CN104480114B - The method of high performance liquid chromatography screening aptamer and aptamer and application - Google Patents
The method of high performance liquid chromatography screening aptamer and aptamer and application Download PDFInfo
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Abstract
The invention discloses a kind of method of high performance liquid chromatography screening aptamer and the method for aptamer and application, wherein high performance liquid chromatography screening aptamer, comprise the following steps：Screening and the nucleic acid library of the aptamer of Docetaxel specific binding from random library RS36；Aptamer is obtained using high performance liquid chromatography screening nucleic acid library；In high performance liquid chromatography, stationary phase is octadecylsilane chemically bonded silica chromatographic column；Mobile phase uses water-acetonitrile gradient elution；Detection wavelength is 260nm；Detector is PDAD.The aptamer screened according to high performance liquid chromatography has the advantages that can be combined with Docetaxel high specific and high-affinity, non-immunogenicity, can chemical synthesis, good biocompatibility, molecular weight it is small, stably, be easy to preservation, can be applied to pharmaceutical carrier, medical separation and purifying, prepare Docetaxel detection probe or target spot probe etc..
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of side of high performance liquid chromatography screening aptamer
Method, further relate to the application of aptamer and aptamer that high performance liquid chromatography screens to obtain.
Background technology
Docetaxel (Docetaxel) belongs to taxanes antineoplastic, mainly act on the tubulin of cell from
And interference cell mitosis, antitumous effect is played, its adverse reaction is mainly shown as allergic reaction, decrease of platelet, the heart
It is dynamic to overrun, low blood pressure, neurotoxicity, gastrointestinal reaction and Skin toxicity etc..Taxotere alcohol-soluble and water-soluble is small, city
Medicine taxotere is sold using Tween-80 solubilising, ethanol hydrotropy, the burden of liver of metabolism can be increased and be possible to trigger allergy anti-
Should, the side effect such as hemolytic reaction.Therefore, taxotere side effect how is reduced, raising clinical efficacy turns into urgently to be resolved hurrily and asked
Topic.Further, since the architectural characteristic of Docetaxel, makes its molar absorption coefficient relatively low, it is difficult to uses simple low cost optical
Spectral method carries out highly sensitive quantitative microanalysis.
Aptamer (aptamer) is obtained by phyletic evolution technology (SELEX) screening of index concentration part,
The single strain oligonucleotide (ssDNA or ssRNA) of energy specific bond target substance.Aptamer is similar with antibody function, still
There are more advantages compared with antibody, such as have higher affinity with specificity, non-immunogenicity, can chemical synthesis,
Cost is low, can be marked, and stability is good, the advantages that being easy to preserve.The target molecule of aptamer is more extensive, including metal
Ion, amino acid, nucleic acid, polypeptide, protein etc., and it is compound complete virion and cell etc. to be extended to from single target
Thing target.
, it is necessary to which the nucleic acid library of preenrichment is sequenced in current conventional aptamer screening technique, then from
The nucleic acid aptamer sequence that affinity is high, selectivity is good is picked out in substantial amounts of sequencing result, this process is in the presence of time-consuming, effect
Rate is low, cost is high, is difficult to the problem of automation.The method of quick-pick Docetaxel aptamer, it will be expected to carry
A kind of method for selecting the aptamer of Docetaxel for high flux, low cost.And the method is also expected to expand in purple
The nucleic acid aptamer sequence of other hydrophobic small molecules medicines such as China fir alcohol, camptothecine is selected.
The content of the invention
The technical problem to be solved in the present invention is overcome the deficiencies in the prior art, there is provided a kind of energy high flux, low cost sieve
The method for selecting Docetaxel aptamer, further correspondingly provide the nucleic acid for screening to obtain using high performance liquid chromatography and be adapted to
Body, the aptamer can be combined with Docetaxel high specific and high-affinity, have non-immunogenicity, can chemistry
Synthesis, good biocompatibility, molecular weight be small, stably, the advantage such as be easy to preserve, can be applied to prepare Docetaxel detection and visit
Pin, Docetaxel target spot probe；As Docetaxel pharmaceutical carrier；Improve the solvability of Docetaxel；It is applied to
The field such as Docetaxel medical separation and purifying, Docetaxel medicine preparation, design and development.
In order to solve the above technical problems, the invention provides a kind of high performance liquid chromatography screening aptamer method,
Comprise the following steps：
(1) screening and the nucleic acid library of the aptamer of Docetaxel specific binding from random library RS36；With
Machine library RS36 has the nucleotide sequence described in SEQ ID NO.1；
(2) nucleic acid library is screened using high performance liquid chromatography and obtains aptamer；The high performance liquid chromatography
In method, stationary phase is octadecylsilane chemically bonded silica chromatographic column；Mobile phase uses water-acetonihile gradient elution；Detection wavelength is
260nm；Detector is PDAD.
