CN101940783A - Application of DNAzyme in preparing medicines for enhancing tumor chemotherapy sensitivity - Google Patents

Abstract

The invention discloses application of DNAzyme in preparing medicines for enhancing tumor chemotherapy sensitivity. The DNAzyme comprises a deoxyribonucleotide sequence 5'-GGCTAGCTACAACGA-3' with catalytic function, and first and second binding sequences, wherein the first and second binding sequences are respectively connected with 3' end and 5' end of the sequence with catalytic function, respectively composed of 7-12 nucleotides, and complemented with 3' end and 5' end sequences of the R*Y cutting site of cell apoptosis inhibiting gene mRNA or pre-mRNA in bcl-2 family. The DNAzyme can specifically recognize and cut the mRNA or the pre-mRNA of the apoptosis inhibiting gene, thereby reducing the expression of the apoptosis inhibiting gene and enhancing the sensitivity of tumor cells to chemotherapeutic medicines.

Description

The application of DNAzyme in the medicine of preparation enhancing chemotherapy of tumors sensitivity

Technical field

The present invention relates to the DNAzyme of a kind of targeted cells apoptogene family, relate in particular to a kind of purposes of DNAzyme, this DNAzyme is by suppressing the expression of specific gene, thereby promotes apoptosis of tumor cells, realizes chemotherapy sensitizing.

Background technology

Malignant tumor is one of major disease of harm humans health.For the treatment of malignant tumor, mainly take the Comprehensive Treatment mode that combines with operative treatment, radiotherapy, chemotherapy etc. at present.Although chemotherapeutics benefits many tumor patients, curative effect still is difficult to satisfactory.Causing the main cause of chemotherapy failure is the constitutional and the acquired drug-resistance of tumor cell.By the curative effect of chemotherapy sensitizing strategy raising chemotherapeutics, reducing its toxicity is the important measures of radical cure tumor.Therefore, low toxicity chemotherapeutic sensitizer efficiently is with a wide range of applications and higher using value, and the R and D of accelerating chemotherapeutic sensitizer will bring new hope for the tumor healing.

It is the apoptosis susceptibility reduction of cell to induced by chemotherapeutic agents that tumor cell produces one of drug-fast important molecule mechanism of chemotherapy.Apoptosis is a kind of body development, emergent and remove the important physiological process of mutant etc.Many pathological changes take place in the body that can cause unusually of this process, comprise tumor, viral infection, nervous system disease etc.Apoptotic approach mainly contains two: one is by the apoptosis enzyme (caspase) in the extracellular signal active cell, and one is to discharge apoptosis enzyme activity factor by mitochondrion to activate caspase.These activatory caspase can cause apoptosis with intracellular important protein degradation.Apoptotic regulation and control relate to many genes, comprise ICE, Apaf-1, Bcl-2, Fas/APO-1, c-myc, p53, ATM etc.

Bcl-2 is an apoptosis suppressor, and coding film integral protein has now found that at least 19 congeners, is referred to as bcl-2 family, and they play regulating and controlling effect in the apoptosis pathway that mitochondrion participates in, can the control line plastochondria in the release of antiapoptotic factors such as cytochrome C.The Bcl-2 family member is contained 1-4 Bcl-2 homeodomain (BH1-4), and have usually a carboxylic end span membrane structure territory (transmembrane region, TM).Wherein BH4 is the peculiar domain of anti-apoptotic proteins, and BH3 is and promotes the relevant domain of apoptosis.Bcl-2 family can be divided into two classes according to function and structure: a class is anti-apoptosis (anti-apoptotic), as: Bcl-2, Bcl-xl, Bcl-w, Mcl-1; One class is (pro-apoptotic) that promotes apoptosis, as: Bax, Bak, Bad, Bid, Bim.Also having a class in pro apoptotic protein is only to contain the BH3 domain, as Bid, Bad.Though Bcl-2 albumen is present on mitochondrial membrane, endoplasmic reticulum and the outer nuclear membrane, mainly is positioned mitochondrial outer membrane, the function of its antagonism pro apoptotic protein.Most of pro apoptotic proteins then mainly are positioned Cytoplasm, in case cell is subjected to inducing of antiapoptotic factors, they can be to the mitochondrion transposition, form transmembrane channel by oligomerization at mitochondrial outer membrane, perhaps open mitochondrial PT hole, thereby cause the antiapoptotic factors in the mitochondrion to discharge, activate caspase, cause apoptosis.Studies show that apoptotic imbalance all appears in many tumors, and be accompanied by the high expressed of anti-apoptotic genes expression, cause the opposing of tumor cell undue growth and chemotherapy.Suppress the anti-apoptotic genes expression expression specifically and be expected to increase the sensitivity of tumor cell chemotherapeutics.

