CN104651501A - Cracking activation type nucleic acid adaptor diagnosis and treatment probe and application thereof - Google Patents
Cracking activation type nucleic acid adaptor diagnosis and treatment probe and application thereof Download PDFInfo
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- CN104651501A CN104651501A CN201510048971.8A CN201510048971A CN104651501A CN 104651501 A CN104651501 A CN 104651501A CN 201510048971 A CN201510048971 A CN 201510048971A CN 104651501 A CN104651501 A CN 104651501A
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
Abstract
The invention discloses a cracking activation type nucleic acid adaptor diagnosis and treatment probe and application of the cracking activation type nucleic acid adaptor diagnosis and treatment probe. The cracking activation type nucleic acid adaptor diagnosis and treatment probe comprises a long chain DNA, a short chain DNA, a fluorescence generation unit and a fluorescence extinguishing unit, wherein the short chain DNA and a part of sequences of the long chain DNA form a double-chain structure; the fluorescence generation unit is modified on the long chain DNA; the fluorescence extinguishing unit is modified on the short chain DNA. The cracking activation type nucleic acid adaptor diagnosis and treatment probe can be applied to detection of tumor living cells and tumors in living objects and has a tumor treatment function after medicines are filled. The cracking activation type nucleic acid adaptor diagnosis and treatment probe is multifunctional, high in specificity, high in sensitivity, relatively high in stability, easy and flexible in design and high in universality; a new guiding thought for building a nucleic acid adapter multifunctional probe is provided.
Description
Technical field
The present invention relates to Nucleic Acid Probe Technique field, particularly relate to one and to split type activation type aptamer diagnosis and treatment probe and application thereof.
Background technology
From 2002, PharmaNetics corporate Chief executive officer Funkhouser proposed diagnoses and treatment (theranostics) first, and diagnoses and treatment medical science becomes the focus of Recent study as optimal treatment pattern medically.Diagnostic and therapeutic system is integrated with diagnostic functions and treatment function, not only can directly diagnose the illness, the position of Real-Time Monitoring medicine and curative effect etc., and the adjustment that also can be successive treatment scheme provides tutorial message.In diagnostic and therapeutic system, realizing quick, high sensitivity, highly selective diagnosis should be prerequisite or even the key of whole system functional realiey.
DNA, as a kind of genetic material, possesses Encoding good, workable, good biocompatibility and stable character flexibly.Meanwhile, it also can as a kind of molecular recognition unit, specific binding target material as antibody.Therefore, DNA is often used to Nanostructure fabrication and applies in the Treatment and diagnosis of disease." Self-assembled; aptamer-tethered DNA nanotrains for targeted transport of molecular drugs in cancer theranostics " " Proceedings of the National Academy of Sciences " 2013
110(20): 7998-8003; Guizhi Zhu, Jing Zheng, Weihong Tan et al. forms double-strand as railway carriage using multiple DNA fragmentation hybridization, and aptamer Sgc8c constructs diagnostic and therapeutic system-nanometer train as locomotive engine.Locomotive engine Sgc8c drives whole system target target tumor locus, and the double stranded section (being rich in ACG/CGT sequence) of vehicle body is then for drug loading Dox.This diagnostic and therapeutic system successfully achieves the drug targeting transport under external Imaging: Monitoring.
But what current most of diagnostic and therapeutic system adopted the imaging of target is " Always on " pattern, and this pattern carrys out imaging target tissue mainly through diagnostic and therapeutic system avidity, accretion rate difference between target tissue and non-target tissue.Because diagnostic and therapeutic system unavoidably exists certain non-specific adsorption at non-target site, because limit this pattern in complex system or live body to the accurate, sensitive of target and imaging fast.Adopt the diagnostic and therapeutic system of activation type imaging pattern due to reach or in conjunction with target before do not produce signal and export, once reach or export in conjunction with signal just can be produced after target, be expected to realize accurate, sensitive and imaging fast.
Summary of the invention
The technical problem to be solved in the present invention overcomes the deficiencies in the prior art, a kind of type activation type aptamer diagnosis and treatment probe (Split Aptamer-based Theranostic Probe that splits of the special response of target tumour cell based on the type aptamer that splits is provided, abbreviate SAT P), additionally provide this application of type activation type aptamer diagnosis and treatment probe in lesion detection and treatment of splitting, there is the advantages such as easy and simple to handle, quick, sensitive, special, cost is low.
For solving the problems of the technologies described above, provide one to split type activation type aptamer diagnosis and treatment probe, the fluorescence quenching unit comprise long-chain DNA, the short chain DNA that can form duplex structure with described long-chain DNA partial sequence, modifying the fluorescence generating unit on described long-chain DNA and modify on described short chain DNA.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, the nucleotides sequence of described long-chain DNA is classified as the nucleotide sequence described in SEQ ID NO.1 or the nucleotide sequence described in SEQ ID NO.2.
Nucleotide sequence wherein described in SEQ ID NO.1 is specifically: 5 '-TAC TGT ACG GTT AGA TCT GCC TGC TCA TCT AAC TGC TGC GCC GCC GGG AAA A-3 ' (L-10).
Nucleotide sequence wherein described in SEQ ID NO.2 is specifically: 5 '-TAC TGT ACG GTT AGA TCT GCC TGC GTT CAT CTA ACT GCT GCG CCG CCG GGA AAA-3 ' (L-12).
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, described long-chain DNA to be connected by catenation sequence to be obtained by the first aptamer fragment and the second aptamer fragment of splitting of splitting.Described short chain DNA and described catenation sequence form duplex structure by the base mispairing effect under base pair complementarity or drug molecule mediate.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, the split nucleotides sequence of fragment of described first aptamer is classified as nucleotide sequence described in SEQ ID NO.3, and the split nucleotides sequence of fragment of described second aptamer is classified as nucleotide sequence described in SEQ ID NO.4.
Nucleotide sequence wherein described in SEQ ID NO.3 is specifically: 5 '-TAC TGT ACG GTT AGA T-3 ' (Apt-b).
Nucleotide sequence wherein described in SEQ ID NO.4 is specifically: 5 '-ATC TAA CTG CTG CGC CGC CGG GAA AA-3 ' (Apt-a).
Above-mentioned splits in type activation type aptamer diagnosis and treatment probe, described catenation sequence is preferably one section and can not splits the fragment that fragment (Apt-a) hybridizes with split fragment (Apt-b) and the second aptamer of the first aptamer, and length is preferably 8 ~ 20bp.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, the nucleotides sequence of described catenation sequence (Linker) is classified as the sequence, poly A sequence or the siRNA sequence relevant to cancer cells that are rich in GC base.The present invention preferred poly A sequence is AAAAAAAAAAAAAAA; But in the present invention, be not limited to this, the length of poly A sequence can adjust according to practical situation, because the effect length of poly A sequence is to probe stability at different temperatures and target stimulation responsiveness.
The siRNA sequence that the present invention is preferably relevant to cancer cells is the nucleotide sequence described in SEQ ID NO.9, be specially: 5 '-UUGAUUAACGCCCAGCGUUdTdT-3 ', but in the present invention, the siRNA sequence relevant to cancer cells is not limited to this, and siRNA sequence that arbitrarily can be relevant to cancer cells all can reach the same or analogous technique effect of the present invention.
When short chain DNA is the relevant siRNA complementary sequence of cancer cells, when catenation sequence is the siRNA sequence relevant to cancer cells, the gene therapy to tumour cell can be realized.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, described in be rich in the sequence of GC base for the nucleotide sequence described in SEQ ID NO.5 or the nucleotide sequence described in SEQ ID NO.6.
Nucleotide sequence wherein described in SEQ ID NO.5 is specifically: 5 '-CTG CCT GCT C-3 ' (Linker-10).