Above-mentioned method, further, A in the mobile phase of high performance liquid chromatography described in step (2) in the step (2)
Xiang Weishui, B phase are acetonitrile, and the gradient elution method is：
0min~5min, mobile phase are 95v/v% water and 5v/v% acetonitrile；
5min~20min, mobile phase are the acetonitrile at the uniform velocity gradient change of 95v/v% water and 5v/v% to 85v/v%'s
The acetonitrile of water and 15v/v%.
Above-mentioned method, further, the column temperature of octadecylsilane chemically bonded silica chromatographic column described in the step (2)
For 25 DEG C.
Above-mentioned method, further, the sample size of high performance liquid chromatography described in the step (2) are 20 μ L.
Above-mentioned method, further, the 5 ' primers used in the step (1) is the nucleosides described in SEQ ID NO.4
Acid sequence；3 ' the primers used is the nucleotide sequences described in SEQ ID NO.5.
The technical concept total as one, present invention also offers the aptamer that a kind of above method screens to obtain,
The nucleotide sequence of the aptamer includes any one in following four sequence：
(1) nucleotide sequence described in SEQ ID NO.2 or the nucleotide sequence described in SEQ ID NO.3；
(2) homology of the nucleotide sequence and described in nucleotide sequence or SEQ ID NO.3 described in SEQ ID NO.2
Sequence more than 60%；
(3) hybridized with the nucleotide sequence described in the nucleotide sequence or SEQ ID NO.3 described in SEQ ID NO.2
Sequence；
(4) transcribed with the nucleotide sequence described in the nucleotide sequence or SEQ ID NO.3 described in SEQ ID NO.2
RNA sequence.
The technical concept total as one, present invention also offers a kind of above-mentioned aptamer as Docetaxel
Application in pharmaceutical carrier.
The technical concept total as one, present invention also offers a kind of above-mentioned aptamer to improve Taxotere
Application in alcohol solvability.
The technical concept total as one, present invention also offers a kind of above-mentioned aptamer to prepare Taxotere
Application in alcohol detection probe, Docetaxel target spot probe.
The technical concept total as one, present invention also offers a kind of above-mentioned aptamer in Docetaxel medicine
Application in thing isolation and purification.
The technical concept total as one, present invention also offers a kind of above-mentioned aptamer in Docetaxel medicine
Prepared by thing, the application in design and development.
When the aptamer of the present invention is applied to prepare Docetaxel detection probe, Docetaxel target spot probe；
As Docetaxel pharmaceutical carrier, when applied to Docetaxel medical separation and purifying, the core of above-mentioned aptamer
A certain position on nucleotide sequence can be phosphorylated, oxygen methylates, methylate, amination, sulfhydrylation or isotopologue.
Meanwhile biotin, digoxin, fluorescent material are can also incorporate on the nucleotide sequence of above-mentioned aptamer
(such as FITC), nano luminescent material, polyethylene glycol, peptide fragment, albumen, enzyme or folic acid mark；Even connect upper radioactivity and control
The property treated material etc..
It is all basic with former aptamer above whether through part substitution or the nucleotide sequence after modification
Same or similar molecular structure, physicochemical property and function, it can be used in and the specific combination of Docetaxel.
Meanwhile the derivative of aptamer of the invention equally has essentially identical or similar point of former aptamer
Minor structure property and function, that is, it can be used in specifically binding with Docetaxel.On foregoing aptamer derivative is
The phosphorothioate backbone that the skeleton of the nucleotide sequence of listed aptamer derives is stated, or above-mentioned listed nucleic acid is fitted
The corresponding lock nucleic acid or peptide nucleic acid that part is transformed into.
Compared with prior art, the advantage of the invention is that：
(1) the invention provides a kind of high performance liquid chromatography screening aptamer method, have instrument requirements it is low,
Universality is strong, label-free, cost is low, automatable advantage, and rapidly a large amount of candidate sequences can be selected.Pass through height
The aptamer that effect liquid phase chromatogram instrument is selected is under the conditions of the mobile phase of acetonitrile-water, and absorption solution of the chromatographic column to sample
Balance is inhaled, sample is in more than 70bar high pressure, and Docetaxel is still combined with aptamer.Therefore efficient liquid is passed through
The Docetaxel aptamer that phase chromatography obtains, have target molecule compared with strong affinity.
(2) aptamer provided by the invention can with high-affinity, combine Docetaxel with high specificity, have steady
It is qualitative it is good, non-toxic, be easy to modification and sequence length it is shorter the advantages that, be advantageous to large-scale industrial production and application.This hair
Bright aptamer also has water-wet behavior, after the medicine Docetaxel with being insoluble in water is combined, can improve Taxotere
Solubility of the alcohol in water, it is expected in the case of without using other cosolvents (such as ethanol), solubilizer (such as tween), reach
To the purpose of clinical practice treatment.