DNAzyme (deoxyribozyme claims DNAzyme again) is a kind of short-movie section single stranded DNA with catalysis that utilizes external molecular evolution technique to obtain, and has catalytic activity and structure identification ability efficiently.DNAzyme will be efficiently the catalytic degradation ability combine with the targeting identification ability of antisense, can close target gene at target from the mRNA level specifically, thereby the goal of regulation and control protein expression is a kind of New Policy of efficiently special target gene therapy.The DNAzyme unique chemical is in the nature deoxy-oligonucleotide, and character is relatively stable; Molecular weight is little, and structure is simple relatively, and is good to the accessibility of substrate; Catalytic efficiency and specificity height, side effect is low; The restriction that target site is selected still less; Be easy to synthesize, cheap.Studies show that, compare with other several methods of closing Disease-causing gene from the mRNA level, DNAzyme can be closed target gene at target from the mRNA level specifically, is a kind of new strategy of target gene therapy efficiently, and in field extensive uses such as antiviral and antitumor.Utilize DNAzyme to suppress disease related gene, for example, oncogene and anti-apoptotic gene are a kind of means of novel targeted therapy tumor.

Summary of the invention

The targeted drug that the purpose of this invention is to provide effective treatment of a class or adjuvant therapy of tumors.

DNAzyme of the present invention strengthens chemotherapy of tumors sensitivity by suppressing the expression of anti-apoptotic genes expression.DNAzyme is a kind of novel gene inhibition agent, and it has the chemical stability of antisense oligonucleotide, have the function of the catalyze cleavage RNA of ribozyme again simultaneously, and its synthetic expense is very low.Based on these unique advantages, this technology has been widely used in the body and external gene regulation (pharmacol.Rev.52 (3) such as Sun: 325-347).

The invention provides a kind of DNAzyme, be used to prepare the medicine that strengthens chemotherapy of tumors sensitivity, it comprises:

One catalysis sequence is deoxynucleoside acid sequence 5 '-GGCTAGCTACAACGA-3;

First binding sequence that connects catalysis sequence 5 ' end, be made up of 7-12 nucleotide, be complementary to 3 ' terminal sequence of the R*Y cleavage site of apoptosis suppressor gene mRNA in the bcl-2 family or pre-mRNA, wherein * represents cut point, R represents A or G, and Y represents U or C; With

Connect second binding sequence of catalysis sequence 3 ' end, form, be complementary to 5 ' terminal sequence of the R*Y cleavage site of apoptosis suppressor gene mRNA in the bcl-2 family or pre-mRNA by 7-12 nucleotide.

Described DNAzyme is a kind of dna molecular, can discern and cut the connecting key of arbitrary purine nucleotides and pyrimidine nucleotide among said target mrna or the pre-mRNA specifically, its structure and mechanism of action as shown in Figure 1, N=G wherein, U, C or A; R*Y is a cleavage site; R=A or G; Y=U or C; 5 ' end binding sequence (being above-mentioned first binding sequence) is complementary to 3 ' terminal sequence of the cleavage site that comprises described Y; 3 ' end binding sequence (being above-mentioned second binding sequence) is complementary to and does not comprise the 5 ' terminal sequence of described R at interior cleavage site.

The Bcl-2 family gene of DNAzyme targeting of the present invention comprises Bcl-2, Bcl-xL, Bcl-w, Bfl-1, brag-1, Mcl-1, apoptosis suppressor genes such as A1.The unconventionality expression of Bcl-2 family gene can cause that cell height propagation and apoptosis are obstructed.The inhibition that the Bcl-2 family gene is expressed can promote the apoptosis of tumor cell, strengthens the sensitivity to chemotherapeutics.First-selected target gene is Bcl-2 and Bcl-xL.SEQ ID No.1 and 2 in representational DNAzyme sequence such as the sequence table.

