CN105296491A - Aptamer AS1411 subjected to chemical modification and application thereof - Google Patents
Aptamer AS1411 subjected to chemical modification and application thereof Download PDFInfo
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Abstract
The invention discloses an aptamer AS1411 subjected to chemical modification and application thereof. A modification strategy of isonucleoside or isonucleoside combined with 2'-deoxyinosine is adopted, and the nucleotide in different positions of the aptamer AS1411 is replaced, so that the partial spatial conformation of the aptamer AS1411 is changed, the space structure is optimized, and the purposes that the molecular stability is improved, the recognition capability for target molecules is strengthened, the acting force for the targets is strengthened, and the biological activity of the aptamer is improved are achieved. Study indicates that the AS1411 modified through the method can be combined with target proteins better, the breast cancer cell proliferation resistant and DNA replication resistant capacities are remarkably strengthened, the inhibitory effect on breast cancer transplantation tumor growth is more obvious, and the difference has statistical significance; the computer simulation shows that the combining capacity of the modified AS1411 with target protein pyrenin is obviously improved; besides, the modified AS1411 can specially regulate some functional miRNA, and can be hopefully developed into a biological network regulating instrument.
Description
Technical field
The present invention relates to a kind of modified aptamer (aptamer) AS1411 and uses thereof, in particular to one by product of utilizing heteronuclear glycosides or different nucleoside combinations 2 '-Hypoxanthine deoxyriboside and carrying out chemically modified to aptamer AS1411 sequence and obtain and uses thereof, the invention belongs to biomedicine field.
Background technology
Aptamer, i.e. aptamer, come from Latin " aptus ", is by the single strain oligonucleotide of 20-60 based composition (RNA) or strand oligodeoxynucleotide (DNA).The multiple target molecules such as its energy Specific binding proteins, small molecules, ion and cell.Aptamer is invented in nineteen ninety by people such as Nobel Prize in medicine winner Szostak and Gold, and it has the advantage that a lot of antibody hardly matches.Aptamer is obtained by SELEX technology screening, the aptamer (Aptamer) utilizing this technology can filter out specificity from random single chain nucleotide sequence storehouse to be combined with target high-affinity.After first Tuerk etc. uses this technology screening to the specific oligonucleotide acid aglucon of specific adsorption phage T4DNA polysaccharase and organic dye molecule, through the development of twenties years, SELEX technology became a kind of important research means and instrument.
In theory, the aptamer that can obtain for any target is screened by SELEX.Utilize SELEX to screen scientist's screening and obtain a series of aptamer closely-related with human diseases, as the aptamer for HIV1-RT, vascular endothelial growth factor (VEGF), paranuclein, zymoplasm, hnRNPA1 albumen etc.Aptamer AS1411 wherein for paranuclein completes phase II clinical trials, and it has obvious therapeutic action to kidney and nonsmall-cell lung cancer, in the middle of carrying out the phase ii clinical trial of acute myeloid leukaemia.Therefore carry out and the research work of aptamer is had very important significance.
The current aptamer overwhelming majority is obtained by SELEX screening.And four important steps in SELEX process: build storehouse, screening, separation, pcr amplification, on screening, whether success all has important impact.At present by reasonably design screening storehouse and optimized Separation method can improve the success ratio of SELEX screening greatly.In SELEX screening, PCR is an important link.To screen the nucleic acid that obtains for template in this link, increase under the effect of archaeal dna polymerase.In the building-up process of DNA, because archaeal dna polymerase has stronger selectivity, natural A, G, C, T tetra-kinds of Nucleotide can only be identified, so screen the combination that the aptamer obtained is generally A, G, C, T.For the better nucleoside analog of some physico-chemical properties as UNA, LNA, 2 '-F-araN etc., archaeal dna polymerase cannot be incorporated in aptamer.This limitation limits the application of SELEX.Carrying out UNA, LNA, 2 '-F-araN, 2 '-O-methyl modification discovery to having screened the aptamer obtained at present, these can be optimized the physico-chemical property of the aptamer screened by SELEX to a certain extent through the aptamers modified or improve the activity of aptamer.
In order to increase the specificity of aptamer and target further, the physico-chemical property optimizing aptamer improves the activity of aptamer, is necessary further to optimize aptamer.And traditional SELEX screening cannot meet this requirement.The identification of aptamer and target is the process that a mutual induction agrees with.Therefore carry out certain fine setting in aptamer local, the interaction with target can be strengthened under the prerequisite keeping nucleic acid total secondary structure, improve specificity and the biological activity of aptamer.