(3) aptamer of the invention has the characteristic for being easy to modification, can be purple by more west by modifying semiochemicals
The UV absorption signal of China fir alcohol is converted into other signals (such as fluorescence), so as to realize the highly sensitive detection of Docetaxel.
(4) present invention is expected to improve the steady of Docetaxel using aptamer and the binding characteristic of Docetaxel
Qualitative, security and solubility, extend drug effect, reduce Docetaxel and the combination of other blood endogenous materials.Selection is originally
The aptamer of invention is pharmaceutical carrier combination Docetaxel, can protect a drug from environment influence, and it is suitable to obtain
Drug release rate, play a role gentle and lasting drug effect, and the adverse drug caused by tween and alcohol can be avoided anti-
Should.Meanwhile the aptamer of the present invention is marked and is modified with beneficial to tracer inside Docetaxel and novel drugs
Exploitation etc..
Brief description of the drawings
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, clear, complete description is carried out to the technical scheme in the embodiment of the present invention.
Fig. 1 is the HPLC figures that the aptamer of the embodiment of the present invention 2 is combined with Docetaxel.
Fig. 2 is the HPLC figures that the aptamer of comparative example 1 of the present invention is combined with Docetaxel.
Fig. 3 is the HPLC figures that the aptamer of comparative example 2 of the present invention is combined with Docetaxel.
Fig. 4 is the mass spectrogram that the aptamer of the embodiment of the present invention 2 is combined with Docetaxel.
Fig. 5 is the circular dichroism spectrogram that the aptamer of the embodiment of the present invention 2 is combined with Docetaxel.
Fig. 6 is human breast carcinoma (MCF- of the amplifying nucleic acid aptamers of the embodiment of the present invention 4 as the pharmaceutical carrier of Docetaxel
7) cell propagation toxicity test result data figure.
Fig. 7 is human breast carcinoma (MDA- of the amplifying nucleic acid aptamers of the embodiment of the present invention 4 as the pharmaceutical carrier of Docetaxel
MB-231) cell propagation toxicity test result data figure.
Fig. 8 is various concentrations Docetaxel Rayleigh scattering experimental result data at 305nm in the embodiment of the present invention 5
Figure.
Fig. 9 is that the amplifying nucleic acid aptamers of the embodiment of the present invention 5 improve Docetaxel solubility, reduces Rayleigh at its 305nm
Scattering experiment result data figure.
Figure 10 is the HPLC datagrams that the amplifying nucleic acid aptamers of the embodiment of the present invention 6 are used to detect Docetaxel probe.
Embodiment
Below in conjunction with Figure of description and specific preferred embodiment, the invention will be further described, but not therefore and
Limit the scope of the invention.
Embodiment
Material and instrument employed in following examples are commercially available.The Docetaxel of the present embodiment is taxanes
Antineoplastic, its chemical name are 5 β, 20- epoxies -1,2 α, 4,7 β, 10 β, 13 α-hexahydroxy taxane -11- alkene -9- ketone -4,
[(2 ' R, 3 ' S)-N- benzoyl -3- phenylisoserine esters, No. CAS is 114977- to 10- diacetate esters -2- benzoic ethers -13-
28-5。
Embodiment 1：
A kind of method of HPLC assisting siftings aptamer, is mainly screened using micro-fluidic chip, specific screening
Process comprises the following steps：
(a) nucleic acid library is optimized：The structure of DNA library divides quinquepartite：Centre is the fixed sequence program of one section of 26 base,
For with fixed probe (BDNA) Complementary hybridization；The both sides of fixed sequence program are two DNA arms, random including 18 bases
Sequence necessary to the PCR amplifications of sequence and 19 bases.Fixed probe be one end indicate biotin with library fixed sequence program
Complementary DNA, the fixation for library.
Random library RS36 has the nucleotide sequence described in SEQ ID NO.1, is specially：
5’-CCGCTTCGCCGTCTCCTAC-NNNNNNNNNNNNNNNNNN-AAAAGTGGGTAGGGCGGGTTGGAAAA-
NNNNNNNNNNNNNNNNNN-CGCTCGTCACCCTTCTCCT-3 ' (notes：N represents any base in A, T, G, C)；
5 ' primers have the nucleotide sequence described in SEQ ID NO.4, are specially：
5’-FAM-CCGCTTCGCCGTCTCCTAC-3’；
3 ' primers have the nucleotide sequence described in SEQ ID NO.5, are specially：
5’-Biotin-AGGAGAAGGGTGACGAGCG-3’；
Fixed probe has the nucleotide sequence described in SEQ ID NO.6, is specially：
5’-Biotin-TACCGCAAAAAAAAACCAACCCGCCCTACCCAC-3’；
(b) primary dcreening operation：By the micro-fluidic surface successively bovine serum albumin(BSA) of modified biological elementization, Avidin, library.