Described DNAzyme generally is made up of 29～39 nucleotide, and the direction from 5 ' to 3 ' is connected by phosphodiester bond.For strengthening the stability of DNAzyme, can carry out the ability that chemical modification is degraded with the antiacid and antienzyme that improves DNAzyme to the phosphodiester bond in its binding sequence, for example, adopt the D2EHDTPA diester linkage, make described first binding sequence and/or second binding sequence respectively contain 1-6 D2EHDTPA diester linkage.Adopt lock nucleic acid (Locked nucleic acids, LNA) and peptide nucleic acid(PNA) (peptide nucleic acids, PNA) modification of form also can improve the stability of DNAzyme.In addition, the enhancing of DNAzyme stability can also realize by binding sequence being carried out other chemical modifications, comprise:

1) to the modification of base, glycosyl and the modification of trunk structure.To the modification of base, for example, methylated base, methylolated base etc.To the modification of glycosyl, for example 2 ', 3 ' ribonucleic acid or DNA (deoxyribonucleic acid) that replaces.Connecting key between ribosyl and the mononucleotide is called the trunk structure of nucleic acid.The modification of trunk structure is comprised that connecting key and other can enhanced stability and the modification of affinity, for example, to the modification of glycosyl structure.Concrete example has, and when being reverse with respect to natural dextroisomer sugar, can use left-handed (L-) isomer deoxyribose in base; Or with 2 ' of glycosyl-hydroxyl with 2 '-halogens, 2 '-O-alkyl, 2 '-O-alkyl-n (O-alkyl) replaces; Perhaps use following connecting key: the methyl acid phosphate diester linkage.DNAzyme of the present invention can have part modifies, or all modifies, or the combination of different modifying.Preferably 2 '-O-methyl is modified.

2) terminal protection: the end of molecule add protecting group because of, to prevent the degraded of molecule.This protection can be 5 ' end, also can be 3 ' end, or two ends is all protected.For example, oppositely connect nucleotide residue endways; Terminal nucleotide is a dideoxy nucleotide; Terminal peptide bond connects; The glycosyl 2 ' of nucleotide or 3 ' connect METHAPHOSPHORIC ACID base, alkyl, aryl, cordycepin (cordycepin), cytosine arabinoside (cytosine arabanoside), alkoxyl (as methoxyl group, ethyoxyl etc.), fluorescein, two pyridyls, cholesterol, biotin, acridine (acridine), rhodamine psoralen (rhodamine psoralen), glyceryl (glyceryl), butanols base, butyl or hexanol base endways.Preferably oppositely connect at 3 ' end 3 '-3 '.

The medicine that can prepare multiple inhibition tumor growth and enhancing chemotherapy of tumors sensitivity with DNAzyme of the present invention as active substance.Described DNAzyme can be formed pharmaceutical composition with chemotherapeutics and use, wherein said chemotherapeutics mainly comprises, but be not limited to: daunorubicin (daunorubicin), dactinomycin (dactinomycin), amycin (doxorubicin), bleomycin (bleomycin), mitomycin (mitomycin), chlormethine (nitrogen mustard), chlorambucil (chlorambucil), melphalan (melphalan, melphalan), cyclophosphamide (cyclophosphamide), Ismipur (6-mercaptopurine), 6-thioguanine (6-thioguanine), cytosine arabinoside (cytarabine), 5-fluorouracil (5-flurouracil), floxuridine (floxuridine), methotrexate (methotrexate), colchicine (colchicine), vincristine (vincristine), vincaleucoblastine (vinblastin), etoposide (etoposide), cisplatin (cisplatin), paclitaxel (Paclitaxel) etc.Can contain various pharmaceutically acceptable excipient, adjuvant in the described pharmaceutical composition.In the described pharmaceutical composition, can contain one or more described DNAzyme, such as containing two kinds, three kinds, four kinds, DNAzyme more than five kinds or five kinds, the apoptosis suppressor gene in the identical or different bcl-2 family of targeting.

DNAzyme provided by the invention for animal or human's dosage is: per kilogram of body weight 1～50mg.

The functional effect of DNAzyme medicine provided by the present invention or pharmaceutical composition can be measured with method well known in the art.These methods comprise that body is interior and external, for example, obtain effective DNAzyme by external selection; In cell culture system, analyze it to the influence of expression of target gene and the influence of pair cell phenotype; In model and the clinical trial, measure the biological effect of medicine or pharmaceutical composition in vivo, comprise drug effect, pharmacology, medicament, toxicity etc.The body inner model comprises the disease animal model and the intact animal of foundation.

Medicine of the present invention or pharmaceutical composition can adopt parenteral, oral or partial administering mode.

Medicine of the present invention or pharmaceutical composition can be prepared by technology well known in the art.The selection of pharmaceutical carrier can be decided according to preparation type and route of administration, and preparation type is spray, suppository, tablet, liquid etc. for example, for example intravenous injection of route of administration, oral, bone marrow injection etc.Excipient can contain several pharmaceutical carriers.In addition, contained stabilizing agent, antiseptic and other composition is generally 0.5%～2% of pharmaceutical composition weight in the pharmaceutical composition.Those skilled in the art can determine suitable administering mode and delivery system.