Nucleotide sequence wherein described in SEQ ID NO.6 is specifically: 5 '-CTG CCT GCG TTC-3 ' (Linker-12).
The nucleotide sequence forming the short chain DNA of duplex structure with catenation sequence complementary pairing should be mutually:
Nucleotide sequence described in SEQ ID NO.7: 5 '-GAG CAG GCA G-3 ' (S-10).
Nucleotide sequence described in SEQ ID NO.8: 5 '-GAA CGC AGG CAG-3 ' (S-12).
The complementary base number of the double stranded section regulating catenation sequence and short chain DNA to be formed or the content of GC pairing, namely change the T of double-strand hybridization
mvalue, the type activation type aptamer diagnosis and treatment probe that can make to split possesses best stability and target stimulation responsiveness under varying experimental conditions.Therefore, when at room temperature carrying out tumour cell test experience, preferred double-chain length is about 10 pairs of nucleotide sequences (containing 7 pairs of GC bases), is namely hybridized by L-10 and S-10 and obtains, now T
mbe worth lower; And when (37 DEG C) detect target tumour cell in live body, preferred double-chain length is about 12 pairs of nucleotide sequences (containing 8 pairs of GC bases), is namely hybridized by L-12 and S-12 and obtain, now T
mbe worth higher.
Preferably, fluorescence generating unit, fluorescence quenching unit should be modified on long-chain DNA and on short chain DNA sequence dna respectively, and both should be close to each other with the condition of satisfied generation FRET (fluorescence resonance energy transfer) (FRET).
Above-mentioned splits in type activation type aptamer diagnosis and treatment probe, and described aptamer fragment preferably refers to the tumor cell specific aptamer screened by the Fas lignand system evolution technology (being called for short SELEX technology) of index concentration.Preferred, described aptamer fragment refers to nucleic acid aptamer sequence people's acute lymphoblastic leukemia T lymphocyte (being called for short CCRF-CEM tumour cell) to specific recognition capability, and the nucleotides sequence of described complete nucleic acid aptamer sequence (Sgc8c) is classified as:
Sgc8c：5’-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3’；
The second aptamer that described complete nucleic acid aptamer sequence is formed after splitting split nucleotide sequence of Segment A pt-b of Segment A pt-a and the first aptamer that splits is respectively:
Apt-a：5’-ATC TAA CTG CTG CGC CGC CGG GAA AA-3’；
Apt-b：5’-TAC TGT ACG GTT AGA T-3’。
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, described fluorescence generating unit is luminescent dye molecule or fluorescent nano particle, and described fluorescence quenching unit is fluorescence quenching, has the functionalized nano material of fluorescence quenching effect.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, described fluorescence dye is fluorescein, tetramethylrhodamine or Cy5; Described fluorescent nano particle is nano SiO 2 particle or the fluorescence quantum of parcel fluorescence dye; Described fluorescence quenching group is DABCYL, BHQ1, BHQ2, BHQ3; The described functionalized nano material with fluorescence quenching effect is gold nano grain, manganese dioxide nano particle, stannic oxide/graphene nano lamella or carbon nanotube.
Those skilled in the art can determine and select the enforcement array mode of fluorescence generating unit and fluorescence quenching unit voluntarily, such as fluorescein and DABCYL, fluorescein and BHQ1, tetramethylrhodamine and BHQ2, Cy5 and BHQ3, tetramethylrhodamine and gold nano grain, Cy5 and gold nano grain, Cy5 and carbon nanotube, the nano SiO 2 particle wrapping up fluorescence dye and BHQ2, fluorescence quantum and BHQ2 etc.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, described in the type activation type aptamer diagnosis and treatment probe that splits also comprise drug molecule, described drug molecule is loaded between the double-strand that described long-chain DNA and short chain DNA forms.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, described drug molecule is Zorubicin (Dox), coralyne (coralyne).
Catenation sequence described in appropriate design, the double-stranded region of formation can be endowed the function of drug loading.When described catenation sequence is for being rich in the sequence of (GC), the preferred Dox of double-stranded region drug loading formed, wherein GC base pair content is preferably 7 to (when catenation sequence length is 10 pairs of nucleotide sequences) or 8 to (when catenation sequence length is 12 pairs of nucleotide sequences); When described catenation sequence is poly A sequence, the preferred coralyne of double-stranded region drug loading of formation, the preferred 8-25 of a sequence length nucleotide sequence.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, described in the type activation type aptamer diagnosis and treatment probe that splits adopt the method comprised the following steps to prepare:
(1) modify in the split junction of fragment of the catenation sequence of long-chain DNA and the first aptamer the long-chain DNA that fluorescence generating unit obtains being modified with fluorescence generating unit; The short chain DNA being modified with fluorescence quenching unit is obtained at 3 ' the terminal modified fluorescence quenching unit of short chain DNA;
The nucleotide sequence being modified with the long-chain DNA of fluorescence generating unit is specifically: 5 '-TAC TGT ACG GTT AGA (Cy5) TCT GCC TGC TCA TCT AAC TGC TGC GCC GCC GGG AAA A-3 ' (Cy5-L-10).
Can also be: 5 '-TAC TGT ACG GTT AGA (Cy5) TCT GCC TGC GTT CAT CTA ACT GCT GCG CCG CCG GGA AAA-3 ' (Cy5-L-12).
The nucleotide sequence being modified with the short chain DNA of fluorescence quenching unit is specifically: 5 '-GAG CAG GCA G-BHQ3-3 ' (BHQ3-S-10).
Can also be: 5 '-GAA CGC AGG CAG-BHQ3-3 ' (BHQ3-S-12).
(2) the long-chain DNA being modified with fluorescence generating unit by the described and described short chain DNA being modified with fluorescence quenching unit is 1: 2 mixing according to mol ratio, in 95 DEG C of water-bath 4 min, then 5 min are on ice placed in rapidly, 40 min are hybridized, the preparation of the type that splits described in completing activation type aptamer diagnosis and treatment probe under placing normal temperature afterwards.
The above-mentioned type activation type aptamer diagnosis and treatment probe that splits, preferably, also comprise the type activation type aptamer diagnosis and treatment probe step of splitting preparing medicine carrying: be to mix at 0.6: 1 in molar ratio by drug molecule and the type activation type aptamer diagnosis and treatment probe that splits, 30 ~ 40min is put in ambient temperatare, make drug molecule embed double-stranded region completely, obtain the type activation type aptamer diagnosis and treatment probe that splits of medicine carrying.
As a total technical conceive, present invention also offers a kind of above-mentioned application of type activation type aptamer diagnosis and treatment probe in tumour cell detects of splitting, can be divided into the detection of tumour cell:
(1) to the specific detection of tumor living cell in damping fluid;
(2) to effective detection of serum tumor viable cell;
(3) the real-time fluorescence imaging of Live Animals in-vivo tumour and In vivo detection.
In above-mentioned application, preferably, to the method for the specific detection of tumor living cell in damping fluid, be specially: the type activation type aptamer diagnosis and treatment probe that splits described in adding in Cell Buffer to be measured (final concentration of the type that splits activation type aptamer diagnosis and treatment probe is preferably 25 nM), after under normal temperature, lucifuge cultivates 60 min, adopt the fluorescent signal of flow cytomery cell immediately, and statistical study is carried out to the fluorescence intensity of the cell mass collected (such as can 10000 count a group), when fluorescence intensity higher than 10 specific cells number account for more than 20% of total cellular score in cell mass time, namely think and have target tumor viable cell to be detected by the described type activation type aptamer diagnosis and treatment probe that splits in damping fluid to be measured.Further, the hybridising region that long-chain DNA and short chain DNA are formed, length is preferably about 10 pairs of nucleotide sequences, wherein GC base pair content be preferably 7 right.