Wherein, library preprocess method：By 1nmol libraries (about 1014Bar DNA), 1.5nmol BDNA be dissolved in 20 μ L's
(component of hybridization buffer is hybridization buffer：0.5mmol/L phosphate buffers, 10.5mmol/L NaCl, 15mmol/L
MgCl2, pH 6.8) in, 95 DEG C of denaturation 10min, it is slowly cooled to room temperature, stands more than 3h, library is hybridized with BDNA, then
Adding 130 μ L combination buffer, (component of combination buffer is：100mmol/L NaCl, 5mmol/L KCl, 2mmol/L
MgCl2, 1mmo/L CaCl2, 20mmol/L Tris-HCl, pH 7.6), it is standby.
Library fixing means：First at room temperature by 150 μ L, 1mg/mL biotinylated BSA by syringe pump with 1 μ
Then L/min flow velocity rinses 10min with 0.01mol/L PBS solution by chip with 5 μ L/min flow velocity；Again with same
Flow velocity be passed through 60 μ L, 1mg/mL Avidin, the same PBS solution with 0.01mol/L is rinsed with 5 μ L/min flow velocity
100min.The DNA library (DNA library after hybridizing) of 150 pretreated μ L is passed through into modification with 0.5 μ L/min flow velocity
The chip crossed, 10min is rinsed with 5 μ L/min flow velocity with combination buffer, then completes the fixation of library in the chips.Then
Object Taxotere alcoholic solution is passed through, the solution flowed out in chip is collected, primary dcreening operation nucleic acid library is obtained, wherein containing DNA
With the compound of Docetaxel.
(c) purify：The primary dcreening operation nucleic acid library that step (b) obtains is entered into performing PCR amplification, amplified production is (i.e. biotinylated
Double-stranded DNA) affinity column filled by Streptavidin microballon separated, then is unwind, formed after desalination time by alkaline denaturation
One-level nucleic acid library.Wherein, PCR reactions amplification condition is：94 DEG C of pre-degeneration 10min, 94 DEG C of denaturation 45sec, 62 DEG C are annealed
30sec, 72 DEG C of extension 30sec, expands 14 circulations；72 DEG C of extension 7min.Using grads PCR, using original nucleic acid library as
Masterplate optimizes annealing temperature, finally gives annealing temperature as 62 DEG C.And often take turns screening product optimal amplification wheel number be all by
Take turns what number optimization obtained.After wheel number optimization, remaining library is expanded under the same conditions.PCR results pass through 3% fine jade
Sepharose electrophoresis is verified that dyestuff is SYBR Glod.Library after PCR is subjected to single-stranded preparation, the sieve for next round
Choosing, single-stranded preparation method：Take 50 μ L Streptavidin microballons to be added in 1.5mL EP pipes, rinsed 3 times with 0.01mol/L PBS
Addition PCR expands the double-stranded products obtained afterwards, is centrifuged after 25 DEG C of concussion incubation 30min and removes supernatant；Continue to use 0.01mol/L PBS
500 μ L 200mmol/L NaOH is added after rinsing 3 times, 25 DEG C of concussions are incubated 10min and carry out single stranded processing；It is collected by centrifugation
The single stranded DNA to dissociate in clear；Collect product and desalting processing is carried out by high performance liquid chromatography, be dissolved in a small amount of water after drying concentration
In, time one-level nucleic acid library is obtained, is quantified with ultraviolet specrophotometer, is preserved in -20 DEG C of refrigerators.
(d) circulate：Initial nucleic acid library is substituted with secondary one-level nucleic acid library obtained above and repeats above-mentioned step (b)
~step (c), every time circulation when should again in micro-fluidic chip modified biological elementization bovine serum albumin bletilla Avidin,
Until obtaining the nucleic acid library for including the aptamer combined with Docetaxel high-affinity and high specific.
(e) screening efficiency is evaluated：Using fluorescence spectrometry screening efficiency, i.e., DNA enrichment condition in screening process.Detection
Signal is 5 ' terminal modified fluorescent dye FAM of DNA library fluorescent value.
The fluorescent value of pretreated DNA library is set to F0 by the present embodiment, and the solution flowed out when modifying library (is contained
Library on unlocked) fluorescent value be set to F1, the fluorescent value for the solution being collected into from chip during competition binding is set to F2, then
Screening efficiency is calculated as follows formula：Screening efficiency=F2/ (F0-F1-F2) × 100%.The sieve of each round is monitored according to this formula
Efficiency is selected, screening efficiency is higher, illustrates that competition is more to the DNA in solution, screening can be monitored by the measure of screening efficiency
Enrichment condition.When screening reaches plateau, cloning and sequencing is carried out to library, obtains the nucleic acid library (nucleic acid of aptamer
About 10 are shared in library14Bar aptamer).