Pharmaceutical composition of the present invention can be made different dosage forms on request, comprises liquid, tablet, pill, granular, powder, ointment, Emulsion, injection or suppository.Any medicine acceptable medium and adjuvant all can use, for example, water, glycerol, oils, ethanol, flavoring agent, antiseptic, food dye etc. are used for liquid preparation, and starch, sugar, diluent, granule, lubricant, binding agent, dispersant etc. are used to prepare solid preparation.

Injection preparation is the aqueous solution of DNAzyme, and solvent is water or the normal saline that meets medicinal standard.Injection liquid can also contain suitable liquid medium, suspending agent and can regulate the preparation of osmotic pressure, antiseptic etc.Actual fabrication method and program are known for a those skilled in the art.

Local application's dosage form can be made Emulsion, dressing, gel, washing liquid, ointment, liquid etc.Can add surfactant, penetrate with the deep layer that increases medicine.These surfactants comprise natural, also can be chemosynthesis, for example different third myristate.

The preparation of spray-type can be adopted and DNAzyme is dissolved in or is suspended in hair spray or hair spray and the solvent, and for example ethanol is as solvent.In local application's dosage form and spray-type, drug ratios (DNAzyme) is generally 0.001%～40% of gross weight.

The preparation of suppository can mix DNAzyme and make with lipid medium, for example cupu oil (theobroma oil), cocoa butter, glycerol, gelatin or polyoxyethylene glycol.

DNAzyme can also be made liposome, to increase the oligonucleotide half-life in vivo.Used lipid comprises, but be not limited to cuorin (Cardiolipin), dimyristoyl phosphatidyl choline (dimyristoyl phosphatidylcholine), dipalmitoyl phosphatidyl choline (dipalmitoyl phosphatidylcholine), dioleoyl phospholipid phatidylcholine (dioleoyl phosphatidylcholine), phosphatidyl glycerol (phosphatidyl glycerol), palmitoyloleoyl phosphatidylcholine, palmitoyloleoyl phosphatidyl glycerol, phosphatidic acid (phosphatidic acid), lysophosphatidic acid (lysophosphatidic acid), Phosphatidylserine (phosphatidyl serine), phosphatidylinositols (phosphatidyl inositol), cholesterol (cholesterol).

When implementing the tumor inner injecting and administering, but dosage unit of administration in per 1～10 day, or 1～10 dosage unit of administration every day, up to healing, or symptom is alleviated.In some cases, administration in 1-24 hour before chemotherapy, dosage is 1～4 dosage unit every day.In some cases, patient is by 2～3 dosage units of doctor's advice medication every day.

Description of drawings

Fig. 1 is the structure and the mechanism of action sketch map of DNAzyme.

The inhibitory action that the DNAzyme DT895 that Fig. 2 has shown targeting Bcl-2 expresses Bcl-2 in prostate gland cancer cell PC3, transitional cell bladder carcinoma cell line T24 and breast cancer cell MDA-MB231, wherein: Cells refers to the situation that pair cell not carries out transfection; TMP refers to only use the situation of transfection reagent TMP transfectional cell; Ctrl refers to irrelevant contrast; DT895 refers to utilize the situation of transfection reagent TMP with the DNAzyme DT895 transfectional cell of 2mM concentration.

The inhibitory action that the DNAzyme DT882 that Fig. 3 has shown targeting Bcl-xL expresses Bcl-xL in prostate gland cancer cell PC3, transitional cell bladder carcinoma cell line T24, lung cell A549, nasopharyngeal carcinoma cell CNE-1 and colon cancer cell HCT116, wherein: Cells refers to the situation that pair cell not carries out transfection; TMP refers to only use the situation of transfection reagent TMP transfectional cell; Ctrl refers to irrelevant contrast; DT882 refers to utilize the situation of transfection reagent TMP with the DNAzyme DT882 transfectional cell of 2mM concentration.

Fig. 4 has shown the interior chemotherapy enhancement effect experimental result of the DNAzyme DT895 body of Bcl-2 among the embodiment 3, and wherein A is the carcinoma of prostate model, and B is a breast cancer model.