In above-mentioned application, preferably, to the detection method of serum tumor viable cell, be specially: the type activation type aptamer diagnosis and treatment probe that splits described in adding in cell serum sample to be measured (containing serum 25%) (concentration of the type that splits activation type aptamer diagnosis and treatment probe is preferably 5 ~ 100 nM), after under room temperature, lucifuge cultivates 60 min, adopt the fluorescent signal of flow cytomery cell immediately, and statistical study is carried out to the fluorescence intensity of the cell mass collected (such as can 10000 count a group), when fluorescence intensity higher than 10 specific cells number account for more than 20% of total cellular score in cell mass time, namely think and have target tumor viable cell to be detected by the described type activation type aptamer diagnosis and treatment probe that splits in test serum.Further, the hybridising region that long-chain DNA and short chain DNA are formed, length is preferably about 10 pairs of nucleotide sequences, wherein GC base pair content be preferably 7 right.
In above-mentioned application, preferably, to the real-time fluorescence imaging of Live Animals in-vivo tumour and the concrete detecting step of In vivo detection be:
To split type activation type aptamer diagnosis and treatment probe and more than 10 times of moving object internal jugular vein to be measured injection 0.3 ~ 0.5 nmol in the random sequence oligonucleotides of type activation type aptamer diagnosis and treatment concentration and probe concentration of splitting, then the fluorescence intensity change at each position in living body fluorescent image system real time record animal body is adopted immediately, when the fluorescent signal observing a certain tissue site (certainly should be got rid of the enhancing of this fluorescent signal apparently higher than during other tissue sites and not be gathered in liver by the type activation type aptamer diagnosis and treatment probe that splits because of passive target, heart, kidney, under the prerequisite that the metabolism related tissue such as bladder cause, this is known for those skilled in the art), namely think and have target tumor to be detected by the described type activation type aptamer diagnosis and treatment probe that splits in this animal body, and tumor locus is positioned at fluorescent signal obviously enhancing place.Further, the hybridising region that long-chain DNA and short chain DNA are formed, length is preferably 12 pairs of nucleotide sequences, wherein GC base pair content be preferably 8 right.
As a total technical conceive, present invention also offers a kind of above-mentioned application of type activation type aptamer diagnosis and treatment probe in preparation tumor of splitting, comprise the following steps:
S1, will without drug buffer, drug containing damping fluid, split containing not medicine carrying the damping fluid of type activation type aptamer diagnosis and treatment probe, and be added to respectively in four cell samples (cell sample is tumor cells specimens) containing the split damping fluid of type activation type aptamer diagnosis and treatment probe of medicine carrying, be placed in cell culture incubator and cultivate 2 h.Preferably, without drug buffer, drug containing damping fluid, the damping fluid of the type activation type aptamer diagnosis and treatment probe that splits containing not medicine carrying, and be 1: 1: 1: 1 containing the split volume ratio of damping fluid of type activation type aptamer diagnosis and treatment probe of medicine carrying.Cell culture incubator conditional, temperature is preferably 37 DEG C, CO
2volumetric concentration is preferably 5%.Preferably, drug containing damping fluid is the damping fluid containing Dox, and the concentration of Dox is 2 μMs; In the type activation type aptamer diagnosis and treatment probe that splits of medicine carrying, the medicine of load is the concentration of Dox, Dox is 2 μMs.
S2, remove 80% cell culture fluid, supplement corresponding fresh cell medium, continue cultivation 48 h in cell culture incubator; Cell culture incubator conditional, temperature is preferably 37 DEG C, CO
2volumetric concentration is preferably 5%.
S3, by through S2 step cultivate after cell carry out activation analysis, obtain respectively with the addition of in the cell sample without drug buffer the absorption value at 490 nm places, with the addition of drug containing molecule damping fluid cell sample in absorption value at 490 nm places, with the addition of containing the type activation type aptamer diagnosis and treatment probe damping fluid that splits of medicine carrying cell sample in absorption value at 490 nm places, with the addition of the absorption value at 490 nm places in the cell sample without the type activation type aptamer diagnosis and treatment probe damping fluid that splits of medicine carrying; When the cell sample of the type activation type aptamer diagnosis and treatment probe process of splitting of medicine carrying is starkly lower than in the absorption at 490 nm places the cell sample not adding drug treating, and with drug treating cell sample the absorption at 490 nm places differ ± 20% time, think that the type activation type aptamer diagnosis and treatment probe that splits of medicine carrying has lethal effect to cell.This cell is preferably tumour cell.
Above-mentioned application, preferably, in S3 step, activation analysis step is carried out according to CellTiter 96 Cell Proliferation Assay specification sheets.
In above-mentioned application method, if the catenation sequence of the type activation type aptamer diagnosis and treatment probe that splits is the nucleotide sequence being rich in GC, can be used for loading anticancer chemotherapeutic agent Zorubicin (doxorubicin, Dox) and realize chemotherapy.In above-mentioned application method, if split, the catenation sequence of type activation type aptamer diagnosis and treatment probe is poly A sequence, with complementary short-chain S(poly A sequence) double stranded region that formed of mispairing for loading anticancer chemotherapeutic agent coralyne (coralyne), realize chemotherapy.In above-mentioned application method, if the catenation sequence of the type activation type aptamer diagnosis and treatment probe that splits is the siRNA sequence relevant to cancer cells, the type activation type aptamer diagnosis and treatment probe that splits of formation may be used for the gene therapy to tumour cell.
Innovative point of the present invention is: probe of the present invention is used in the method for tumour cell detection, when without target tumour cell, the catenation sequence be modified with in the long-chain DNA of the short chain DNA of fluorescence quenching unit and the fluorescence generating unit of modification is hybridized, form duplex structure that is stable, rigidity, now because of near there is FRET in fluorescence generating unit and fluorescence quenching unit, causes fluorescent signal faint; When after introducing target tumour cell, because Apt-a, Apt-b part of target cell induction long-chain DNA is close, this inductive capacity is better than the ability of short chain DNA in conjunction with long-chain DNA, and therefore duplex structure is destroyed, and short chain DNA then separates from long-chain DNA.Now, fluorescence generating unit and fluorescence quenching unit away from, fluorescent signal recovers.
Compared with prior art, the invention has the advantages that:
(1) the present invention takes full advantage of based on type nucleic acid aptamer probe of splitting advantage flexible and changeable in design, construct dexterously and possess high stable, highly sensitive signal is changed the mechanism characteristic, the type activation type aptamer diagnosis and treatment probe that splits of set oncotherapy function and tumor imaging function, for the diagnostic and therapeutic system design based on nucleic acid provides new thinking, and establish the tumour cell test-and-treat technology simultaneously based on the type activation type aptamer diagnosis and treatment probe that splits.
(2) based in SATP Cell Measurement Technique, the present invention utilizes the type aptamer fragment of splitting of splitting in type activation type aptamer diagnosis and treatment probe as Signal analysis element, fluorophor mark double-stranded region as signal generator component, by generation that is highly sensitive for the identification of aptamer to target tumour cell, that be converted to fluorescent signal with high specificity.Avoid existing based in the detection method of single fluorescence or radio-labeling aptamer, for overcoming the loaded down with trivial details washing process that non-specific adsorption signal carries out, shortening detection time, having expanded detection system applicatory.
(3) the present invention is that the application of aptamer in lesion detection, imaging and Therapy study provides brand-new means and thinking.This splits, and the detection analysis operation of type activation type aptamer diagnosis and treatment probe to tumor living cell and live tumor tissue is simple, quick, sensitive, special, cost is low, there are important scientific value and wide market outlook, have huge Social benefit and economic benefit.
Accompanying drawing explanation
For making the object of the embodiment of the present invention, technical scheme and advantage clearly, below in conjunction with the accompanying drawing in the embodiment of the present invention, clear, complete description is carried out to the technical scheme in the embodiment of the present invention.
