In the fluorescence analysis method used in the embodiments of this invention, a fluorescence quenching group BHQ1 was added to the 3' end of deoxyribonuclease 17NEM, naming it 17NEMQ for ease of description. When this deoxyribonuclease is used in other analytical methods, other labeling methods can be used, or labeling may not be required. Figure 4 The diagram shows the structure of the deoxyribozyme 17NEMQ and its substrate designed in this embodiment, where X1 is the nucleotide residue indicated by 1 or 1'.

[0109] In this embodiment, deoxyribonuclease 17EV1 was also synthesized as a control, and a BHQ1 group was introduced at the 3'-end, denoted as 17EV1Q, with the following structural formula: Figure 4 As shown, it is the deoxyribozyme with the highest known catalytic activity. In addition, other deoxyribozymes 8-17, 17E, Mg5, and GR5M4 were synthesized as controls in this example. They have a BHQ1 group introduced at their 3' end and are therefore represented as 8-17Q, 17EQ, Mg5Q, and GR5M4Q, respectively, with structures as shown. Figure 6 As shown.

[0110] The molecular weight was identified by ESI-MS, as shown in Table 2. The difference between the measured and calculated values ​​was within the error range, indicating that the synthesized sequence was correct.

[0111] Table 2. Mass spectrometry identification results of deoxyribonucleases

[0112]

[0113] Example 2: Detection of catalytic activity of deoxyribozymes

[0114] Prepare three reaction systems:

[0115] (1) 50mM HEPES (pH 7.0) buffer: Weigh 2.838g of HEPES and dissolve it in 200mL of sterile water, and adjust the pH to 7.0 with NaOH.

[0116] Magnesium chloride hexahydrate or calcium chloride dihydrate was added to a 50 mM HEPES (pH 7.0) buffer solution to prepare solutions containing different concentrations of Ca. 2+ or Mg 2+ The reaction buffer solution is stored at 4°C for later use.

[0117] (2) 50mM MES (pH 6.0) buffer: Weigh 2.1325g MES and dissolve it in 200mL of sterile water, and adjust the pH to 6.0 with NaOH.

[0118] Zinc chloride was added to a 50 mM MES (pH 6.0) buffer solution to prepare solutions containing different concentrations of Zn. 2+ The reaction buffer solution is stored at 4°C for later use.

[0119] (3) 50mM HEPES-100mM NaNO3 (pH 7.0) buffer: Weigh 2.838g HEPES and 1.6998g NaNO3 and dissolve them in 200mL of sterile water, and adjust the pH to 7.0 with NaOH.

[0120] Lead nitrate was added to a 50mM HEPES-100mM NaNO3 (pH 7.0) buffer solution to prepare solutions containing different concentrations of Pb. 2+ The reaction buffer.

[0121] Heavy metal ions such as mercuric nitrate, chromium chloride, cadmium chloride, barium chloride, silver nitrate, cobalt chloride, nickel chloride, manganese chloride, ferrous chloride, ferric chloride, copper chloride, or thallium nitrate are added to a 50mM HEPES-100mM NaNO3-pH 7.0 buffer solution to prepare a reaction buffer solution containing 5μM heavy metal ions. The buffer solution is then stored at 4℃ for later use.

[0122] Depending on the reaction system, appropriate buffer solutions were selected to dissolve the deoxyribozymes obtained in Example 1 into 100 μM-10 μM stock solutions, and the substrate nucleic acid 3'BHQ1-d(TCC TAG AT)-r(AG)-d(CCG AGG TA)-FAM-5' into 10 μM-1 μM stock solutions. 2 μL of the deoxyribozyme stock solution and 2 μL of the substrate nucleic acid stock solution were added to each reaction buffer to initiate the reaction. The reaction was carried out at 37.0 °C, and fluorescence scanning was started simultaneously. A Tecan 1000-pro microplate reader was used, with an excitation wavelength of 496 nm (slit width 5.0 nm) and an emission wavelength of 524 nm (slit width 10.0 nm).

[0123] 2.1 Deoxyribonuclease 17NEMQ in HEPES-Ca 2+ Reactions in the system

[0124] In 50 mM HEPES (pH 7.0) buffer, the concentration ratio of deoxyribonuclease to substrate nucleic acid was 10:1 (1 μM deoxyribonuclease: 100 nM substrate nucleic acid), Ca 2+ The concentration gradients were: 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM, 0.156 mM, and 0.078 mM. 17NEMQ and other deoxyribozymes were detected at 0.156 mM Ca2+. 2+The catalytic reaction changes over time in the presence of Ca, and the changes of 17NEMQ and 17EV1Q at different Ca concentrations. 2+ The changes in the catalytic reaction over time in the presence of the following are as follows: Figure 7 As shown. The results show that in HEPES-Ca 2+ In the reaction system, the reaction of 17NEMQ is faster than that of 17EV1Q and other deoxyribozymes.