Docetaxel is small-molecule drug, preliminary to infer that base really in connection be no more than 30.To surveying
Sequence result carries out sequence analysis, according to library designs scheme, fixed sequence program and PCR primer sequence be present in all sequences and
Length is longer, therefore two arms of sequence are split, and leaves out primer portion sequence, only random library part is carried out efficient
The binding ability examination of liquid chromatography.
(f) high performance liquid chromatography is screened：The aptamer in nucleic acid library is subjected to high performance liquid chromatography sieve respectively
Choosing, investigate the binding ability of aptamer and Docetaxel.Specifically method is：
Test sample：Prepare aptamer final concentration of 3 μM, final concentration of 6 μM of Taxotere alcoholic solution, with reference to slow
It is 100mmol/L NaCl, 5mmol/L KCl, 2mmol/L MgCl to rush solution2, 1mmo/L CaCl2With 20mmol/L Tris-
HCl mixed solution, pH 7.6.
Chromatographic condition is：Chromatographic column uses octadecylsilane chemically bonded silica as filler；Mobile phase is：A：Water, B：Acetonitrile；
Elution requirement is：0min → 5min → 20min, water：95v/v% → 85v/v% → 85v/v%, acetonitrile：5v/v% → 5v/v%
→ 15v/v%；Flow velocity：1mL/min, detector：PDAD；Detection wavelength：260nm；Column temperature：25℃；Sample introduction
Amount：20μL.
Embodiment 2
A kind of aptamer 1 for the Docetaxel for screening to obtain by the method for embodiment 1, the core of the aptamer 1
Nucleotide sequence has the nucleotide sequence described in SEQ ID NO.2, specially 5 '-TTGTTTCTCTGTCGATTA-3 '.
Embodiment 3
A kind of aptamer 2 for the Docetaxel for screening to obtain by the method for embodiment 1, the core of the aptamer 2
Nucleotide sequence has the nucleotide sequence described in SEQ ID NO.3, specially 5 '-ATTAGCTGTCTCTTTGTT-3 '.
The nucleotide sequence of above-described embodiment 2 and the aptamer of embodiment 3 is selected from naturally occurring or artificial synthesized
Sequence, or the same sequence in any other source.
Embodiment 2 and 3 is only the preferred nucleic acid aptamers that the present invention filters out, and according to the screening technique of the present invention, may be used also
To screen the aptamer with following nucleotide sequence：
(1) with embodiment 2 or 3 listed by aptamer nucleotide sequence homology more than 60% (such as can will
Above-mentioned nucleic acid aptamer sequence is deleted or the nucleotides of increase partial complementarity)；
(2) with embodiment 2 or 3 listed by aptamer the sequence that is hybridized of nucleotide sequence；
(3) RNA sequence of the nucleotide sequence transcription of aptamer listed by above-described embodiment 2 or 3.
Comparative example 1
A kind of aptamer 3, the nucleotide sequence of the aptamer 3 have the nucleotides described in SEQ ID NO.7
Sequence, specially 5 '-TGCCTTTCTGTTTCTT TG-3 '.
Comparative example 2
A kind of aptamer 4, the nucleotide sequence of the aptamer 4 have the nucleotides described in SEQ ID NO.8
Sequence, specially 5 '-CGGAGGTGCCTTTCCTAC-3 '.
The high-efficient liquid phase chromatogram (HPLC) of embodiment 2, the aptamer of comparative example 1 and 2 is analyzed respectively, with
Verify the accuracy of high performance liquid chromatography screening aptamer.
Fig. 1 is the HPLC figures of the aptamer (5 '-TTGTTTCTCTGTCGATTA-3 ') of embodiment 1, by investigation result
Fig. 1 understands that Docetaxel and aptamer 1 have stronger combination, extend the retention time of aptamer, make it
Chromatographic peak is split into two chromatographic peaks.
Fig. 2 is the HPLC figures of the aptamer (5 '-TGCCTTTCTGTTTCTTTG-3 ') of comparative example 1, by investigation result
Fig. 2 understands that Docetaxel exists with aptamer 3 to be combined to a certain degree, its chromatographic peak is turned into acromion.
Fig. 3 is the HPLC figures of the aptamer (5 '-CGGAGGTGCCTTTCCTAC-3 ') of comparative example 2, by investigation result
Fig. 3 understands that Docetaxel and the binding ability of aptamer 4 are weaker or are not combined, its chromatographic peak peak shape symmetrically without dividing or
Acromion phenomenon.
It was found from Fig. 1 to Fig. 3 analysis result, the aptamer selected by high performance liquid chromatograph is in acetonitrile-water
Mobile phase under the conditions of, and chromatographic column balances to the adsorption-desorption of sample, and sample be in more than 70bar high pressure, more west purples
China fir alcohol is still combined with aptamer.Therefore the Docetaxel aptamer obtained by high performance liquid chromatography, it is right
Target molecule has compared with strong affinity.