Fig. 1 be split in the embodiment of the present invention 1 type activation type aptamer diagnosis and treatment probe SATP be combined with target tumour cell before and after structural representation (wherein solid line represents double-strand forming region, dotted line represents that split Segment A pt-a, dotted line of the second aptamer represents that the first aptamer splits Segment A pt-b).
Fig. 2 splits type activation type aptamer diagnosis and treatment probe SATP to the Cleaning Principle figure of tumour cell in the embodiment of the present invention 1.
Fig. 3 is based on the Cleaning Principle figure of single fluorescently-labeled nucleic acid aptamer probe to tumour cell in conventional art.
Fig. 4 is the type activation type aptamer diagnosis and treatment probe SATP strategy feasibility fluorescence spectrum phenogram in damping fluid that splits in the embodiment of the present invention 3.
Fig. 5 is split in the embodiment of the present invention 4 type activation type aptamer diagnosis and treatment probe SATP-10 and the fluorescence spectrum phenogram of type activation type contrast probe SCTP-10 in physiological buffer that split.
Fig. 6 splits type activation type aptamer diagnosis and treatment probe SATP-10 and the type activation type contrast probe SCTP-10 that splits to the detected result comparison diagram of different tumour cell damping fluid in the embodiment of the present invention 4.
Fig. 7 be in the embodiment of the present invention 5 different types of probe to the detection sensitivity comparative result figure of CCRF-CEM tumour cell in damping fluid.
Fig. 8 splits type activation type aptamer diagnosis and treatment probe SATP-10 to the specific detection result figure of tumour cell different in serum in the embodiment of the present invention 6.
Fig. 9 is the fluorescence investigation figure of type activation type aptamer diagnosis and treatment probe SATP-10 Drug loading capacity of splitting in the embodiment of the present invention 7.
Figure 10 kills and wounds specificity comparison diagram without medicine carrying type activation type aptamer diagnosis and treatment probe SATP-10, the medicine carrying type activation type aptamer diagnosis and treatment probe SATP-Dox and free Dox that splits that splits to different tumour cell in the embodiment of the present invention 8.
Figure 11 is the fluorescent stability investigation figure of type activation type aptamer diagnosis and treatment probe SATP-12 in serum that split in the embodiment of the present invention 9.
Figure 12 is the tumour In vivo detection Comparative result figure of type activation type aptamer diagnosis and treatment probe SATP-12 and the single fluorescence labeling probe Cy5-L-12 of tradition in tumor bearing nude mice body that split in the embodiment of the present invention 10, and the position wherein indicated by circle is the position at CCRF-CEM tumour place.
Figure 13 is the nude mice imaging results comparison diagram of type activation type aptamer diagnosis and treatment probe SATP-12 to not lotus knurl, lotus CCRF-CEM tumour, lotus Ramos tumour that split in the embodiment of the present invention 11, and the type activation type contrast probe SCTP-12 that splits is to the nude mice imaging results comparison diagram of CCRF-CEM tumour.
Embodiment
Below in conjunction with Figure of description and concrete preferred embodiment, the invention will be further described, but protection domain not thereby limiting the invention.The material adopted in following examples and instrument are commercially available.
embodiment 1:
With reference to Fig. 1: one is split type activation type aptamer diagnosis and treatment probe (SATP-10), comprises DNA long-chain 1, DNA short chain 2, medicine 3, fluorescence quenching unit 4, fluorescence generating unit 5.Wherein DNA long-chain 1 to be connected by catenation sequence 13 to be obtained by the first aptamer fragment (Apt-b) 11 and the second aptamer fragment (Apt-a) 12 of splitting of splitting, and DNA short chain 2 can mediate time base mispairing by base pair complementarity or drug molecule with catenation sequence 13 and form duplex structure.3 ' end of DNA short chain 2 is modified with fluorescence quenching unit 4, corresponds, be modified with fluorescence generating unit 5 in the split junction of fragment (Apt-b) 11 of catenation sequence 13 and the first aptamer.
In the present embodiment, fluorescence generating unit is Cy5; Fluorescence quenching unit is BHQ3.
First aptamer fragment 11 of splitting has nucleotide sequence described in SEQ ID NO.3, is specially:
5’-TAC TGT ACG GTT AGA T-3’（Apt-b）。
Second aptamer fragment 12 of splitting has nucleotide sequence described in SEQ ID NO.4, is specially:
5’-ATC TAA CTG CTG CGC CGC CGG GAA AA-3’（Apt-a）。
Junction fragment 13 is DNA of one section of 10 length of nucleotides, has the nucleotide sequence described in SEQ ID NO.5, is specially:
5’-CTG CCT GCT C-3’（Linker-10）。
When the first aptamer split fragment 11 and the second aptamer split fragment 12 be connected to the two ends of junction fragment 13 time, form DNA long-chain 1, this DNA long-chain 1 has the nucleotide sequence described in SEQ ID NO.1, is specially:
5’-TAC TGT ACG GTT AGA TCT GCC TGC TCA TCT AAC TGC TGC GCC GCC GGG AAA A-3’（L-10）。
Modify in the split junction of fragment (Apt-b) 11 of the catenation sequence of DNA long-chain 1 and the first aptamer the DNA long-chain that Cy5 obtains being modified with fluorescence generating unit, concrete nucleotides sequence is classified as:
5’-TAC TGT ACG GTT AGA （Cy5）TCT GCC TGC TCA TCT AAC TGC TGC GCC GCC GGG AAA A-3’（ Cy5-L-10）。
DNA short chain 2 can with junction fragment 13 complementary pairing in DNA long-chain 1, and DNA short chain 2 has the nucleotide sequence described in SEQ ID NO.2, is specially:
5’- GAG CAG GCA G -3’（S-10）。
Obtain at the 3 ' terminal modified BHQ3 of DNA short chain 2 the DNA short chain being modified with fluorescence quenching unit, concrete nucleotides sequence is classified as: 5 '-GAG CAG GCA G-BHQ3-3 ' (BHQ3-S-10).
When there is not tumour target cell in reaction system, DNA short chain 2 is hybridized with the catenation sequence 13 of DNA long-chain 1, form duplex structure that is stable, rigidity, make the first aptamer split fragment 11 and the second aptamer split fragment 12 cannot near being formed in conjunction with conformation.The fluorescent signal now modifying the Cy5 near-infrared fluorescent group in the middle of DNA long-chain 1, by the BHQ3 fluorescence quenching group absorptions modified in short chain one end, makes fluorescent signal be in "Off" state.Meanwhile, drug molecule is still loaded between double-strand, cannot discharge to play treatment function.
When there is tumour target cell in reactive system, inducibility due to tumour target cell is better than the binding ability of DNA short chain 2 and DNA long-chain 1, DNA short chain 2 is separated with DNA long-chain 1, duplex structure destroys, and the first aptamer splits, fragment 11 and the second aptamer fragment 12 of splitting is close, be combined with each other formation hairpin structure, thus produce the recovery of obvious fluorescence, state that fluorescent signal is in " opening ".Meanwhile, drug molecule part discharges between double-strand, and then enters cells play treatment function.Therefore the type activation type aptamer diagnosis and treatment probe that splits of the present invention can realize specific detection to target and treatment simultaneously.
Simultaneously with reference to Fig. 2: because CCRF-CEM tumour cell can specific induction Apt-a, Apt-b be formed in conjunction with conformation, and its inductive capacity is greater than the hybridization power of double-strand, therefore do not have fluorescent signal when CCRF-CEM tumour cell to be in "Off" state all the time, therefore detection background is lower.So just achieve special, the high-sensitive real-time analysis diagnosis of CCRF-CEM tumour cell height.