[0125] 2.2 Deoxyribonuclease 17NEMQ in HEPES-Mg 2+ system reaction

[0126] In 50 mM HEPES (pH 7.0) buffer, the concentration of deoxyribonuclease was 1 μM, the concentration of substrate was 100 nM, and the solution was prepared in Mg2+. 2+ The physiological concentration of 1.1 mM was used as the catalytic reaction condition. Various deoxyribonucleases reacted at 1.1 mM Mg... 2+ The changes in the catalytic reaction over time in the presence of the following are as follows: Figure 8 As shown in the figure. The results indicate that 17NEMQ exhibits the fastest catalytic reaction compared to 17EV1Q and other deoxyribozymes.

[0127] 2.3 Deoxyribonuclease 17NEMQ at physiological concentrations of Ca 2+ / Mg 2+ Reactions in the system

[0128] In 50 mM HEPES (pH 7.0) buffer, the concentration of deoxyribonuclease was 1 μM, the concentration of substrate nucleic acid was 100 nM, and the concentration of Ca2+ was within physiological limits. 2+ / Mg 2+ As catalytic reaction conditions, deoxyribonucleases 17NEMQ and 17EV1Q were subjected to different concentrations of Ca... 2 + / Mg 2+ The changes in the catalytic reaction over time in the presence of the following are as follows: Figure 9 As shown, both 17NEMQ and 17EV1Q underwent rapid catalytic reactions under physiological concentrations of divalent ions, thus both can be applied to intracellular target RNA cleavage and have potential value as gene therapy drugs. The results also showed that the catalytic reaction of 17NEMQ was faster than that of 17EV1Q.

[0129] 2.4 Deoxyribonuclease 17NEMQ in MES-Zn 2+ system reaction

[0130] In 50 mM MES (pH 6.0) buffer: the concentration ratio of deoxyribonuclease to substrate nucleic acid is 10:1 (1 μM deoxyribonuclease: 100 nM substrate), Zn 2+The concentration gradients are: 20 mM, 10 mM, 5 mM, 2.5 mM, 1 mM, 0.5 mM, 0.25 mM, 0.125, 0.05 mM, 5 μM, 500 nM, 50 nM.

[0131] Deoxyribonucleases 17NEMQ and 17EV1Q in 0.25 mM Zn 2+ The catalytic reaction changes over time in the presence of Zn, and the changes of 17NEMQ and 17EV1Q at different Zn concentrations. 2+ The changes in the catalytic reaction over time in the presence of the following are as follows: Figure 10 As shown. The results show that at 0.25 mM Zn 2+ Under the given reaction conditions, 17NEMQ reacts faster than 17EV1Q and other deoxyribonucleases.

[0132] 2.5 Deoxyribonuclease 17NEMQ in HEPES-Pb 2+ system reaction

[0133] In 50 mM HEPES-100 mM NaNO3 (pH 7.0) buffer, the ratio of deoxyribonuclease to substrate nucleic acid is 10:1 (1 μM deoxyribonuclease: 100 nM substrate nucleic acid) or 100:1 (1 μM deoxyribonuclease: 10 nM substrate nucleic acid), Pb 2+ The concentration gradients were 2 μM, 1 μM, 500 nM, 200 nM, 100 nM, 50 nM, and 25 nM.

[0134] Deoxyribonucleases 17NEMQ and 17EV1Q at different concentrations of Pb 2+ The changes in the catalytic reaction over time in the presence of the following are as follows: Figure 11 As shown. The results indicate that the catalytic reactions of the two ratios of deoxyribozymes with the substrate nucleic acid are basically the same, at various concentrations of Pb. 2+ In the catalytic system, the catalytic reaction of 17NEMQ is faster than that of 17EV1Q.