In order to verify the reliability of the method for high performance liquid chromatography screening aptamer, the present invention employs electricity simultaneously
Spraying ionization mass spectrometry and circular dichroism detector are verified.
Investigate 1：Using electrospray ionization mass spectrometry, the mixing to Docetaxel and the aptamer of embodiment 2 is molten
Liquid is detected.
According to atomic chart (IUPAC 2007standard atomic weights), the nucleic acid of patent offer of the present invention
The composite molecular weight of aptamers 1 and Docetaxel is 6270.4.The nucleic acid obtained by electrospray ionization mass spectrometry is adapted to
(when the part mass spectra peak of selection amplifies, instrument occurs slightly the composite molecular weight of body 1 and Docetaxel for 6268.7
Drift).According to third party's examining report, electrospray ionization mass spectrometry error is 0.03%.Therefore, measurement error allows in method
In error range, it was demonstrated that the aptamer 1 that this patent provides can be combined with Docetaxel with high-affinity, as a result see figure
4。
Investigate 2：Aptamer 1 (5 '-TTGTTTCTCTGTCGATTA-3 ') using circular dichroism detector to embodiment 2
Change of configuration after structure and aptamer 1 are combined with Docetaxel is characterized.
The concentration of fixed nucleic acid aptamers 1 is 20 μM, is separately added into final concentration of 0 μM, 5 μM, 10 μM, 15 μM of more west purples
China fir alcoholic solution.Cushioning liquid is 100mmol/L NaCl, 5mmol/L KCl, 2mmol/L MgCl2, 1mmo/L CaCl2With
20mmol/L Tris-HCl mixed solution, pH 7.6.As a result Fig. 5 is seen, from circular dichroism detector experimental result, nucleic acid is fitted
Negative peak of the circular dichroism spectrogram of ligand 1 at 251nm increases with the concentration of Docetaxel, angle signal enhancing, and ripple
About 2nm blue shift occurs for peak.Illustrate that Docetaxel makes the configuration of aptamer 1 change, in a manner of embedded and originally
The aptamer 1 that embodiment 2 is provided combines.
Embodiment 4
A kind of aptamer 1 (5 '-TTGTTTCTCTGTCGATTA-3 ') of Docetaxel of embodiment 2 is used as more
The application of the pharmaceutical carrier of western taxol, specifically using cell propagation toxicity test method, comprise the following steps：
It is to combine the Docetaxel of buffer preparation series concentration and aptamer 1, cushioning liquid
100mmol/L NaCl, 5mmol/L KCl, 2mmol/L MgCl2, 1mmo/L CaCl2With 20mmol/L Tris-HCl mixing
Solution, pH 7.6.The human breast carcinoma (MCF-7) and human breast carcinoma (MDA-MB-231) cell of exponential phase are chosen, with 2 ×
104The concentration of individual cells/well is inoculated in 96 porocyte culture plates (the training base per the μ L of hole 200), is cultivated in cell culture incubator
Night.Then cell culture fluid is discarded, is cleaned twice with D-Hanks, it is respectively 0nM, 60nM, 300nM to add 50 μ L concentration,
1500nM, 3000nM, 6000nM Docetaxel and aptamer 1 and Docetaxel compound are (more western in compound
The ratio of the molar concentration of taxol and aptamer and Docetaxel is 1: 1), add 250 μ L contains 15% calf
1640 training bases of serum, the final concentration for making Docetaxel and aptamer 1 and Docetaxel compound is respectively 0nM,
10nM, 50nM, 250nM, 500nM and 1000nM.96 orifice plates are placed in 37 DEG C, 5%CO in cell culture incubator2Under the conditions of cultivate
After 48h, per hole add concentration be 5mg/mL 3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides (tetrazolium bromide,
MTT) the μ L of solution 20, continue to be incubated 4h, terminate culture.Culture supernatant is discarded, 170 μ L DMSO are added per hole, and (dimethyl is sub-
Sulfone), concussion is incubated 10min, crystal is fully dissolved.Selected absorbing wavelength is 490nm, and the suction in each hole is measured using ELIASA
The hole cell survival rate that the complex concentration of shading value, wherein Docetaxel and aptamer 1 and Docetaxel is 0nM
It is assumed that 100%, the hole cell survival rate for being not added with cell is assumed to 0, and each identical treatment group does 5 holes.In terms of following equation
Cell survival rate is calculated, as a result sees Fig. 6 and Fig. 7.
It was found from from Fig. 6 and Fig. 7, the Docetaxel aptamer 1 of the embodiment of the present invention 2 can be used as Taxotere
The carrier of alcohol is used to treat, and does not suppress the activity of Docetaxel, and is expected in aptamer and folic acid, antibody target
To the effect that targeted therapy is realized in the case of molecular modification, the exploitation for new drug.