Heteronuclear glycosides refers to that a base analog is moved to the nucleosides of other position by 1 ' of glycosyl.Be similar to natural ribose, according to the difference of glycosyl configuration, heteronuclear glycosides can be divided into again D-and L-two kinds, and wherein D-form is identical with natural nucleus glycoside type, and L-configuration is then contrary.Because the base of heteronuclear glycosides is shifted, the local conformation of the aptamer causing heteronuclear glycosides to mix changes.When the site that heteronuclear glycosides mixes is the action site of aptamer and target, the change of Base space conformation can cause changing with the space length of target, when the heteronuclear glycosides contrary with two kinds of configurations is incorporated into the binding site of nucleic acid and target respectively, will cause a kind ofly increasing with the operating distance of target, active reduction, the operating distance of the aptamer that another kind of configuration heteronuclear glycosides mixes and target shortens, active increase.Therefore heteronuclear glycosides is incorporated in aptamer, by adjustment aptamer local space conformation, the reactive force strengthened with target can be reached, improve the bioactive object of aptamer further.This modification strategy of heteronuclear glycosides is different with SELEX, SELEX screening is from numerous unordered nucleotide sequence, search out the nucleic acid that a certain bar specifically can be combined with target specificity, and it is then optimization to existing aptamer that heteronuclear glycosides modifies object that strategy acts on.But their congruence, namely obtains the aptamer that can be combined with target high specific by various method.SELEX is the screening at a kind of initial stage, it is the optimization in a kind of later stage that heteronuclear glycosides modifies strategy, and heteronuclear glycosides modifies strategy and the supplementary and perfect each other method of screening aptamer of SELEX screening method.
The present invention utilizes the aptamer AS1411 of the modification strategy of heteronuclear glycosides or different nucleoside combinations 2 '-Hypoxanthine deoxyriboside to paranuclein to carry out chemically modified, has searched out and can improve the aptamer with target avidity further.Also prove that the aptamer after chemically modified policy optimization of the present invention can significantly improve the biological activity to target by experiment in vitro and experimentation on animals.Therefore, this modification strategy further being promoted, by being expected to, various different aptamer being optimized, thus obtain with target specificity stronger, active better aptamer.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of product by obtaining after utilizing heteronuclear glycosides or different nucleoside combinations 2 '-Hypoxanthine deoxyriboside to carry out chemically modified to aptamer AS1411 sequence and uses thereof, the present invention carries out chemically modified by adopting heteronuclear glycosides or different nucleoside combinations 2 '-Hypoxanthine deoxyriboside to aptamer, change the space conformation of aptamer and target bound fraction base, thus reach the interaction strengthening aptamer and target, strengthen aptamer to the specificity of target, and improve the bioactive object of aptamer.
In order to achieve the above object, the technical solution used in the present invention is as follows:
A kind of aptamer AS1411 through chemically modified of the present invention, the sequence of the aptamer AS1411 before modification is as shown in SEQIDNo.1, it is characterized in that, heteronuclear glycosides is mixed in one or more nucleosides sites of nucleic acid aptamer sequence AS1411, the structural formula of described heteronuclear glycosides is as shown in Formula I or Formulae II, heteronuclear glycosides shown in Formula I is the heteronuclear glycosides of L-configuration, heteronuclear glycosides shown in Formulae II is the heteronuclear glycosides of D-form, wherein, Base is VITAMIN B4 A, thymus pyrimidine T, guanine G or cytosine(Cyt) C
The corresponding position of natural nucleus glycoside in aptamer AS1411 sequence is replaced to carry out coupling with the heteronuclear glycosides shown in Formula I or Formulae II.
In the present invention, preferably, while heteronuclear glycosides is mixed in one or more nucleosides sites of aptamer AS1411 sequence, 2 '-Hypoxanthine deoxyriboside is mixed in the one or more nucleosides sites being also included in aptamer AS1411 sequence, and the structural formula of described 2 '-Hypoxanthine deoxyriboside is as shown in Formulae II I:
The corresponding position of natural nucleus glycoside in aptamer AS1411 sequence is replaced to carry out coupling with 2 '-Hypoxanthine deoxyriboside shown in the heteronuclear glycosides shown in Formula I or Formulae II and Formulae II I.
D/L configuration heteronuclear glycosides has following features compared with natural nucleus glycoside: a. base is displaced to 2 ' position from 1 ' of sugared ring; The raw upset of sugared environment-development of b.L configuration heteronuclear glycosides, local conformation change compares D configuration nucleosides greatly.Causing under the constant prerequisite of aminoacid sequence, can there is certain change in the aptamer conformation compared with natural aptamer adopting D/L heteronuclear glycosides to modify.When this decorating site is in the region be combined with target, then can play the effect regulating and be combined with target.Moiety site mixes heteronuclear glycosides, finally reaches the bonding force of raising and target, strengthens bioactive object.The effect of 2 '-Hypoxanthine deoxyriboside and the effect of heteronuclear glycosides similar, but it reaches the object of adjustment local space conformation mainly through adjustment base.