[0135] 2.6 Dependence of deoxyribonuclease 17NEMQ on heavy metal ions

[0136] In a 50 mM HEPES-100 mM NaNO3 (pH 7.0) buffer, deoxyribonuclease (1 μM) and substrate nucleic acid (10 nM) were added, followed by the addition of 5 μM Hg(NO3)2, 5 μM CrCl3, 5 μM CdCl2, 5 μM BaCl2, 5 μM AgNO3, 5 μM CoCl2, 5 μM NiCl2, 5 μM MnCl2, 5 μM FeCl2, 5 μM FeCl3, 5 μM CuCl2, or 5 μM TlNO3. The effect of polyvalent ions on the deoxyribonuclease reaction was evaluated. The catalytic reaction of deoxyribonuclease 17NEMQ in the presence of 5 μM metal ions over time is shown in the figure. Figure 12 As shown in the figure. The results indicate that the catalytic reaction of deoxyribozyme 17NEMQ is very significant in the presence of 5 μM metal ions, while for 17EV1Q, metal ions can hardly drive its catalytic reaction. Therefore, 17NEMQ is a deoxyribozyme with broad ion dependence.

[0137] The embodiments described above only illustrate one implementation of the present invention, and the steps and implementation methods are described in detail. However, this should not be construed as limiting the scope of the present invention. It should be particularly noted that those skilled in the art can make many modifications, improvements, and extensions without departing from the design and concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims. SEQUENCE LISTING <110> Military Medical Research Institute of the Academy of Military Sciences of the Chinese People's Liberation Army <120> Deoxyribonucleases and their uses <130> IDC210007 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Deoxyribonuclease <220> <221> misc_feature <222> (22)..(22) <223> Nucleotide residues represented by Formula 1 or 1' <400> 1 aggatctatc tcagcgagac gnaggctcca t 31 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Deoxyribonuclease <400> 2 aggatctatc cgagccggac gaggctccat 30 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Deoxyribonuclease <400> 3 aggatctatc tcagcgagac gaaggctcca t 31 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Deoxyribonuclease <400> 4 aggatctatc cgagccggtc gaaggctcca t 31 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Deoxyribonuclease <400> 5 aggatctatc cgagccggac gaaggctcca t 31 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Deoxyribonuclease <400> 6 aggatctatc cgagccggcc gaaggctcca t 31 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Substrate <220> <221> misc_feature <222> (9) (10) <223> Ribonucleic acid <220> <221> misc_feature <222> (9) (10) <223> Ribonucleotides <400> 7 tcctagatga ccgaggta 18

Claims

1. The deoxyribozyme or its derivative shown in Formula 17NE, 5'-d(N) m -dT 2.1 -d(CTCAGCGAGACGXA)-d(N) m’ -3' 17NE Where: d (N) m and d(N) m’ These represent the substrate nucleic acid recognition arms at the 5' and 3' ends of the deoxyribonuclease, respectively. Their base sequences are complementary to the substrate nucleic acid sequences. The composition and length of their base sequences are determined according to the substrate nucleic acid sequences. Each N is an independent nucleotide residue, and m and m' represent the number of N, each being a natural number greater than or equal to 6. dT 2.1 Represents a deoxythymidine residue, which pairs with the cleavage site rG or dG of the substrate nucleic acid sequence; X is a nucleotide residue represented by formula I or I', where R is (CH2). n R', where R' is an amino group, and n is 1, 2, 3, 4, 5, 6, 7, or 8. The derivative differs from the deoxyribonuclease only in that its 5' or 3' end is labeled with a marker, and it retains the function of the deoxyribonuclease. The marker is a substance capable of generating a detection signal.

2. The deoxyribozyme or its derivative according to claim 1, wherein the marker is a substance capable of generating fluorescence, a substance capable of quenching fluorescence signals, a substance capable of generating radioactive signals, or a substance capable of generating electrical signals.

3. The deoxyribonuclease or its derivative according to claim 1, wherein the substance capable of generating fluorescence is 6-carboxyfluorescein, the substance capable of quenching fluorescence signals is a fluorescence quenching molecule, and the substance capable of generating radioactive signals is a radioactive isotope.

4. The deoxyribonuclease or its derivative according to claim 3, wherein the substance capable of quenching the fluorescence signal is BHQ1, and the substance capable of generating a radioactive signal is radioactive. 32 P.

5. The deoxyribonuclease or its derivative according to claim 1, wherein n is 2, 3, 4, 5 or 6.

6. The deoxyribozyme or its derivative according to claim 5, wherein n is 2, 3 or 4.

7. The deoxyribozyme or its derivative according to claim 6, wherein n is 3.

8. The deoxyribozyme or its derivative according to claim 1, wherein the catalytic domain of the deoxyribozyme is 5'-d(CTCAGCGAGACGXA)-3', and the base positions in its sequence are labeled as 5'-d(C 3 T 4 C 5 A 6 G 7 C 8 G 9 A 10 G 11 A 12 C 13 G 14 X 15 A 15.0 The catalytic domain is divided into three parts: 1) consisting of three pairs of complementary bases, namely C 3 T 4 C 5 With G 11 A 10 G 9 The stem formed between them; 2) Terminal ring structure A 6 G 7 C 8 ;3) Large ring structural domain A 12 C 13 G 14 X 15 A 15.0 .