Embodiment 5
A kind of aptamer 1 (5 '-TTGTTTCTCTGTCGATTA-3 ') of the Docetaxel of embodiment 2 is improving
Application in Docetaxel solvability, investigated using the Rayleigh intensity of Docetaxel at measure 305nm.Tool
The application process of body comprises the following steps：
Application process one：The aptamer 1 of the Docetaxel of embodiment 2 is configured to final concentration of 50 μM of nucleic acid
Liquid solution is adapted to, is then dissolved in cushioning liquid, cushioning liquid is 100mmol/L NaCl, 5mmol/L KCl, 2mmol/L
MgCl2, 1mmo/L CaCl2With 20mmol/L Tris-HCl mixed solution, pH 7.6.Final concentration of 10mM is prepared with DMSO
Docetaxel.Docetaxel is gradually added into aptamer solution, after 4~8 hours combine balance, utilization is glimmering
Light instrument, the Rayleigh scattering signal of 305nm transmittings measure aptamer and Docetaxel complex solution is excited with 305nm.
From DrugBank database retrievals, the solubility of Docetaxel is 12.7mg/L, i.e., about 15.72 μM.
Experimental result is shown in Fig. 8, as can be known from Fig. 8：With the rise of Taxotere determining alcohol in solution to be measured, at 305nm
Rayleigh scattering signal gradually strengthen.And there is flex point at 200 μM to 300 μM, illustrate the state generation of now solution
Rapidly change, insoluble state is gradually converted into from the state of dissolving, is illustrated under the auxiliary of aptamer, Taxotere
The solubility of alcohol is lifted between 200 μM to 300 μM.As a result illustrate, aptamer provided by the present invention can be used for improving
The solubility of Docetaxel.
Application process two：The Docetaxel aptamer 1 of embodiment 2 is configured to cushioning liquid final concentration of
2mM aptamer solution, wherein cushioning liquid are 100mmol/L NaCl, 5mmol/L KCl, 2mmol/L MgCl2,
1mmo/L CaCl2With 20mmol/L Tris-HCl mixed solution, pH 7.6.Docetaxel is suspended in cushioning liquid
It is configured to final concentration of 50 μM of Docetaxel suspension.It is molten that aptamer is gradually added into Docetaxel suspension
Liquid, after 4~8 hours combine balance, using luminoscope, excite 305nm transmittings measure aptamer and more west purple with 305nm
The Rayleigh scattering signal of China fir alcohol complex solution.
Experimental result is shown in Fig. 9, as can be known from Fig. 9：With the rise of nucleic acid in solution to be measured adaptation bulk concentration, at 305nm
Rayleigh scattering signal gradually weaken.Illustrate that the state of Docetaxel suspension changes, in the auxiliary of aptamer
Under, the insoluble particles in Docetaxel suspension gradually decrease.As a result illustrate, aptamer provided by the present invention can use
In the solubility for improving Docetaxel.
Embodiment 6
It is prepared by a kind of aptamer 1 (5 '-TTGTTTCTCTGTCGATTA-3 ') of the Docetaxel of embodiment 2
Application in Docetaxel detection probe, Docetaxel target spot probe, HPLC methods specifically are used, specifically include following step
Suddenly：
Respectively with buffer preparation test sample 1 and test sample 2, cushioning liquid is 100mmol/L NaCl, 5mmol/L
KCl, 2mmol/L MgCl2, 1mmo/L CaCl2, 20mmol/L Tris-HCl, pH 7.6.Test sample 1 is final concentration of 3 μM of core
The solution of sour aptamers, test sample 2 are the solution of final concentration of 3 μM of aptamers and 6 μM of Docetaxel compound.Color
Spectral condition is：Chromatographic column uses octadecylsilane chemically bonded silica as filler；Mobile phase is：A：Water, B：Acetonitrile；Elution requirement
For：0min → 5min → 20min, water：95v/v% → 85v/v% → 85v/v%, acetonitrile：5v/v% → 5v/v% → 15v/
V%；Flow velocity：1mL/min, detector：PDAD；Detection wavelength：260nm；Column temperature：25℃；Sample size：20μ
L.As seen from Figure 10, when no Docetaxel is present, aptamer is an independent chromatographic peak to experimental result,
And as the addition of Docetaxel, the chromatographic peak of originally aptamer are split into acromion.This experimental result can illustrate,
In the presence of Docetaxel, obvious change occurs for the chromatographic peak of aptamer, and this aptamer may be used as
Detect the probe of Docetaxel.
A certain position on the nucleotide sequence of the aptamer of embodiment 2 or embodiment 3 can be phosphorylated, oxygen methyl
Change, methylate, amination, sulfhydrylation or isotopologue.Phosphorylation, oxygen methylate, methylated, amination, sulfhydrylation or same position
Docetaxel aptamer after elementization equally has following purposes：(1) purposes in for pharmaceutical carrier；(2) exist
Improve the purposes of drug solubility；(3) in drug design and the purposes in exploitation；(4) in medical separation and the purposes in purifying；
(5) purposes in Docetaxel detection probe is prepared；(6) purposes in Docetaxel is prepared as target spot probe.