Fig. 3 is based on the Cleaning Principle figure of single fluorescently-labeled aptamer probe to tumour cell in conventional art.As shown in Figure 3, no matter whether there is target tumor in system to be detected, fluorescent signal based on single fluorescently-labeled aptamer probe all can be detected, target tumor is analyzed and also needs to carry out loaded down with trivial details washing process, thus be difficult to realize detecting in real time, efficiently, easily target tumor.
embodiment 2
With reference to Fig. 1: one is split type activation type aptamer diagnosis and treatment probe (SATP-12), comprises DNA long-chain 1, DNA short chain 2, medicine 3, fluorescence quenching unit 4, fluorescence generating unit 5.Wherein DNA long-chain 1 to be connected by catenation sequence 13 to be obtained by the first aptamer fragment (Apt-b) 11 and the second aptamer fragment (Apt-a) 12 of splitting of splitting, and DNA short chain 2 mediate time base mispairing with catenation sequence 13 by base pair complementarity or drug molecule and forms duplex structure.3 ' end of DNA short chain 2 is modified with fluorescence quenching unit 4, corresponds, be modified with fluorescence generating unit 5 in the split junction of fragment (Apt-b) 11 of catenation sequence 13 and the first aptamer.
In the present embodiment, fluorescence generating unit is Cy5; Fluorescence quenching unit is BHQ3.
First aptamer fragment 11 of splitting has nucleotide sequence described in SEQ ID NO.3, is specially:
5’-TAC TGT ACG GTT AGA T-3’（Apt-b）。
Second aptamer fragment 12 of splitting has nucleotide sequence described in SEQ ID NO.4, is specially:
5’-ATC TAA CTG CTG CGC CGC CGG GAA AA-3’（Apt-a）。
Junction fragment 13 is one section of DNA containing 12 nucleotide sequences, has the nucleotide sequence that SEQ ID NO.6 states, is specially:
5’-CTG CCT GCG TTC-3’（Linker-12）。
When the first aptamer split fragment 11 and the second aptamer split fragment 12 be connected to the two ends of junction fragment 13 time, form DNA long-chain 1, this DNA long-chain 1 has the nucleotide sequence described in SEQ ID NO.2, is specially:
5’-TAC TGT ACG GTT AGA TCT GCC TGC GTT CAT CTA ACT GCT GCG CCG CCG GGA AAA-3’（L-12）。
Modify in the split junction of fragment (Apt-b) 11 of the catenation sequence of DNA long-chain 1 and the first aptamer the DNA long-chain that Cy5 (being positioned in T base) obtains being modified with fluorescence generating unit, concrete nucleotides sequence is classified as:
5’-TAC TGT ACG GTT AGA （Cy5）TCT GCC TGC GTT CAT CTA ACT GCT GCG CCG CCG GGA AAA-3’（Cy5-L-12）。
DNA short chain 2 can with junction fragment 13 complementary pairing in DNA long-chain 1, and DNA short chain 2 has the nucleotide sequence described in SEQ ID NO.2, is specially:
5’- GAA CGC AGG CAG -3’（S-12）。
Obtain at the 3 ' terminal modified BHQ3 of DNA short chain 2 the DNA short chain being modified with fluorescence quenching unit, concrete nucleotides sequence is classified as: 5 '-GAA CGC AGG CAG-BHQ3-3 ' (BHQ3-S-12).
Embodiment 1 and embodiment 2 are only the preferred embodiments of the present invention, and in the present invention, the catenation sequence 13 as long-chain DNA1 can also be rich in the sequence of GC base, poly A sequence or the siRNA sequence relevant to cancer cells; The selected type aptamer that splits also can be the specific nucleic acid aptamers of other tumour cells; Being modified at the split fluorescence generating unit 5 of junction of fragment (Apt-b) 11 of the catenation sequence of DNA long-chain and the first aptamer can be luminescent dye molecule, as fluorescein, tetramethylrhodamine or Cy5; Or fluorescent nano particle, as wrapped up nano SiO 2 particle or the fluorescence quantum of fluorescence dye.Be modified at DNA short chain 23 ' the fluorescence quenching unit 4 held can be fluorescence quenching group, as DABCYL, BHQ1, BHQ2 or BHQ3; Or for having the functionalized nano material of fluorescence quenching effect, gold nano grain, manganese dioxide nano particle, stannic oxide/graphene nano lamella or carbon nanotube; All can implement, and reach same or analogous technique effect.
embodiment 3
Investigate the fluorescence spectrum of type activation type aptamer diagnosis and treatment probe SATP in damping fluid that split to characterize, the probe adopted in this example is the SATP-10 of embodiment 1.
1, sample is prepared: (composition of binding buffer liquid is Dulbecco ' s PBS, 4.5g/L glucose, 5 mM MgCl with binding buffer liquid
2) configure 5 testing samples respectively:
The Cy5-L-10 of sample 1:50 nM.
The mixture of sample 2:50 nM Cy5-L-10 and 50 nM BHQ3-S-10.
The mixture of sample 3:50 nM Cy5-L-10 and 100 nM BHQ3-S-10.
Sample 4:50 nM Cy5-L-10,50 nM BHQ3-S-10,50 nM compete the mixture of chain cDNA.
Sample 5(buffer): binding buffer liquid.
2, hybridize: respectively above-mentioned 5 samples are placed in 95 DEG C of water-bath 4 min, then are placed in 5 min on ice, be placed on room temperature 40 min, long-chain Cy5-L-10 and short chain BHQ3-S-10 is fully hybridized, and the whole process of all samples takes lucifuge process.
The nucleotide sequence of above-mentioned DNA chain is as follows respectively:
Cy5-L-10：5’-TAC TGT ACG GTT AGA （Cy5）TCT GCC TGC TCA TCT AAC TGC TGC GCC GCC GGG AAA A-3’。
BHQ3-S-10：5’- GAG CAG GCA G -BHQ3-3’。
cDNA：5’-CAG TTA GAT GAG CAG GCA G-3’。
3, fluoroscopic examination: get above-mentioned five the sample spectrophotofluorometers of 200 μ L (Hitachi Japan F-7000 type) respectively and record fluorescence spectrum, wherein excitation wavelength is 620 nm, and emission wavelength is 640 ~ 740 nm.The fluorescence spectrum of record is with reference to Fig. 4.As seen from Figure 4, adopt load to have the short chain DNA(BHQ3-S-10 of fluorescence quenching unit 4) and load have the long-chain DNA(Cy5-L-10 of fluorescence generating unit 5) ratio when being 2: 1, background is zero substantially; In the blank physiological buffer not having target compound, the more single fluorescently-labeled aptamer probe Cy5-L-10 of the type that splits activation type aptamer diagnosis and treatment probe SATP-10 declines to a great extent, and has lower detection background.
embodiment 4
Investigate and split type activation type aptamer diagnosis and treatment probe SATP to the specificity of tumour cell in damping fluid, the type activation type aptamer diagnosis and treatment probe that splits adopted in this example is the SATP-10 of embodiment 1.Concrete investigation method is:
(1) the type activation type aptamer diagnosis and treatment probe that splits is prepared:
The preparation of SATP-10: be dispersed in binding buffer liquid at 2: 1 according to mol ratio by BHQ3-S-10 and Cy5-L-10, at 95 DEG C of water-bath 4 min, be placed in 5 min on ice again, be placed on room temperature 40 min fully to hybridize, obtain the type activation type aptamer diagnosis and treatment probe SATP-10 that splits.
The preparation of SCTP-10: be dispersed in buffer damping fluid at 2: 1 according to mol ratio by BHQ3-S-10 and Cy5-LC-10, at 95 DEG C of water-bath 4 min, be placed in 5 min on ice again, be placed on room temperature 40 min fully to hybridize, obtain splitting type activation type contrast probe.