9. The deoxyribozyme or a derivative thereof according to claim 1, wherein m and m' are each independently 6, 7, 8, 9, 10, 11 or 12.

10. The deoxyribozyme or a derivative thereof according to claim 1, wherein m and m' are each independently 6, 7, 8 or 9.

11. The deoxyribozyme or its derivative according to any one of claims 1-10, wherein the substrate nucleic acid is 3'-(N'). x -G-rA-(N') y The -5' symbol represents an RNA or a DNA-RNA-DNA chimeric structure containing at least one RNA unit rA, where G represents rG or dG, 3'-G-rA-5' is the cleavage site of the deoxyribonuclease, rA does not participate in base pairing, and rG or dG interacts with dT in the deoxyribonuclease. 2.1 Pairing, (N') x It is the substrate nucleic acid recognition arm (N) that can interact with the 5' end of the deoxyribozyme. m This forms a DNA or RNA sequence with complementary base pairings, (N') y It is the substrate nucleic acid recognition arm (N) that can interact with the 3' end of the deoxyribozyme. m’ A DNA or RNA sequence is formed by complementary base pairing, where each N' is an independent nucleotide residue, x and y represent the number of N's, and x and y are each independent natural numbers greater than or equal to 6.

12. The deoxyribonuclease or its derivative according to claim 11, wherein the substrate nucleic acid is synthetic RNA or DNA-RNA-DNA, or The substrate nucleic acid is RNA derived from plasmid expression or RNA derived from living organisms.

13. The deoxyribozyme or a derivative thereof according to claim 11, wherein x and y are each independently 6, 7, 8, 9, 10, 11 or 12.

14. The deoxyribozyme or a derivative thereof according to claim 11, wherein x and y are each independently 6, 7, 8 or 9.

15. The deoxyribozyme or a derivative thereof according to claim 11, wherein x>m, y>m', (N') x The partial base sequence adjacent to G-rA in the middle and (N) m Complementary, (N') y The partial base sequence adjacent to G-rA in the middle and (N) m’ Complementary; or x and m are equal, y and m' are equal, (N') x With d(N) m The bases are completely complementary, (N') y With d(N) m’ The bases are completely complementary.

16. The deoxyribonuclease or its derivative according to any one of claims 1-10, wherein the sequence of the deoxyribonuclease is: d(AGG ATC TAT CTC AGC GAG ACG XA GGC TCC AT), The sequence of the deoxyribonuclease derivative is: d(AGG ATC TAT CTC AGC GAG ACG XA GGC TCCAT)-BHQ1, Where X is a nucleotide residue represented by formula 1 or 1'. 。 17. Use of the deoxyribonuclease or a derivative thereof according to any one of claims 1-16 in the preparation of a sensor or chip for detecting metal ions, wherein the metal ion is Mg. 2+ Ca 2+ Zn 2+ Pb 2+ Mn 2+ Cd 2+ Ba 2+ Co 2+ Ni 2+ Fe 2 + Fe 3+ Cr 3+ Hg 2+ Cu 2+ Tl + or Ag + .

18. A chip or sensor, said chip or sensor comprising: a) The deoxyribozyme or a derivative thereof as described in any one of claims 1-16, and b) The substrate nucleic acid as described in any one of claims 1-16, or a substrate nucleic acid capable of being cleaved by the deoxyribonuclease or its derivative as described in any one of claims 1-16, or The chip or sensor contains: a) The deoxyribozyme or a derivative thereof as described in any one of claims 1-16, and c) Metal ions, wherein the metal ions are selected from Mg 2+ Ca 2+ Zn 2+ Pb 2+ Mn 2+ Cd 2+ Ba 2+ Co 2+ Ni 2+ Fe 2+ Fe 3 + Cr 3+ Hg 2+ Cu 2+ Tl + Ag + and any combination thereof.

19. The chip or sensor of claim 18, wherein the 5' or 3' end of the substrate nucleic acid in b) is labeled with a marker, said marker being a substance capable of generating a detection signal.

20. The chip or sensor of claim 19, wherein the marker is a substance capable of generating fluorescence, a substance capable of quenching fluorescence signals, a substance capable of generating radioactive signals, or a substance capable of generating electrical signals.