In a particular embodiment of the present invention, be selected from through the aptamer AS1411 of chemically modified the group that following sequence forms:
1) 6 of the AS1411 sequence shown in SEQIDNo.1 mix the heteronuclear glycosides shown in Formula I and replace natural nucleus glycoside to carry out coupling in corresponding position and the aptamer (AS1411-6L) that obtains, and wherein, Base is thymus pyrimidine T;
2) 12 of the AS1411 sequence shown in SEQIDNo.1 mix the heteronuclear glycosides shown in Formulae II and replace natural nucleus glycoside to carry out coupling in corresponding position and the aptamer (AS1411-12D) that obtains, and wherein, Base is thymus pyrimidine T; And
3) 6 and 12 of the AS1411 sequence simultaneously shown in SEQIDNo.1 mix the heteronuclear glycosides replacement natural nucleus glycoside shown in Formula I and Formulae II respectively and carry out coupling in corresponding position and the aptamer (AS1411-6L/12D) that obtains, wherein, Base is thymus pyrimidine T; And
4) while 6 and 12 of the AS1411 sequence shown in SEQIDNo.1 mix the heteronuclear glycosides shown in Formula I and Formulae II respectively, 24 in AS1411 sequence are mixed 2 ' shown in Formulae II I-Hypoxanthine deoxyriboside, the aptamer (AS1411-6L/12D/24dI) that replacement natural nucleus glycoside carries out coupling in corresponding position and obtains, wherein, Base is thymus pyrimidine T.
For AS411-6L, the above 6 of AS1411 sequence shown in SEQIDNo.1 mixes the heteronuclear glycosides (Base is thymus pyrimidine T) shown in Formula I and replaces natural nucleus glycoside to carry out coupling in corresponding position referring to that different thymidine replacement natural nucleus glycoside (T) of holding the 6th Nucleotide (i.e. " the T ") position started to mix the L configuration shown in Formula I at AS1411 (nucleotides sequence is classified as 5 '-GGTGGTGGTGGTTGTGGTGGTGGTGG-3 ' (shown in SEQIDNo.1)) nucleotide sequence 5 ' carries out coupling in corresponding position, and all the other by that analogy.
Research proves, AS1411-6L, AS1411-6L/12D that heteronuclear glycosides provided by the invention is modified have the features such as stable in physicochemical property; In addition AS1411-6L, AS1411-6L/12D, AS1411-6L/12D/24dI have biological activity more better than natural parental sequences.
Be applied to by the Activity Results that heteronuclear glycosides is incorporated in AS1411 in MCF-7 cancerous cell line, protein level and cell levels result show: mix the different thymidine shown in Formula I at 6 (AS1411-6L) of AS1411 positive-sense strand or mix the different thymidine shown in Formulae II at AS1411 positive-sense strand 12 (AS1411-12D) and simultaneously mix the different thymidine (AS1411-6L/12D) shown in Formula I and Formulae II respectively 6 and 12 of AS1411 positive-sense strand and replace natural nucleus glycoside AS1411 after the modification that coupling obtains is carried out in corresponding position can significantly improve bonding force and the anticancer proliferation activity of itself and paranuclein.Different nucleoside combinations 2 '-Hypoxanthine deoxyriboside is carried out chemically modified discovery to AS1411, 6 in AS1411 sequence are mixed the different thymidine shown in Formula I, the different thymidine shown in Formulae II is mixed at 12, simultaneously mixing 2 ' shown in III-Hypoxanthine deoxyriboside at 24 replaces the aptamer AS1411-6L/12D/24dI of natural nucleus glycoside after the modification that coupling obtains is carried out in corresponding position can improve the bonding force of itself and paranuclein further and suppress MCF-7 cancer cell multiplication vigor, experimentation on animals also shows that AS1411-6L/12D/24dI can show the growth suppressing mouse tumor.In experimentation on animals, find that AS1411-6L, AS1411-12D and AS1411-AS1411-6L/12D/24dI also can suppress the growth of mouse tumor-tissue significantly, computer simulation show AS1411-AS1411-6L/12D/24dI and target protein binding ability significantly improve.
Therefore, further, the invention allows for the described purposes of aptamer AS1411 in preparation treatment mammary cancer or leukemic medicine or reagent through chemically modified.Wherein, preferably, described treatment mammary cancer comprises anti-breast cancer cell proliferation and DNA replication dna, and suppresses breast cancer transplantable tumor growth.And
The medicine of the described aptamer AS1411 through chemically modified in preparation mammary cancer or leukemia early detection or reagent and as the purposes in bio-networks regulatory molecule.
Compared to prior art, the invention has the advantages that:
1. the AS1411 that heteronuclear glycosides provided by the invention is modified or different nucleoside combinations 2 '-Hypoxanthine deoxyriboside is modified, has the features such as stable in physicochemical property; Product self has better biological activity more fit than natural acid, and the AS1411 biological activity increase rate of combined modification is the highest in known report.
2., by the aptamer that comprehensive investigation heteronuclear glycosides or different nucleoside combinations 2 '-Hypoxanthine deoxyriboside mix in different loci, improve the external activity of aptamer.In addition, results of animal also shows that the aptamer that heteronuclear glycosides or different nucleoside combinations 2 '-Hypoxanthine deoxyriboside are modified can improve in the restraining effect of animal level to cancerous tissue.For the clinical application of the aptamer of heteronuclear glycosides modification provides important directive significance.
