CN110143997A - A kind of operating method of expansible synthetic oligonucleotide peptide conjugate - Google Patents
A kind of operating method of expansible synthetic oligonucleotide peptide conjugate Download PDFInfo
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- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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Abstract
A kind of method that the present invention discloses synthetic oligonucleotide peptide conjugate comprising following steps: azido oligonucleotides and DBCO- peptide and highly purified a, are prepared；B, reaction prepares: DBCO- peptide being dissolved in DMSO and obtains DBCO- peptide solution, azido oligonucleotides is dissolved in the mixed liquor of water or water and DMSO and obtains azido oligonucleotide solution；C, efficient liquid phase chromatographic analysis first coupling reaction: is carried out to azido oligonucleotides, the position at conjugate peak is determined again, sample is extracted to reaction product and carries out efficient liquid phase chromatographic analysis, determine the position at the peak of reaction product after coupling reaction, the area at the peak of unreacted azido oligonucleotides, carries out gradually stacking reaction later.The present invention be suitble to large-scale production, without being further purified, it is at low cost, avoid product degradation.
Description
Technical field
The present invention relates to organic chemistry, biochemistry, technical field of molecular biology, more particularly to a kind of expansible conjunction
At the operating method of oligonucleotides peptide conjugate.
Background technique
Oligonucleotides peptide conjugate (Peptide-oligonucleotide conjugates, OPCs) has simultaneously because of it
The structure of standby oligonucleotides and polypeptide is widely used in from nanotechnology to drug delivery with activity and with its unique property
With the every field such as antisense technology.
Polypeptide can identify the special site of albumen, have penetrability, the base-pair of such characteristic and oligonucleotides to cell
The combination of recognition capability, enable OPCs molecule be widely used (Leonetti, Degols et al., 1990；
Soukchareun,Tregear et al.,1995).OPCs is initially used to antisense technology, i.e., by by a small DNA sequence
Column, which hybridize to the region mRNA, makes it encode out desired genetic fragment.OPCs is highly suitable for the research of this type, because
The polypeptide combined on OPCs molecule can identify specific cell type, and oligonucleotides can be carried out transmembrane transport, can
Selectively hybridize to inhibit its translation with mRNA in the cell.
Since initial OPCs molecule is by as antisense agent, and there are many new applications, has extended to biology and received
Rice technology.One type work is in the progress of structural DNA nanotechnology, which will using the nucleic acid moiety of OPCs molecule
Single polypeptide is directed to the specific position on DNA nanostructure surface.For example, c-myc polypeptide can be 20 alkali with a length
The short single strand dna of base is coupled, and obtained conjugated molecules are complementary with the DNA probe sequence on two dimension DNA nano-array surface
(Williams,Lund et al.,2007).After the completion of hybridization, tiling formula array generates a nanoscale uniform intervals 64 and receives
The pattern of parallel lines of rice, when being detected with the anti-c-mys antibody of homology, antibody can be identified specifically
Nano-array outwardly c-myc peptide.It is this to show that the technology of peptide is referred to as nanometer and shows on DNA nanostructure surface.Other one
A example is that the OPCs displayed on DNA nanotube is used as template, so that DNA nanotube ground surface is uniformly distributed nanogold
Grain (Williams, Lund et al., 2007).In short, the two examples prove that OPCs molecule can be accurate in nanometer level
Encoding human material and inorganic material.
The method for having several different production OPCs molecules at present be reported (Roberts and Szostak, 1997；
Venkatesan and Kim,2006；Moroder, Steger et al., 2009), wherein most of be directed to linear, solid-phase
Synthesis step, that is, continuously coupling synthesizes (Grandas, Marchan et directly on solid phase carrier by polypeptide and oligonucleotides
al., 2007).Polypeptide sequence is linked on solid phase carrier first, in one hydroxyl of modification on C-terminal group, then passes through di(2-ethylhexyl)phosphate
First core Gan Danti of linkage, then successively synthesizes remaining oligonucleotide sequence.Continuous synthesis is needed according to polypeptide
The characteristics of synthesizer peptide and oligonucleotide synthesizer, is specifically designed, and the chemical protecting group being related to also must while be compatible with polypeptide
With the solid phase chemistries of oligonucleotides (Venkatesan and Kim, 2006).