The nucleotide sequence of above-mentioned DNA chain is as follows respectively:
Cy5-L-10：5’-TAC TGT ACG GTT AGA（Cy5）TCT GCC TGC TCA TCT AAC TGC TGC GCC GCC GGG AAA A-3’。
Cy5-LC-10：5’- NNN NNN NCG GTT AGA（Cy5）TCT GCC TGC TCA TCT AAC TGN NNN NNN NNN NNN NNN N-3’。
BHQ3-S-10：5’- GAG CAG GCA G –BHQ3-3’。
First get the above-mentioned ready type activation type aptamer diagnosis and treatment probe SATP-10 that splits of 200 μ L 50nM, the type that splits activation type contrast probe SCTP-10 spectrophotofluorometer (Hitachi Japan F-7000 type) records fluorescence spectrum, wherein excitation wavelength is 620 nm, emission wavelength is 640 ~ 740 nm, and fluorescence spectrum figure is see Fig. 5.As seen from Figure 5, in the blank physiological buffer not having target compound, comparatively SATP-10 background is high for SCTP-10, may be that the long-chain Cy5-LC-10 two ends sequence pair formation double-stranded region in SCTP-10 has certain inhibition.
(2) tumour cell damping fluid is prepared:
Trial-product 1: disperse 1.5 × 10 in the binding buffer liquid of 100 μ L
5individual CCRF-CEM tumour cell obtains tumour cell damping fluid sample, adds the SATP-10 of the above-mentioned preparation of 100 μ L 50 nM, mix in forgoing neoplasms Cell Buffer sample.
Reference substance 1: disperse 1.5 × 10 in the binding buffer liquid of 100 μ L
5individual CCRF-CEM tumour cell obtains tumour cell damping fluid sample, adds the SCTP-10 of the above-mentioned preparation of 100 μ L 50 nM, mix in forgoing neoplasms Cell Buffer sample.
Reference substance 2: according to the negative control damping fluid of preceding method preparation containing Ramos cell.
Reference substance 3: according to the negative control damping fluid of preceding method preparation containing human liver cancer cell (being called for short SMMC-7721 cell).
Reference substance 4: according to the negative control cell damping fluid of preceding method preparation containing Human normal hepatocyte (being called for short L02 cell).
(3) fluorescent signal is detected: SCTP-10, SATP-10 are hatched 60 min with trial-product 1, reference substance 1, reference substance 2, reference substance 3, reference substance 4 lucifuge under normal temperature respectively, FACSCalibur flow cytometer (Becton-Dickinson, the U.S.) is used to detect the fluorescent signal of cell at FL4 passage immediately.Detected result as shown in Figure 6.As seen from Figure 6, SATP-10 and SCTP-10 all can not produce stronger fluorescent signal to negative control cells such as Ramos cell, SMMC-7721 cell, L02 cells, and wherein the fluorescence of SCTP-10 is by force because the background fluorescence of itself is strong compared with SATP-10.But stronger fluorescence can be there is and recovers in the type activation type aptamer diagnosis and treatment probe SATP-10 that splits of the present invention after being combined with CCRF-CEM tumour cell, and the type activation type contrast probe SCTP-10 that splits still can not produce stronger fluorescent signal, the high degree of specificity that this type activation type aptamer diagnosis and treatment probe that splits demonstrating the present embodiment detects different tumour cell.
embodiment 5
Investigate and split type activation type aptamer diagnosis and treatment probe to the detection sensitivity of tumour cell in damping fluid, the probe adopted in this example is the SATP-10 of embodiment 1.
With binding buffer liquid, (composition of binding buffer liquid is Dulbecco ' s PBS, 4.5g/L glucose, 5 mM MgCl
2) configure 4 testing samples respectively:
Testing sample 1:50 nM Cy5-L-10+100 nM BHQ3-S-10(abbreviate SAT P-10).
Testing sample 2:50 nM Cy5-L-10+100 nm S-10(is called for short Cy5-SATP-10).
Testing sample 3:50 nM Cy5-L-10.
The original aptamer Sgc8c(that testing sample 4:50 nM Cy5 marks is called for short Cy5-Sgc8c).
Experimental group: it is 1.5 × 10 that above-mentioned for 100 μ L 4 testing samples are added concentration respectively
5in the CCRF-CEM positive tumor cell damping fluid of individual/100 μ L, after mixing, under normal temperature, lucifuge hatches 60 min, uses FACSCalibur flow cytometer (Becton-Dickinson, the U.S.) to detect the fluorescent signal of cell immediately.
Control group: it is 1.5 × 10 that above-mentioned for 100 μ L 4 testing samples are added concentration respectively
5in the damping fluid of the Ramos negative cells of individual/100 μ L, after mixing, under normal temperature, lucifuge hatches 60 min, uses FACSCalibur flow cytometer (Becton-Dickinson, the U.S.) to detect the fluorescent signal of cell immediately.According to detecting that the fluorescent signal of FL4 passage calculates the signal-to-background ratio of each probe, formula is: (F
(CCRF-CEM+ probe)-F
cCRF-CEM)/ (F
(Ramos+ probe)-F
ramos).Calculation result as shown in Figure 7, as seen from Figure 7: 1. probe Cy5-L-10 is substantially the same relative to the signal-to-background ratio of Cy5-Sgc8c, illustrate after original aptamer Sgc8c is transformed, improved Cy5-L-10 is compared to original aptamer, and recognition capability does not change substantially; 2. probe Cy5-SATP-10 is substantially the same relative to the signal-to-background ratio of Cy5-Sgc8c, and what complementary short-chain was described adds the recognition capability that can not affect probe Cy5-SATP-10 and probe SATP-10; 3. the signal-to-background ratio of probe SATP-10 is far away higher than probe Cy5-SATP-10, Cy5-L-10, Cy5-Sgc8c, illustrates that the type activation type aptamer diagnosis and treatment probe that splits possesses the ability detecting low concentration target tumor.
embodiment 6:
Investigate and split type activation type aptamer diagnosis and treatment probe to the specificity of serum tumor cell, the probe adopted in this example type activation type aptamer diagnosis and treatment probe that splits is the SATP-10 of embodiment 1.The preparation of SATP-10 is with reference to embodiment 4.
Be 1.5 × 10 in concentration
5mice serum, the concentration of the CCRF-CEM positive tumor cell of individual/100 μ L are 1.5 × 10
5the Ramos negative tumor cells of individual/100 μ L mice serum in, add the probe SATP-10 that 100 μ L concentration are the above-mentioned preparation of 100 nM respectively, 60 min are hatched in room temperature lucifuge after mixing, use FACSCalibur flow cytometer (Becton-Dickinson immediately, the U.S.) detect the fluorescent signal of cell, detected result is as shown in Figure 8.As seen from Figure 8, even in this COMPLEX MIXED system of serum, the type activation type aptamer diagnosis and treatment probe that splits of the present embodiment still can realize detecting the high specific of target CCRF-CEM tumour cell.
embodiment 7:
The investigation of the type that splits activation type aptamer diagnosis and treatment probe Drug loading capacity, the probe adopted in this example type activation type aptamer diagnosis and treatment probe that splits is the SATP-10 of embodiment 1.The preparation of SATP-10 is with reference to embodiment 4.