3. the AS1411 modified through heteronuclear glycosides provided by the invention can significantly improve the bonding force with target, find that AS1411-6L/12D can significantly improve the ability suppressing MCF-7 cancerous cell line propagation by the experiment of cell levels and experimentation on animals, this AS1411 clinical application afterwards for the modification of heteronuclear glycosides provides further-tests support.The AS1411-6L/12D/24dI modified through different nucleoside combinations 2 '-Hypoxanthine deoxyriboside can significantly improve and the bonding force of target (in known report, increase rate is the highest), has the activity of the growth at the strongest suppression mouse tumor of animal level.Computer simulation show, compared to parental sequences, AS1411-6L/12D/24dI and target protein binding ability significantly improve, and specificity can regulate some Breast Cancer-Specific function miRNA, as bio-networks regulation and control instrument.
Accompanying drawing explanation
Fig. 1 is natural and the CD spectrum experiment result figure of the AS1411 of heteronuclear glycosides modification;
Fig. 2 is the breast cancer cell Inhibit proliferaton experimental result picture of AS1411 and unit point heteronuclear glycosides modified outcome;
Fig. 3 is the AS1411 breast cancer cell Inhibit proliferaton experimental result picture that dibit point heteronuclear glycosides and the combination of different nucleoside combinations 2 '-Hypoxanthine deoxyriboside are modified;
Fig. 4 is AS1411 leukemia cell's Inhibit proliferaton experimental result picture that dibit point heteronuclear glycosides and the combination of different nucleoside combinations 2 '-Hypoxanthine deoxyriboside are modified;
Fig. 5 is that AS1411 animal level activity evaluation result is modified in dibit point heteronuclear glycosides and the combination of different nucleoside combinations 2 '-Hypoxanthine deoxyriboside;
Fig. 6 is that different the nucleoside combinations 2 '-Hypoxanthine deoxyriboside combination of AS1411 and dibit point modifies AS1411 with paranuclein in conjunction with Computer simulation results;
The MicroArray experimental result that Fig. 7 is AS1411, AS1411 is modified in the combination of different nucleoside combinations 2 '-Hypoxanthine deoxyriboside.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The solid phase synthesis of the AS1411 that embodiment 1 heteronuclear glycosides or different nucleoside combinations 2 '-Hypoxanthine deoxyriboside are modified
The synthesis of DNA adopts ApplliedBiosystemsmodel394DNA solid phase synthetic instrument.
The phosphorus acylated monomer of normal deoxynucleoside (dT, dGAc, dABz, dCAc) is bought from Shanghai JiMa pharmacy Technology Co., Ltd; 2 '-Hypoxanthine deoxyriboside phosphoramidite monomer (compound VI) is bought from Shanghai Zhi Yan Science and Technology Ltd. (Shanghai).CPG (CPG-dG), CAP-A and CAP-B, oxidation I2 liquid, Cl
3cCOOH buys from Beijing AudioCodes biotechnology company; The 5-second sulfydryl 1H-tetrazole solution of 0.25M is bought from Shanghai Zhi Yan Science and Technology Ltd. (Shanghai).
According to document (HWYu, LRZhang, JCZhuo, LTMa, LHZhang, Bioorg.Med.Chem., 1996,40,609-614) method, the heteronuclear glycoside compound shown in Formula I and Formulae II is prepared into respectively the heteronuclear glycosides phosphoramidite monomer shown in Formula I V and chemical formula V.That is: monomeric compound I, II is dry in a vacuum respectively, add the 1H-tetrazole of 3.5 times of equivalents, dried in vacuo overnight, add 5ml anhydrous pyridine, under condition of ice bath, add the sub-phosphorus reagent of 3.5 equivalents, react rear solvent evaporated, under anhydrous and oxygen-free condition, adopt silicagel column to be separated, finally obtain white solid, be i.e. compound IV, V.
Wherein, Base is selected from VITAMIN B4 A, thymus pyrimidine T, guanine G or cytosine(Cyt) C.