There are also segment coupling methods, it is a kind of method more convenient and fast than linear continuous synthesis.It is being respectively synthesized polypeptide
After oligonucleotides, two molecules are chained up by covalent chemical bond as an OPCs molecule, and is covalently attached
Group in chemical structure and in nature can there are many selections, generated at present generally by click chemistry method, such as sulfydryl
With the addition reaction of maleimide, the addition reaction of alkynyl and azido.
Whether the OPCs product that segment coupling method or continuous synthesis obtain is kept during producing OPCs
The structural intergrity of oligonucleotides is vital.Conventional production design requirement creates an absolutely not DNAse/
The production environment of RNAse pollution, but reagent, equipment and maintenance cost are quite high.And production process may require reagent it is long when
Between a possibility that contacting aqueous solution, inevitably increasing OPCs degradation.Further, due to oligonucleotide synthesis method sheet
The degree of polymerization that body determines is uncertain, participate in functional group's (such as azido) of coupling reaction molar concentration be it is uncertain, lead
Final products are caused not exclusively to there are problems that serious low-purity due to coupling reaction.Generally require through a kind of following method or
A variety of methods are combined to purify: ION PAIR Reverse Phase chromatography, ion-exchange chromatography, gel electrophoresis, dialysis, ultrafiltration.These
Method is complicated, costly and inefficient, is unsuitable for being mass produced.
Summary of the invention
It is an object of the present invention to be directed to the above-mentioned deficiency of the prior art, propose it is a kind of be suitble to large-scale production, without into
One step purifying, expansible synthetic oligonucleotide peptide conjugate at low cost, avoiding product degradation operating method.
The present invention solves its technical problem, the technical solution adopted is that, propose a kind of expansible synthetic oligonucleotide peptide
The operating method of conjugate comprising following steps:
A, azido oligonucleotides and DBCO- peptide and highly purified are prepared；
B, reaction prepare: DBCO- peptide is dissolved in DMSO and obtains DBCO- peptide solution, by azido oligonucleotides be dissolved in water or
Azido oligonucleotide solution is obtained in the mixed liquor of water and DMSO；
C, coupling reaction: (c1), efficient liquid phase chromatographic analysis is carried out to azido oligonucleotides, the area at starting peak is A0,
The amount for taking DBCO- peptide solution is V0Be added in azido oligonucleotide solution reaction and obtain reaction product, wherein DBCO- peptide with
The molar ratio of azido oligonucleotides is 1:1.00-1.25；(c2), it determines the position at conjugate peak: sample is extracted to reaction product
Product simultaneously carry out efficient liquid phase chromatographic analysis, determine the position at the peak of reaction product after coupling reaction, and unreacted azido is few
The area at the peak of nucleotide is A0；(c3), it gradually stacking reaction: (1) first time stacking reaction: is added into reaction product
The amount of DBCO- peptide solution is V1,；(2) second of stacking reaction: continue to add the amount of DBCO- peptide solution
For V2,；(3) n-th stacking reaction: the amount for continuing to add DBCO- peptide solution is Vn,；Wherein, after each stacking reaction, sample is extracted to product and is carried out efficient
Liquid-phase chromatographic analysis obtains the area A at the peak of unreacted azido oligonucleotidesn+1, until An+1No longer reduce, terminates folded
Reaction is added to obtain final product.