21. The chip or sensor of claim 20, wherein the substance capable of generating fluorescence is 6-carboxyfluorescein, the substance capable of quenching fluorescence signals is a fluorescence quenching molecule, and the substance capable of generating radioactive signals is a radioactive isotope.

22. The chip or sensor of claim 21, wherein the substance capable of quenching the fluorescence signal is BHQ1, and the substance capable of generating a radioactive signal is radioactive. 32 P.

23. A method for detecting metal ions for non-diagnostic purposes, comprising: (1) Provide the sample to be tested; (2) Contact the sample to be tested with the deoxyribonuclease or its derivative as described in any one of claims 1-16 and the substrate nucleic acid as described in any one of claims 1-16 or the substrate nucleic acid that can be catalytically cleaved by the deoxyribonuclease or its derivative as described in any one of claims 1-16; (3) Measure the lysis of the substrate nucleic acid. The metal ions are selected from Mg 2+ Ca 2+ Zn 2+ Pb 2+ Mn 2+ Cd 2+ Ba 2+ Co 2+ Ni 2+ Fe 2+ Fe 3+ Cr 3+ Hg 2+ Cu 2+ Tl + Ag + and any combination thereof.

24. The method of claim 23, wherein the 5' or 3' end of the substrate nucleic acid is labeled with a marker, said marker being a substance capable of generating a detection signal.

25. The method of claim 24, wherein the marker is a substance capable of generating fluorescence, a substance capable of quenching fluorescence signals, a substance capable of generating radioactive signals, or a substance capable of generating electrical signals.

26. The method of claim 25, wherein the substance capable of generating fluorescence is 6-carboxyfluorescein, the substance capable of quenching fluorescence signals is a fluorescence quenching molecule, and the substance capable of generating radioactive signals is a radioactive isotope.

27. The method of claim 26, wherein the substance capable of quenching the fluorescence signal is BHQ1, and the substance capable of generating the radioactive signal is radioactive. 32 P.

28. A method for detecting nucleic acids for non-diagnostic purposes, comprising: (1) Provide the sample to be tested; (2) Contact the sample to be tested with the deoxyribonuclease or its derivative as described in any one of claims 1-16 and metal ions; (3) Measure the lysis of nucleic acids in the sample to be tested. The metal ions are selected from Mg 2+ Ca 2+ Zn 2+ Pb 2+ Mn 2+ Cd 2+ Ba 2+ Co 2+ Ni 2+ Fe 2+ Fe 3+ Cr 3+ Hg 2+ Cu 2+ Tl + Ag + Or any combination thereof.

29. A kit comprising the deoxyribonuclease or a derivative thereof as described in any one of claims 1-16.

30. The kit of claim 29, further comprising: The substrate nucleic acid as described in any one of claims 1-16, or a substrate nucleic acid capable of being catalytically cleaved by the deoxyribonuclease or its derivative as described in any one of claims 1-16, or Metal ions, wherein the metal ions are selected from Mg 2+ Ca 2+ Zn 2+ Pb 2+ Mn 2+ Cd 2+ Ba 2+ Co 2+ Ni 2+ Fe 2+ Fe 3+ Cr 3+ Hg 2+ Cu 2+ Tl + Ag + and any combination thereof.

31. The kit of claim 30, wherein the 5' or 3' end of the substrate nucleic acid is labeled with a marker, said marker being a substance capable of generating a detection signal.

32. The kit of claim 31, wherein the marker is a substance capable of generating fluorescence, a substance capable of quenching fluorescence signals, a substance capable of generating radioactive signals, or a substance capable of generating electrical signals.

33. The kit of claim 32, wherein the substance capable of generating fluorescence is 6-carboxyfluorescein, the substance capable of quenching fluorescence signals is a fluorescence quenching molecule, and the substance capable of generating radioactive signals is a radioactive isotope.

34. The kit of claim 33, wherein the substance capable of quenching the fluorescence signal is BHQ1, and the substance capable of generating a radioactive signal is radioactive. 32 P.

35. The kit according to any one of claims 29-34, further comprising a buffer substance.

36. The kit of claim 35, wherein the buffer substance is selected from 4-hydroxyethylpiperazine ethanesulfonic acid, morpholine ethanesulfonic acid, and tris(hydroxymethyl)aminomethane.

37. Use of the deoxyribonuclease or its derivative according to any one of claims 1-16 in the preparation of gene therapy drugs.

38. A composition comprising the deoxyribonuclease or a derivative thereof as described in any one of claims 1-16, and a pharmaceutically acceptable carrier or excipient.

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