Synthesis scale: ~ 1.0 μm of ol
The preparation of 1H-tetrazole solution: argon shield lower-weighing, adds anhydrous acetonitrile, is made into 0.5M1H-tetrazole solution;
The preparation of the phosphorus acylated monomer solution of nucleosides: argon shield lower-weighing, adds anhydrous acetonitrile, is made into 0.12M solution;
The preparation of the phosphorus acylated monomer solution of the heteronuclear glycosides: { 5S-[3-O-(4 getting 0.231mmol under argon shield, 4 '-dimethoxytriphenylmethyl)-methyl]-4R-O-[(2-cyanoethyl-N, N '-di-isopropyl)-phosphoramidite base]-3S-(thymine base-1-yl) }-tetrahydrofuran (THF) (the heteronuclear glycoside compound shown in Formula I V), add the anhydrous acetonitrile of 2.5ml, be made into the solution of 0.092M; { 5R-[the 3-O-(4 of 0.244mmol is got under argon shield, 4 '-dimethoxytriphenylmethyl)-methyl]-4S-O-[(2-cyanoethyl-N, N '-di-isopropyl)-phosphoramidite base]-3R-(thymine base-1-yl) }-tetrahydrofuran (THF) (the heteronuclear glycoside compound shown in chemical formula V), add the anhydrous acetonitrile of 2.5ml, be made into the solution of 0.098M;
The preparation of 2 '-Hypoxanthine deoxyriboside phosphoramidite monomer solution: argon shield lower-weighing 2 '-Hypoxanthine deoxyriboside phosphoramidite monomer, adds anhydrous acetonitrile, be made into the solution of 0.12M;
The AS1411 sequence that to have adopted the method for solid phase synthesis to synthesize natural and heteronuclear glycosides is modified.On DNA synthesizer, be incorporated in synthesized sequence simultaneously replace natural nucleus glycoside phosphoramidite monomer to carry out coupling in corresponding position by the heteronuclear glycosides phosphoramidite monomer described in one or several or by the 2 '-Hypoxanthine deoxyriboside phosphoramidite monomer described in the heteronuclear glycosides phosphoramidite monomer described in one or several and one or several, every coupling nucleosides is a circulation, and each circulation comprises four reactions: de-DMT, coupling, closed, oxidation.
Wherein, paranuclein nucleic acid aptamer sequence (AS1411) modify before sequence as shown in SEQIDNo.1.
To synthesize AS411-6L, the 6th Nucleotide (i.e. " the T ") place that AS1411 sequence 5 ' end starts shown in SEQIDNo.1 mixes the heteronuclear glycosides (wherein Base is selected from thymus pyrimidine T) shown in Formula I and replaces natural nucleus glycoside to carry out coupling in corresponding position, and all the other by that analogy.
Synthesis step: weigh about 33mgCPG-dC/CPG-dG at every turn and load in synthesis post, according to the standard step of ABi394 nucleic acid synthesizer, each synthesis totally 84 steps.Normal nucleotide monomer coupling 3 times, each 180 seconds, different nucleoside monomers coupling 3 times, each 300 seconds, amounted to coupling time 15 minutes.
The cutting of DNA, deprotection: after end of synthesis, take off CPG, and it be 30% strong aqua and massfraction is 25% aqueous methylamine solution that equal-volume adds massfraction; constant temperature 60 DEG C; shaking table oscillatory reaction 60 minutes, cuts down oligonucleotide from CPG and removes part protecting group, centrifugal drying.Then add appropriate amount of purified water to dissolve, on the whizzer of 10,000 4 thousand revs/min centrifugal 10 minutes, supernatant is taken out, centrifugal drying, preserve at product is placed-80 DEG C.
The separation and purification of DNA: mixture dissolves with pure water, HPLC (SephedaxG-25, the aqueous solution of 50% acetonitrile) desalination, centrifugal drying.Then HPLC mode purifying, adopts following condition: Dionex-DNAPac-PA200 anion-exchange column gradient elution, 0-40min, 10%-30%A liquid (B liquid: 0.02Mtris-HClO
4damping fluid, pH is 8.0, containing 10% (referring to volume fraction) MeCN; A liquid: 0.4mol/LNaClO
4inB liquid), flow velocity 1.2mL/min.Lyophilize products therefrom, DEPC water redissolves, and HPLC (SephedaxG-25) desalination, lyophilize, obtains target single stranded DNA ,-80 DEG C of preservations.
According to the method described above, 3,6,9,12,13,15 or 24 of having synthesized respectively in AS1411 sequence mix the heteronuclear glycosides shown in Formula I or Formulae II or combine the 2 '-Hypoxanthine deoxyriboside (wherein Base is selected from thymus pyrimidine T) shown in chemical formula III, replace the AS1411 sequence of natural nucleus glycoside after the modification that coupling obtains is carried out in corresponding position.
The essential property research of the AS1411 sequence after embodiment 2 modification
1. sample ID: 3,6,9,12,13,15 of AS1411 sequence or 24 are mixed the heteronuclear glycosides (wherein Base is selected from thymus pyrimidine T) shown in Formula I or Formulae II, replace the AS1411 sequence of natural nucleus glycoside after the modification that coupling obtains is carried out in corresponding position, prepared by embodiment 1 method.
2.CD spectroscopic analysis
Dissolve AS1411 sequence sample with PBS, all samples, after 95 DEG C of sex change 5min, is slowly down to room temperature, to be measured.CD sweep limit is from 200-400nm, and homo(io)thermism is 25 DEG C, scanning interval 0.2nm, sweep velocity 100nm/min, and each sample repeats 3 scanning, adopts instrument to carry the smoothing process of software.Experimental result is shown in Fig. 1.