Present invention application click chemistry realizes segment coupling reaction.In order to avoid heavy metal element sews oligonucleotides peptide
The adverse effect that composition powder downstream uses must avoid 1,3- dipole [3+2] ring based on copper catalytic reagent from the beginning
Addition reaction (1,3-dipolar cycloaddition).The present invention advocates 1,3- dipole [3+2] ring promoted using ring strain
Addition reaction, especially dibenzazepine cyclooctyne base (DBCO) obtain 1,2,3-triazoles by cycloaddition with azido (N3)
Group.The method can ensure that oligonucleotides peptide conjugate product can be common to living cells, organism and non-living body experiment sample
In this.DBCO group exclusively marks the molecule containing azido.Within the scope of physiological temp and physiology pH value, DBCO group is not
It can be reacted with active group sulfydryl naturally occurring in molecular biosciences, amido or hydroxyl.
Click chemistry reaction principle is as follows:
Preferably, the concentration of the DBCO- peptide solution is in 1-10mM, the concentration of the azido oligonucleotide solution is in 0.1-
1.0mM。
Preferably, the high performance liquid chromatography uses UV detector, the UV detector Detection wavelength is set as
210nm。
Preferably, carrying out vacuum freeze drying i.e. to final product can be directly used in next step in fact after step e
It tests.
Preferably, in stepb, azido oligonucleotides being dissolved in the mixed liquor of water or water and DMSO and obtaining concentration
In the azido oligonucleotide solution of 0.1mM-100mM, wherein in water and DMSO mixed liquor, the mixed volume ratio of water and DMSO are
1:1-5。
Preferably, the molar ratio of DBCO- peptide and azido oligonucleotides is 1:1 in step (c1).
Preferably, the mixed volume ratio of water and DMSO are 1:1 in water and DMSO mixed liquor.
Preferably, the molar ratio of DBCO- peptide and azido oligonucleotides is 1:1 in step (c1).
The invention has the following beneficial effects:
1. oligonucleotides is easy to be degraded by DNA enzymatic or RNA enzyme, method of the invention can be to avoid the pollution problem of external enzyme.
2. using click chemistry standard measure, immediately completing coupling reaction.DBCO group is exclusively marked containing azido
Molecule.Within the scope of physiological temp and physiology pH value, DBCO group will not be with active group naturally occurring in molecular biosciences
Sulfydryl, amido or hydroxyl are reacted.The one-to-one reaction efficiency of this species specificity is up to 95% or more.
3. gradually being operated to stacking reaction under the guidance of efficient liquid phase chromatographic analysis, and optimize.By minority
(general 2 times) gradually overlap-add operation is measured up to theoretic chemical reaction than 1:1, is finally reached effective superposition of 95-99%
Reaction end.The time of efficient liquid phase chromatographic analysis sample is the required time that gradually stacking reaction operates every time.Therefore, sharp
Production efficiency can be steadily improved with the successive optimization end reaction of HPLC analytical method, and is expanded to extensive
Reaction.
4. effectively reducing OPCs additional purification step after the reaction was completed, since reaction almost carries out, react
By-product belongs to small molecule, will not generate any influence to downstream reaction, it is not necessary that carry out further purification work.
5. oligonucleotides is marked using azido group, and polypeptide is marked using DBCO in stacking reaction operation of the invention
Note.If oligonucleotides is marked using DBCO, polypeptide is marked using azido group, in this way exchanges labelling groups, this optimization side
Method is also applicable in completely.
6. labelling groups can be according to circumstances placed on times in polypeptide or nucleic acid sequence by the stacking reaction operating method
Meaning site, labelling groups do not influence final coupling reaction effect in any site.Different from linear synthesis OPCs method, this is folded
The experimental technique for adding operation method to use standard in the solution can be completed, and special equipment or reagent are not needed.
7. yield can be with popularization.This stacking reaction operation preparation is illustrated in the embodiment of the present invention from micro-
Gram product is conjugated to the oligonucleotides peptides of tens milligrams of magnitudes.