Be add different ratios (SATP-10/Dox in anticancer drugs, doxorubicin (Dox) sample of 2 μMs toward 10 200 μ L concentration respectively, mol ratio) above-mentioned SATP-10, be respectively: 0,0.1,0.2,0.3,0.4,0.6,0.8,1.0,1.3,1.6.After mixing, ambient temperatare puts 30 min, and recording Dox(excitation wavelength with spectrophotofluorometer (Hitachi Japan F-7000 type) is 480 nm, and emission wavelength is 500 ~ 700 nm) fluorescence spectrum, result is as shown in Figure 9.Because Dox embeds after in the GC base pair of double-strand, autofluorescence can be extinguished, therefore the fluorescence demonstrated in Fig. 9 along with the increase Dox of probe SATP-10 concentration weakens gradually; When the concentration of probe SATP-10 is increased to a certain degree, free Dox amount seldom, therefore continues to increase concentration and probe concentration, and the fluorescent weakening trend of DOX slows down gradually until constant.Now illustrate that Dox substantially all embeds in probe.Here being called for short medicine carrying probe is SATP-Dox.
embodiment 8:
Application during the type that splits activation type aptamer diagnosis and treatment probe kills and wounds tumor cell in vitro, the type activation type aptamer diagnosis and treatment probe that splits adopted in this example is the SATP-10 of embodiment 1.
Preparation SATP-10: prepare the SATP-10 probe that concentration is 3.6 μMs according to the treatment process in embodiment 4.
Preparation SATP-Dox: prepare according to the processing mode in embodiment 7 the medicine carrying probe SATP-Dox that concentration is 3.6 μMs respectively, wherein Dox concentration is 2 μMs.
With 96 orifice plates for carrier, in hole, add 100 μ L be respectively dispersed with 5 × 10
4the serum-free RPMI 1640 of individual CCRF-CEM positive tumor cell trains base.The hole being added with CCRF-CEM positive tumor cell is divided into four groups, often organizes 3 holes.Respectively toward often organize in hole add above-mentioned 3.6 μMs of preparing of 50 μ L without medicine carrying probe SATP-10, the medicine carrying probe SATP-Dox of 3.6 μMs, free Dox, buffer of 2 μMs.After mixing, be positioned over 37 DEG C, 5% (volume ratio) CO
2cell culture incubator in cultivate 2 h.Remove the supernatant of 80% afterwards, then add equivalent containing 15%(volume ratio) RPMI 1640 of foetal calf serum trains base and continues cultivation 48 h.Then in each hole, add 20 μ L CellTiter 96 Cell Proliferation Assay(U.S. Promega) continue cultivation 2 h, the absorption at 490nm place is detected afterwards by microplate reader.The survival rate of each porocyte in 100% calculating different treatment situation is set to the mean value of the CCRF-CEM cell hole light absorption value not dealing with (namely only having added buffer).Meanwhile, Ramos cell is processed simultaneously.Without medicine carrying probe SATP-10, medicine carrying probe SATP-Dox and free Dox specificity comparative result is killed and wounded as shown in Figure 10 to different tumour cell.As seen from Figure 10: 1. without the CCRF-CEM cell of medicine carrying probe SATP-10 process or the survival rate of Ramos cell all up to more than 90%, illustrating does not have lethal effect without medicine carrying probe SATP-10 to cell; 2. the CCRF-CEM cell of free Dox process or the survival rate of Ramos cell all only have about 30%, illustrate that free Dox has lethal effect to cell, and kill and wound and do not have selectivity; 3. the survival rate of the CCRF-CEM cell of medicine carrying probe SATP-Dox process only has about 30%, and the survival rate of Ramos cell is up to 90%, illustrates that the lethal effect of medicine carrying probe SATP-Dox has high selectivity.
embodiment 9:
The fluorescent stability of the type that splits activation type aptamer diagnosis and treatment probe in serum is investigated, and the type activation type aptamer diagnosis and treatment probe that splits adopted in this example is the SATP-12 in embodiment 2.
The preparation of SATP-12: be dispersed in binding buffer liquid at 2: 1 according to mol ratio by BHQ3-S-12 and Cy5-L-12, at 95 DEG C of water-bath 4 min, be placed in 5 min on ice again, be placed on room temperature 40 min fully to hybridize, obtain the type activation type aptamer diagnosis and treatment probe SATP-12 that splits.
The unmarked random probe of 250 nM probe SATP-12 and 625nM is added, incubation at 37 DEG C in 160 μ L mice serums.
By the fluorescence intensity of spectrophotofluorometer (Hitachi Japan F-7000 type) monitoring experiment group, excitation wavelength 620 nm, emission wavelength 672 nm, monitoring 8500s(about 2.5 h, live body middle probe analytic metabolism when 2.5 h is complete).Adopt monitoring result as shown in figure 11.As seen from Figure 11, along with the prolongation of the time of cultivation, the fluorescence of probe SATP-12 rises gradually, this may be the nuclease in serum to caused by the degraded of probe, also may be because the combination of some nonspecific proteins and probe makes its configuration change, thus make fluorescence obtain recovery to a certain extent.In addition, at 37 DEG C, the transformation period of the present embodiment probe SATP in mice serum is approximately 28 minutes, and this stability is enough to the needs meeting inside and outside tumour cell detection.
embodiment 10:
The application of the type that splits activation type aptamer diagnosis and treatment probe in vivo tumor detects, the type activation type aptamer diagnosis and treatment probe that splits adopted in this example is the SATP-12 in embodiment 2, and SATP-12 adopts the method for embodiment 9 to prepare.
Experimental group: first 200 μ L are about contained 1 × 10
7the subcutaneous injection of individual eugonic CCRF-CEM cell suspension enters the right fore back of BALB/c male nude mouse in 3 ~ 4 week age, grows and obviously can become knurl after 3 ~ 4 weeks.Choose the tumor bearing nude mice with suitable size tumour subsequently, be not random nucleic acid fragment and the 0.35 nmole SATP-12 of any mark with tail vein injection about 300 μ L containing 4.5 nmole.
Control group: the single fluorescently-labeled aptamer probe Cy5-L-12 preparing 0.35 nmole according to the method for embodiment 3, injected in tumor bearing nude mice body with tail vein by Cy5-L-12, all the other operations are identical with experimental group.
Adopt Maestro simultaneously
tMthe fluorescence intensity of whole body optical imaging system (CRI, the U.S.) Real-Time Monitoring experimental group, control group tumor locus.The detected result of the present embodiment as shown in figure 12, the type that splits activation type aptamer diagnosis and treatment probe SATP-12 can observe obvious fluorescent signal at tumor locus in about 5 minutes in injection CCRF-CEM tumor bearing nude mice body, and along with time lengthening, the fluorescent signal contrast gradient of this tumor locus and other non-target tissues increases gradually; By about 30 minutes, tumor locus still maintained higher fluorescent signal contrast gradient, effectively achieved the live body targeted imaging to target tumor and detection.By comparison compared with single fluorescently-labeled aptamer probe Cy5-L-12 in injection CCRF-CEM tumor bearing nude mice body after, fluorescent signal spreads all over mouse systemic fast, and reduces gradually along with time lengthening fluorescent signal; But cause because background is too high that the signal of target tumor locus and background signal contrast gradient are not high, detection sensitivity is poor, thus be unfavorable for the early diagnosis of tumour.
embodiment 11:
Investigate the tumour In vivo detection specificity of the type activation type aptamer diagnosis and treatment probe that splits, the type activation type aptamer diagnosis and treatment probe that splits adopted in this example is the SATP-12 in embodiment 2, and corresponding contrast probe is SCTP-12.
SATP-12 adopts the method for embodiment 9 to prepare.The preparation method of SCTP-12 is: be dispersed in binding buffer liquid at 2: 1 by BHQ3-S-12 and Cy5-LC-12 according to mol ratio, at 95 DEG C of water-bath 4 min, then is placed in 5 min on ice, be placed on room temperature 40 min fully to hybridize.The nucleotides sequence of Cy5-LC-12 is classified as: 5 '-NNN NNN NCG GTT AGA(Cy5) TCT GCC TGC GTT CAT CTA ACT GN NNN NNN NNN NNN NNN N-3 '.