Biotin-binding, digoxin, fluorescence on the nucleotide sequence of the aptamer of embodiment 2 or embodiment 3
Matter, nano luminescent material, polyethylene glycol, peptide fragment, albumen or enzyme mark, or the tumor targeted molecular such as folic acid mark.Using biology
Element, digoxin, fluorescent material, nano luminescent material, polyethylene glycol, peptide fragment, albumen or enzyme mark, or the cancer target point such as folic acid
Docetaxel aptamer after son mark equally has following purposes：(1) purposes in for pharmaceutical carrier；(2)
Improving the purposes of drug solubility；(3) in drug design and the purposes in exploitation；(4) in medical separation and the use in purifying
On the way；(5) purposes in Docetaxel detection probe is prepared；(6) use in Docetaxel is prepared as target spot probe
On the way.
The skeleton of the nucleotide sequence of the nucleotide sequence of the aptamer of embodiment 2 or embodiment 3 can also derive
Phosphorothioate backbone, above-mentioned aptamer can also be transformed into corresponding lock nucleic acid or peptide nucleic acid, have following purposes：(1) exist
For the purposes in pharmaceutical carrier；(2) purposes (3) of drug solubility is being improved in drug design and the purposes in exploitation；(4)
In medical separation and the purposes in purifying；(5) purposes in Docetaxel detection probe is prepared；(6) more west purples are being prepared
China fir alcohol is used as the purposes in target spot probe.
The above described is only a preferred embodiment of the present invention, any formal limitation not is made to the present invention.Though
So the present invention is disclosed as above with preferred embodiment, but is not limited to the present invention.It is any to be familiar with those skilled in the art
Member, in the case where not departing from the Spirit Essence of the present invention and technical scheme, all using in the methods and techniques of the disclosure above
Appearance makes many possible changes and modifications to technical solution of the present invention, or is revised as the equivalent embodiment of equivalent variations.Therefore,
Every content without departing from technical solution of the present invention, the technical spirit according to the present invention is to made for any of the above embodiments any simple
Modification, equivalent substitution, equivalence changes and modification, still fall within technical solution of the present invention protection in the range of.
Claims (6)
1. the method that high performance liquid chromatography screens aptamer, it is characterised in that comprise the following steps：
（1）Screening and the nucleic acid library of the aptamer of Docetaxel specific binding from random library RS36；It is described
Random library RS36 has the nucleotide sequence described in SEQ ID NO.1；
（2）The nucleic acid library is screened using high performance liquid chromatography and obtains aptamer；In the high performance liquid chromatography,
Stationary phase is octadecylsilane chemically bonded silica chromatographic column；Mobile phase uses water-acetonihile gradient elution；Detection wavelength is 260nm；
Detector is PDAD；The step（2）Described in high performance liquid chromatography mobile phase in A phases be water, B phases are
Acetonitrile, the gradient elution method are：
0min~5min, mobile phase are 95 v/v% water and 5 v/v % acetonitrile；
5min~20min, mobile phase be 95 v/v% water and 5 v/v % acetonitrile at the uniform velocity gradient change to 85 v/v% water and
15 v/v % acetonitrile；
The step（2）Described in octadecylsilane chemically bonded silica chromatographic column column temperature be 25 DEG C.
2. according to the method for claim 1, it is characterised in that the step（2）Described in high performance liquid chromatography sample introduction
Measure as 20 μ L.
3. a kind of methods described of claim 1 or 2 screens obtained aptamer, it is characterised in that the aptamer
Nucleotide sequence include following sequence in any one：
The nucleotide sequence described in nucleotide sequence or SEQ ID NO.3 described in SEQ ID NO.2.
4. the aptamer described in a kind of claim 3 is fitted as the application in Docetaxel pharmaceutical carrier, the nucleic acid
The nucleotides sequence of part is classified as the nucleotide sequence described in SEQ ID NO.2.
5. application of the aptamer in Docetaxel solvability is improved described in a kind of claim 3, the nucleic acid
The nucleotides sequence of aptamers is classified as the nucleotide sequence described in SEQ ID NO.2.
6. application of the aptamer in Docetaxel detection probe is prepared described in a kind of claim 3, the nucleic acid
The nucleotides sequence of aptamers is classified as the nucleotide sequence described in SEQ ID NO.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410852323.3A CN104480114B (en) | 2014-12-31 | 2014-12-31 | The method of high performance liquid chromatography screening aptamer and aptamer and application |
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| β-葡萄糖醛酸苷酶对甘草酸键选择性的改造及其 DNA 适配体筛选;矫丽媛;《中国优秀硕士论文全文数据库》;20040228;第10页 * |
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