Fig. 5 has shown the interior chemotherapy enhancement effect experimental result of the DNAzyme DT882 body of Bcl-xL among the embodiment 4, and wherein A is the carcinoma of prostate model, and B is a breast cancer model

The specific embodiment

Below by embodiment the present invention is described in further detail, but the scope that does not limit the present invention in any way.

Embodiment 1: the DNAzyme of targeting bcl-2 and bcl-xL mRNA is to the inhibitory action of expression of target gene

Bcl-2 and Bcl-xL are high expressed in many tumors, comprise pernicious cutaneous tumor, nasopharyngeal carcinoma, breast carcinoma, colon cancer, carcinoma of prostate, ovarian cancer, lymphoma etc.Targeting suppresses Bcl-2 or the Bcl-xL expression can promote apoptosis of tumor cells, strengthens chemosensitivity.

In the present embodiment, by scanning to bcl-2 and bcl-xLmRNA, find out AU and GU cleavage site, and design DNAzyme, carry out the enzyme-substrate complex thermodynamic stability and analyze (Δ G ° kcal/mol), the evaluation of external cutting power filters out two respectively DNAzyme DT895 and the DT882 of targeting bcl-2 and bcl-xL.Its sequence is:

DT895：5’-CCCAGTTCAGGCTAGCTACAACGACCCGTCCCT-3’(SEQ?ID?No.1)

DT882：5’-TTTTTATAAGGCTAGCTACAACGAAGGGATGGG-3’(SEQ?ID?No.2)

Further DT895 and DT882 have been carried out chemical modification and (, introduced 3 D2EHDTPA diester linkages respectively at 5 ' end and 3 ' end; 3 ' end is introduced a T who oppositely connects) after, respectively with DT895 and the transfection of DT912 DNAzyme to different tumor cells, extract cell protein, utilize Western blot to analyze the depression effect that DNAzyme is expressed bcl-2 and bcl-xL.As shown in Figure 2, the DT895 DNAzyme can suppress the expression of Bcl-2 in prostate gland cancer cell PC3, transitional cell bladder carcinoma cell line T24 and breast cancer cell MDA-MB231.As shown in Figure 3, the DT882 DNAzyme can suppress Bcl-xL at prostate gland cancer cell PC3, bladder cancer cell lines T24, lung cancer cell line A549, and colon carcinoma cell line HCT116 and nasopharyngeal carcinoma cell are the expression among the CNE1.

Embodiment 2: the external chemotherapy sensitizing effect of the DNAzyme of targeting Bcl-2 and bcl-xL

The DNAzyme of having confirmed targeting Bcl-2 and Bcl-xL can suppress target gene expression specifically in cell after, respectively DT895 and the transfection of DT882 DNAzyme are entered a series of tumor cells, observation DNAzyme itself and DNAzyme and anti-cancer medicine paclitaxel (Taxol) share the influence to apoptosis of tumor cells.Flow cytometer is adopted in the apoptosis analysis, measures the ratio of sub-G1 cell mass (apoptotic cell group) with the PI colouring method.As shown in table 1, the DNAzyme of targeting Bcl-2 or Bcl-xL use separately (2mM) but equal inducing tumor cell generation apoptosis.As the DNAzyme (2mM) of targeting Bcl-2 or Bcl-xL and Taxol (50nM) when share, the apoptosis ratio that is subject to processing cell significantly strengthens, and this short chemosensitivity effect all produces effect to different tumors.Contrast is irrelevant oligonucleotide.

The external chemotherapy sensitizing effect of table 1. targeting Bcl-2 and Bcl-xL DNAzyme

Embodiment 3: chemotherapy enhancement effect in the DNAzyme DT895 body of targeting Bcl-2

In order further to analyze the chemotherapy sensitizing effect of DNAzyme in vivo, we have used human prostata cancer and breast cancer transplantable tumor mouse model, and the DNAzyme DT895 of checking targeting Bcl-2 is in animal body to the influence of tumor growth.Experiment divides four groups: normal saline contrast (saline); Use DNAzyme (DNAzyme) separately; Use paclitaxel (Taxol) separately; DNAzyme and paclitaxel share (DNAzyme+Taxol).Every group is 8 Balb/C nude mices, every nude inoculation 1 * 10 ⁶Tumor cell (being suspended in 0.1ml Matrigel).Gross tumor volume measures twice weekly, by length * wide * height * π/6 formula volume calculated.When gross tumor volume reaches 100-200mm ³The time, by operation method the Alzet osmotic pumps is implanted mouse peritoneal, the opening of pump is towards tumor.Alzet osmotic pumps (model1002) is that (1.5 * 0.6cm), interior dress 0.5ml DNAzyme liquid discharges totally 14 days to capsule shape with 12.5mg/kg/d dosage.Paclitaxel is with 25mg/kg dosage intraperitoneal administration, weekly (200 μ l).The experimental result (see figure 4) shows that the DNAzyme of targeting Bcl-2 and chemotherapeutics share the curative effect that can improve chemotherapeutics significantly, thereby has further proved the radiotherapy sensitization effect of DNAzyme.