First 200 μ L are about contained 1 × 10
7the subcutaneous injection of individual eugonic CCRF-CEM cell suspension enters the right fore back of BALB/c male nude mouse in 3 ~ 4 week age, grows and obviously can become knurl after 3 ~ 4 weeks.Same process obtains the nude mice of lotus Ramos tumour.Control group 1: get the not tumor bearing nude mice with suitable size, is not the random nucleic acid fragment of any mark and the SATP-12 of 0.35 nmole with tail vein injection about 300 μ L containing 4.5 nmole.Positive group: the nude mice of getting the lotus CCRF-CEM tumour with suitable size tumour, is not the random nucleic acid fragment of any mark and the SATP-12 of 0.35 nmole with tail vein injection about 300 μ L containing 4.5 nmole.Control group 2: the nude mice of getting the CCRF-CEM tumour with suitable size tumour, is not the random nucleic acid fragment of any mark and the SCTP-12 of 0.35 nmole with tail vein injection about 300 μ L containing 4.5 nmole.Control group 3: the nude mice of getting the Ramos tumour with suitable size tumour, is not the random nucleic acid fragment of any mark and the SATP-12 of 0.35 nmole with tail vein injection about 300 μ L containing 4.5 nmole.
Adopt Maestro simultaneously
tMwhole body optical imaging system (CRI, the U.S.) fluorescence intensity of Real-Time Monitoring tumor locus, monitoring result as shown in figure 13, as seen from Figure 13, the In vivo detection of type activation type aptamer diagnosis and treatment probe to tumour that split of the present embodiment possesses high degree of specificity, only can at CCRF-CEM tumor locus generation hyperfluorescenceZeng Yongminggaoyingguang signal.
<110> Hunan University
<120> splits type activation type aptamer diagnosis and treatment probe and application thereof
<130> without
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 52
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(52)
<223> experimentally requires and designs, the long-chain DNA(L-10 as the type activation type aptamer diagnosis and treatment probe that splits)
<400> 1
tactgtacgg ttagatctgc ctgctcatct aactgctgcg ccgccgggaa aa 52
<210> 2
<211> 54
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(54)
<223> experimentally requires and designs, the long-chain DNA(L-12 as the type activation type aptamer diagnosis and treatment probe that splits)
<400> 2
tactgtacgg ttagatctgc ctgcgttcat ctaactgctg cgccgccggg aaaa 54
<210> 3
<211> 16
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(16)
<223> experimentally requires and designs, using fragment (Apt-b) of splitting as first aptamer of long-chain DNA in the type activation type aptamer diagnosis and treatment probe that splits
<400> 3
tactgtacgg ttagat 16
<210> 4
<211> 26
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(26)
<223> experimentally requires design, to split fragment (Apt-a) as second aptamer of long-chain DNA in the type activation type aptamer diagnosis and treatment probe that splits
<400> 4
atctaactgc tgcgccgccg ggaaaa 26
<210> 5
<211> 10
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(10)
<223> experimentally requires and designs, using as the catenation sequence (Linker-10) in long-chain DNA
<400> 5
ctgcctgctc 10
<210> 6
<211> 12
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(12)
<223> experimentally requires and designs, as the catenation sequence (Linker-12) of long-chain DNA in the type activation type aptamer diagnosis and treatment probe that splits
<400> 6
ctgcctgcgt tc 12
<210> 7
<211> 10
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(10)
<223> experimentally requires and designs, as the type activation type aptamer short-and-medium chain DNA of diagnosis and treatment probe (S-10) that splits
<400> 7
gagcaggcag 10
<210> 8
<211> 12
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(12)
<223> experimentally requires and designs, as the type activation type aptamer short-and-medium chain DNA of diagnosis and treatment probe (S-12) that splits
<400> 8
gaacgcaggc ag 12
<210> 9
<211> 23
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (1)..(23)
<223> experimentally requires and designs, as the siRNA sequence relevant to cancer cells
<400> 9
uugauuaacg cccagcguud tdt 23
Claims (10)
1. the type that splits activation type aptamer diagnosis and treatment probe, it is characterized in that, the fluorescence quenching unit comprise long-chain DNA, the short chain DNA that can form duplex structure with described long-chain DNA partial sequence, modifying the fluorescence generating unit on described long-chain DNA and modify on described short chain DNA.
2. the type activation type aptamer diagnosis and treatment probe that splits according to claim 1, it is characterized in that, described long-chain DNA to be connected by catenation sequence to be obtained by the first aptamer fragment and the second aptamer fragment of splitting of splitting, and described short chain DNA and described catenation sequence form duplex structure by base pair complementarity.
3. the type activation type aptamer diagnosis and treatment probe that splits according to claim 2, it is characterized in that, the nucleotides sequence of described long-chain DNA is classified as the nucleotide sequence described in SEQ ID NO.1 or the nucleotide sequence described in SEQ ID NO.2; The split nucleotides sequence of fragment of described first aptamer is classified as nucleotide sequence described in SEQ ID NO.3, and the split nucleotides sequence of fragment of described second aptamer is classified as nucleotide sequence described in SEQ ID NO.4.
4. the type activation type aptamer diagnosis and treatment probe that splits according to claim 2, it is characterized in that, the nucleotides sequence of described catenation sequence is classified as the sequence, poly A sequence or the siRNA sequence relevant to cancer cells that are rich in GC base.
5. the type activation type aptamer diagnosis and treatment probe that splits according to claim 4, is characterized in that, described in be rich in the sequence of GC base for the nucleotide sequence described in SEQ ID NO.5 or the nucleotide sequence described in SEQ ID NO.6; The base number of described poly A sequence is 8 ~ 30; The nucleotides sequence of the described siRNA sequence relevant to cancer cells is classified as the nucleotide sequence described in SEQ ID NO.9.
6. the type activation type aptamer diagnosis and treatment probe that splits according to claim 1, it is characterized in that, described fluorescence generating unit is luminescent dye molecule or fluorescent nano particle, and described fluorescence quenching unit is fluorescence quenching group or the functionalized nano material for having fluorescence quenching effect.
7. the type activation type aptamer diagnosis and treatment probe that splits according to claim 6, it is characterized in that, described fluorescence dye is fluorescein, tetramethylrhodamine or Cy5; Described fluorescent nano particle is nano SiO 2 particle or the fluorescence quantum of parcel fluorescence dye; Described fluorescence quenching group is DABCYL, BHQ1, BHQ2 or BHQ3; The described functionalized nano material with fluorescence quenching effect is gold nano grain, manganese dioxide nano particle, stannic oxide/graphene nano lamella or carbon nanotube.
8. the type activation type aptamer diagnosis and treatment probe that splits according to any one of claim 1 to 7, it is characterized in that, the described type activation type aptamer diagnosis and treatment probe that splits also comprises drug molecule, and described drug molecule is loaded between the double-strand of described long-chain DNA and short chain DNA composition.
9. the type activation type aptamer diagnosis and treatment probe that splits according to claim 8, it is characterized in that, described drug molecule is Zorubicin, coralyne.
10. the application that the type activation type aptamer diagnosis and treatment probe that splits according to any one of a claim 1 to 9 detects at tumour cell and prepares in antitumor drug.
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|---|---|---|---|
| CN201510048971.8A CN104651501A (en) | 2015-01-30 | 2015-01-30 | Cracking activation type nucleic acid adaptor diagnosis and treatment probe and application thereof |
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| CN108273056A (en) * | 2018-02-01 | 2018-07-13 | 中国科学院长春应用化学研究所 | A kind of modified gold nano-material/nucleic acid probe nanometer system and preparation method thereof, application |
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| CN111789961B (en) * | 2020-08-26 | 2022-03-29 | 西南大学 | Nano probe for nucleolin cross-linking induction of tumor cell apoptosis and preparation method and application thereof |
| CN113930482A (en) * | 2021-10-26 | 2022-01-14 | 湖南大学 | Three-dimensional DNA walker and application thereof in tumor exosome detection |
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