The avidity of that embodiment 3SPR measures the modification of heteronuclear glycosides and that heteronuclear glycosides combined deoxidation inosine is modified AS1411 sequence and paranuclein albumen
1. sample ID: AS1411-6L, AS1411-12D, AS1411-13L, AS1411-6L/12D, AS1411-6L/12D/24dI, is prepared by embodiment 1 method.
2. method
(1) by paranuclein proteopexy at CM5 chip.
A certain amount of paranuclein albumen is made into respectively the solution of a series of concentration with the sodium acetate soln of suitable pH value, optimization protein is at the best coupling condition of chip surface.Concrete operations are as follows: PBS damping fluid is flowing working fluid, temperature 25 DEG C, flow velocity 10 μ L/min, steady baseline.EDC and NHS mixed solution is flow through chip surface 7min with the flow velocity of 5 μ L/min, the carboxyl of activating surface.Be mixed with the paranuclein protein solution of different concns respectively, inject chip surface, find and be coupled to the most suitable concentration of chip surface.Then, close unreacted activated carboxyl with diethanolamine hydrochloride (1mmol/L, pH8.5), the time is about 2-3h.Be blank passage with passage 1, passage 2 carries out next step research as experimental group.
(2) AS1411 that heteronuclear glycosides is modified and different nucleoside combinations 2 '-Hypoxanthine deoxyriboside is modified and the interaction of paranuclein egg
The AS1411 sample that heteronuclear glycosides is modified and different nucleoside combinations 2 '-Hypoxanthine deoxyriboside is modified is mixed with respectively the solution (PBS damping fluid) of a series of concentration gradients within the scope of 0.02 ~ 100 μM, with 30 μ L/min, 3min, Dissociation time is that the flow velocity of 5min flows through chip surface, real time record itself and paranuclein in conjunction with situation.After reaction terminates, with the NaOH of different concns as regenerated liquid, the RU value according to sensing figure changes, and evaluates its elute effect, therefrom selects suitable regeneration soln, reusing for chip.
(3) dynamic experiment
According to the result of above-mentioned experiment screening, suitable different concns gradient is selected to carry out dynamic experiment, by the data Scatchard curve calculation aptamer that records and the interactional equilibrium dissociation constant of target proteins (Kd) value.The concentration of regenerated liquid NaOH is chosen to be 10mM.
(4) data processing
All experimental datas adopt the Biaevaluation analysis software of BIAcore3000 to carry out Treatment Analysis.
3. experimental result
The avidity experimental result of the AS1411 that unit point heteronuclear glycosides is modified and paranuclein albumen is in table 1.Dibit point heteronuclear glycosides modifies AS1411 and the combination of different nucleoside combinations 2 '-Hypoxanthine deoxyriboside modifies the avidity experimental result of AS1411 and paranuclein albumen in table 2.
The avidity test experiments result of the AS1411 that the natural and unit point D/L heteronuclear glycosides of table 1. is modified and target
The AS1411 that table 2. is natural, the different nucleoside combinations of dibit point D/L is modified, different the nucleoside combinations 2 '-Hypoxanthine deoxyriboside combination of dibit point D/L is modified and the avidity test experiments result of target
The above results shows, compared to the AS1411 sequence of unmodified, AS1411 sequence after the inventive method is modified and the bonding force of paranuclein are significantly improved, particularly dibit point heteronuclear glycosides modifies the AS1411 sequence of AS1411 and different nucleoside combinations 2 '-Hypoxanthine deoxyriboside combination modification, compared to the AS1411 sequence that unit point heteronuclear glycosides is modified, there is the higher ability be combined with target.
The AS1411 that embodiment 4 heteronuclear glycosides and different nucleoside combinations 2 '-Hypoxanthine deoxyriboside are modified is on the Inhibit proliferaton determination of activity of MCF-7 cancer cells and on Normocellular impact
1. sample ID: AS1411-3D, AS1411-3L, AS1411-6D, AS1411-6L, AS1411-15D, AS1411-15L, AS1411-9D, AS1411-9L, AS1411-12D, AS1411-12L, AS1411-13D, AS1411-13L, AS1411-24D, AS1411-24L, AS1411-6L/12D, AS1411-6L/12D/24dI, prepared by embodiment 1 method.
2. method
By MCF-7 breast cancer cell bed board in 96 orifice plates, with suitable serum for substratum, cell concn is made to be 1.5 × 10
3be advisable in individual/hole.After 16 hours, make cell attachment, respectively aptamers sample (or PBS in contrast) is directly joined in substratum, make the final concentration of the oligonucleotide of aptamers sample be 0.75 μM.In 2-4 subsequently days, the dosage of aptamers sample is reduced by half, joins in substratum.Cell counting Kit CCK-8 is used to count cell quantity every day after plating cells.In whole experimentation, not replaced medium.Experimental result as shown in Figures 2 and 3, result shows for MCF-7 breast cancer cell, AS1411-6L, AS1411-12D, AS1411-6L/12D (6L/12D), AS1411-6L/12D/24dI (6L/12D/24dI) administration group cell absorbancy OD value are starkly lower than AS1411 administration group, AS1411-6L, AS1411-12D, AS1411-6L/12D (6L/12D) are described, AS1411-6L/12D24dI (6L/12D/24dI) cell growth can produce more obvious restraining effect.More than experiment shows that AS1411-6L/12D, AS1411-6L/12D/24dI can significantly suppress MCF-7 breast cancer cell growth.