Embodiment 4: chemotherapy enhancement effect in the DNAzyme DT882 body of targeting Bcl-xL

Similar to Example 3, we have used human prostata cancer and breast cancer transplantable tumor mouse model, and the DNAzyme DT882 of checking targeting Bcl-xL is in animal body to the influence of tumor growth.Experiment divides four groups: normal saline contrast (saline); Use DNAzyme (DNAzyme) separately; Use paclitaxel (Taxol) separately; DNAzyme and paclitaxel share (DNAzyme+Taxol).Every group is 8 Balb/C nude mices, every nude inoculation 1 * 10 ⁶Tumor cell (being suspended in 0.1ml Matrigel).Gross tumor volume measures twice weekly, by length * wide * height * π/6 formula volume calculated.When gross tumor volume reaches 100-200mm ³The time, by operation method the Alzet osmotic pumps is implanted mouse peritoneal, the opening of pump is towards tumor.Alzet osmotic pumps (model1002) is that (1.5 * 0.6cm), interior dress 0.5ml DNAzyme liquid discharges totally 14 days to a kind of capsule shape with 12.5mg/kg/d dosage.Paclitaxel is with 25mg/kg dosage intraperitoneal administration, weekly (200 μ l).The experimental result (see figure 5) shows that the DNAzyme of targeting Bcl-xL and chemotherapeutics share the curative effect that can improve chemotherapeutics significantly, thereby has further proved the radiotherapy sensitization effect of DNAzyme.

Claims (9)

1. the application of DNAzyme in the medicine of preparation enhancing chemotherapy of tumors sensitivity is characterized in that described DNAzyme comprises:

One catalysis sequence is deoxynucleoside acid sequence 5 '-GGCTAGCTACAACGA-3 ';

Connect first binding sequence of catalysis sequence 5 ' end, form, be complementary to 3 ' terminal sequence of the R*Y cleavage site of apoptosis suppressor gene mRNA in the bcl-2 family or pre-mRNA by 7-12 nucleotide; With

Connect second binding sequence of catalysis sequence 3 ' end, form, be complementary to 5 ' terminal sequence of the R*Y cleavage site of apoptosis suppressor gene mRNA in the bcl-2 family or pre-mRNA by 7-12 nucleotide;

In R*Y, * represents cut point, and R represents A or G, and Y represents U or C.

2. application as claimed in claim 1 is characterized in that, described DNAzyme is the oligonucleotide that is formed by connecting by 29-39 nucleotide.

3. application as claimed in claim 1 is characterized in that, described apoptosis suppressor gene is Bcl-2, Bcl-xL, Bcl-w, Bfl-1, brag-1, Mcl-1 or A1.

4. application as claimed in claim 3 is characterized in that, described DNAzyme is the nucleotide sequence shown in the SEQ ID No.1 or 2 in the sequence table.

5. application as claimed in claim 1 is characterized in that, described DNAzyme has a kind of chemical modification at least in first and second binding sequences.

6. application as claimed in claim 5 is characterized in that, respectively contains 1-6 D2EHDTPA diester linkage in first binding sequence of described DNAzyme and/or second binding sequence.

7. application as claimed in claim 5 is characterized in that described chemical modification is selected from: end oppositely connects, ribosyl 2 '-O-methyl, lock nucleic acid and peptide nucleic acid(PNA).

8. application as claimed in claim 1, it is characterized in that, described DNAzyme and chemotherapeutics are formed pharmaceutical composition, contain one or more described DNAzyme in this pharmaceutical composition, the apoptosis suppressor gene in the identical or different bcl-2 family of targeting.

9. application as claimed in claim 8, it is characterized in that described chemotherapeutics is selected from one or more in the following medicine: daunorubicin, dactinomycin, amycin, bleomycin, mitomycin, chlormethine, chlorambucil, melphalan, cyclophosphamide, Ismipur, 6-thioguanine, cytosine arabinoside, 5-fluorouracil, floxuridine, methotrexate, colchicine, vincristine, vincaleucoblastine, etoposide, cisplatin and paclitaxel.

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