By K562 leukemia cell's bed board in 96 orifice plates, with suitable serum for substratum, cell concn is made to be 1.0 × 10
3be advisable in individual/hole.Respectively aptamers sample (or PBS in contrast) is directly joined in substratum, make the final concentration of the oligonucleotide of aptamers sample be 0.75 μM.In 1-2 subsequently days, the dosage of aptamers sample is reduced by half, joins in substratum.Cell counting Kit CCK-8 is used to count cell quantity every day after plating cells.In whole experimentation, not replaced medium.Experimental result as shown in Figure 4, result shows for K562 leukemia cell, AS1411-6L/12D/24dI (6L/12D/24dI) administration group cell absorbancy OD value is starkly lower than AS1411 administration group, illustrates that AS1411-6L/12D24dI (6L/12D/24dI) cell growth can produce more obvious restraining effect.More than experiment shows that AS1411-6L/12D/24dI can significantly suppress K562 leukemic cell growth.
The AS1411 that embodiment 5 heteronuclear glycosides and different nucleoside combinations 2 '-Hypoxanthine deoxyriboside are modified tests the tumor-inhibitory growth of mouse
1, sample ID: AS1411-12D (12D), AS1411-6L12D (6L/12D), AS1411-6L12D24dI (6L/12D/24dI), prepared by embodiment 1 method.
2, method
By being 5 × 10 to mouse in subcutaneous vaccination concentration
6the MCF-7 cell of individual cell/200 μ L sets up heterograft, and each site is set up at the right back side of nude mice.Inoculate after 5 days, when gross tumor volume reaches about 100mm
3time, mouse is divided at random 5 groups (n=6) namely: blank (PBS), contrast (AS1411), AS1411-12D (12D), AS1411-6L12D (6L/12D), AS1411-6L12D24dI (6L/12D/24dI).To the mouse of built vertical tumor model, treated by the mode of abdominal injection aptamer, injected dose is equivalent to 2.5OD/ mouse/sky.In injection 1-4 days every days once, inject once respectively the 6th day and the 8th day, inject six times altogether.Gross tumor volume uses following formulae discovery:
V＝L×W2/2
Wherein, L and W represents length and the width of the tumour using vernier callipers to measure in experimentation respectively.The weight of results of regular determination nude mice, to carry out the investigation of toxicity.As can be seen from Figure 5, the aptamers AS1411-6L/12D (6L/12D) that dibit point heteronuclear glycosides is modified and the aptamers AS1411-6L/12D/24dI (6L/12D/24dI) that different nucleoside combinations 2 '-Hypoxanthine deoxyriboside is modified obviously can delay tumor growth, by test constantly gross tumor volume, after modifying, AS1411 group is compared with natural A S1411 group, gross tumor volume obviously reduces, there is statistical significance (p < 0.01), and nude mice body weight has no obvious decline, namely without overt toxicity.
The Computer simulation results of the AS1411 sequence that different the nucleoside combinations 2 '-Hypoxanthine deoxyriboside combination of embodiment 6 dibit point is modified
1. sample ID: AS1411, AS1411-6L/12D/24dI are prepared by embodiment 1 method.
2. method:
Manual construction AS1411, AS1411-6L/12D/24dI respectively with the complex model of paranuclein, after the molecular dynamics simulation of 10ns, system reaches balance.Average structure analysis shows that four bases in loop district are combined by hydrogen bond, electrostatic and hydrophobic interaction with albumen interface amino acid.After the molecular dynamics simulation of 10ns, system reaches balance.Binding pattern analysis shows: 12 heteronuclear glycosides with D configuration replace, change the local conformation of four bases in loop district, add interaction amino acid whose with albumen interface, especially very strong electrostatic interaction and interaction of hydrogen bond is added, thus enhance the binding ability with paranuclein albumen, shown in Fig. 6.
The MicroArray experimental result of the AS1411 that different the nucleoside combinations 2 '-Hypoxanthine deoxyriboside combination of embodiment 7 dibit point is modified
1. sample ID: AS1411, AS1411-6L/12D/24dI, is prepared by embodiment 1 method.
2. method:
A. sample RNA extracting: by MCF-7 Breast Cancer Cell according to 1 × 10
7individual cells/well is laid on incubated overnight in 6 orifice plates.In nutrient solution, within second day, add AS1411 or AS1411-6L/12D/24dI that final concentration is 20 μMs, or equal-volume PBS is as blank.After culturing cell 72h, sucking-off nutrient solution adds the RNA extraction agent TRIzol of 1ml, extracted total RNA after cracking.
B.RNA quality examination: use Nanodrop to measure RNA in the absorption value of spectrophotometer 260nm, 280nm and 230nm, assesses purity with calculating concentration.Carry out Denaturing Agarose Gel electrophoresis with formaldehyde electrophoresis reagents, detect RNA purity and integrity.RNAQC is provided to report.
C. prepare fluorescence labeling probe: adopt miRCURYArrayPower labelling kit, with marker enzyme by Hy3 or Hy5 fluorophor mark miRNA, can obtain for the fluorescent probe with chip hybridization.
D. chip hybridization: use MAUI hybridization instrument by the probe that marked and miRCURY chip hybridization at the standard conditions.E. IMAQ and data analysis: the fluorescence intensity using GenePix4000B chip scanner scanning chip, and experimental result is converted to digital data preservation, use software kit to carry out analytic operation to raw data.
Fig. 7 result shows, after filtering out administration, compared to the discrepant miR of blank group MCF-7 tumour cell group down-regulated expression totally 76 (AS1411-6L/12D/24dI>AS1411>PB S, fold>2.5), wherein 7 have document support.After filtering out administration, compared to the discrepant miR of blank group tumour cell group up-regulated totally 35 (AS1411-6L/12D/24dI<AS1411<PBS, fold>2.5), wherein 4 have document support.
Claims (7)
1. the aptamer AS1411 through chemically modified, the sequence of the aptamer AS1411 before modification is as shown in SEQIDNo.1, it is characterized in that, heteronuclear glycosides is mixed in one or more nucleosides sites of nucleic acid aptamer sequence AS1411, the structural formula of described heteronuclear glycosides is as shown in Formula I or Formulae II, heteronuclear glycosides shown in Formula I is the heteronuclear glycosides of L-configuration, heteronuclear glycosides shown in Formulae II is the heteronuclear glycosides of D-form, wherein, Base is VITAMIN B4 A, thymus pyrimidine T, guanine G or cytosine(Cyt) C
The corresponding position of natural nucleus glycoside in aptamer AS1411 sequence is replaced to carry out coupling with the heteronuclear glycosides shown in Formula I or Formulae II.
2. the aptamer AS1411 through chemically modified according to claim 1, it is characterized in that, while heteronuclear glycosides is mixed in one or more nucleosides sites of aptamer AS1411 sequence, 2 '-Hypoxanthine deoxyriboside is mixed in the one or more nucleosides sites being also included in aptamer AS1411 sequence, and the structural formula of described 2 '-Hypoxanthine deoxyriboside is as shown in Formulae II I:
The corresponding position of natural nucleus glycoside in aptamer AS1411 sequence is replaced to carry out coupling with 2 '-Hypoxanthine deoxyriboside shown in the heteronuclear glycosides shown in Formula I or Formulae II and Formulae II I.
3. the aptamer AS1411 through chemically modified according to claim 1 and 2, it is characterized in that, the nucleic acid aptamer sequence after modification is selected from the group that following sequence forms:
1) 6 of the AS1411 sequence shown in SEQIDNo.1 mix the heteronuclear glycosides shown in Formula I and replace natural nucleus glycoside to carry out coupling in corresponding position and the aptamer that obtains, and wherein, Base is thymus pyrimidine T;
2) 12 of the AS1411 sequence shown in SEQIDNo.1 mix the heteronuclear glycosides shown in Formulae II and replace natural nucleus glycoside to carry out coupling in corresponding position and the aptamer that obtains, and wherein, Base is thymus pyrimidine T;
3) 6 and 12 of the AS1411 sequence simultaneously shown in SEQIDNo.1 mix the heteronuclear glycosides replacement natural nucleus glycoside shown in Formula I and Formulae II respectively and carry out coupling in corresponding position and the aptamer that obtains, and wherein, Base is thymus pyrimidine T; And
4) while 6 and 12 of the AS1411 sequence shown in SEQIDNo.1 mix the heteronuclear glycosides shown in Formula I and Formulae II respectively, 24 in AS1411 sequence are mixed 2 ' shown in Formulae II I-Hypoxanthine deoxyriboside, the aptamer that replacement natural nucleus glycoside carries out coupling in corresponding position and obtains, wherein, Base is thymus pyrimidine T.
4. the purposes of aptamer AS1411 in preparation treatment mammary cancer or leukemic medicine or reagent through chemically modified described in any one of claim 1-3.
5. purposes as claimed in claim 4, is characterized in that described treatment mammary cancer comprises anti-breast cancer cell proliferation and DNA replication dna, and suppresses breast cancer transplantable tumor growth.
6. the aptamer AS1411 through chemically modified described in any one of claim 1-3 is in the medicine prepared in mammary cancer or leukemia early detection or the purposes in reagent.
7. the aptamer AS1411 through chemically modified described in any one of claim 1-3 is as the purposes in bio-networks regulatory molecule.
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