Detailed description of the invention
Fig. 1 is the chromatogram that high performance liquid chromatography verifies azido oligonucleotides in embodiment 1；
Fig. 2 is the chromatogram of the DBCO- peptide of high performance liquid chromatography verifying after purification in embodiment 1；
Fig. 3 is the high-efficient liquid phase chromatogram of operating time point 0 in embodiment 1；
Fig. 4 is the high-efficient liquid phase chromatogram of operating time point 1 in embodiment 1；
Fig. 5 is the high-efficient liquid phase chromatogram of operating time point 2 in embodiment 1；
Fig. 6 is the high-efficient liquid phase chromatogram of operating time point 3 in embodiment 1；
Fig. 7 is the high-efficient liquid phase chromatogram of operating time point 4 in embodiment 1；
Fig. 8 is the high-efficient liquid phase chromatogram of operating time point 5 in embodiment 1；
Fig. 9 is the chromatogram of the DBCO- peptide of high performance liquid chromatography verifying after purification in embodiment 2；
Figure 10 is the high-efficient liquid phase chromatogram of operating time point 1 in embodiment 2；
Figure 11 is the high-efficient liquid phase chromatogram of operating time point 2 in embodiment 2；
Figure 12 is the high-efficient liquid phase chromatogram of operating time point 3 in embodiment 2；
Figure 13 be in embodiment 3 high performance liquid chromatography verifying azido oligonucleotides double-strand in antisense strand chromatogram；
Figure 14 is the chromatogram that high performance liquid chromatography verifies the positive-sense strand in the double-strand of azido oligonucleotides in embodiment 3；
Figure 15 is the chromatogram of the DBCO- peptide of high performance liquid chromatography verifying after purification in embodiment 3；
Figure 16 is the high-efficient liquid phase chromatogram of operating time point 0 in embodiment 3；
Figure 17 is the high-efficient liquid phase chromatogram of operating time point 1 in embodiment 3；
Figure 18 is the high-efficient liquid phase chromatogram of operating time point 2 in embodiment 3；
Figure 19 is the high-efficient liquid phase chromatogram of operating time point 3 in embodiment 3；
Figure 20 is the high-efficient liquid phase chromatogram of operating time point 4 in embodiment 3；
Figure 21 is the high-efficient liquid phase chromatogram of operating time point 5 in embodiment 3；
Figure 22 is the high-efficient liquid phase chromatogram of operating time point 6 in embodiment 3；
Figure 23 is the high-efficient liquid phase chromatogram of reaction product in comparative example 1；
Figure 24 is the high-efficient liquid phase chromatogram of reaction product in comparative example 2.
Specific embodiment
Following is a specific embodiment of the present invention in conjunction with the accompanying drawings, technical scheme of the present invention will be further described,
However, the present invention is not limited to these examples.
Condition is used when high performance liquid chromatography (HPLC) analysis used in following embodiment and comparative example.
Chromatographic column: Ascentis Express C18,50x2.1 mm, 2.7 μm
Mobile phase: A=10mM TEAA aqueous solution
B=10mM TEAA acetonitrile solution
Flow velocity: 0.8ml/min
Temperature: 25 DEG C
Detector: 210nm, bw 4nm
Embodiment 1:
The present embodiment double center chain RNA(dsRNA) it may be separated well under special high-efficient liquid phase chromatogram condition.Because
Antisense strand is not marked with azido, so it keeps intact during the reaction, peak height and chromatographic retention dimension
It holds constant.The example is reacted with the double-stranded RNA of 50mg in total with DBCO- peptide, and final products need to carry out at annealing
Reason becomes the double-stranded RNA containing polypeptide coupling again.
A kind of operating method of expansible synthetic oligonucleotide peptide conjugate comprising following steps:
A, passed through by the way that DBCO- maleimide is connected to preparation DBCO- peptide, azido on polypeptide cysteine side chain thiol
The end 5' or 3' that chemical method is covalently attached to oligonucleotides prepares azido oligonucleotides, DBCO- peptide and azido few nucleosides
Acid is purified by high performance liquid chromatography.
B, reaction prepares: DBCO- peptide 29.3mg being dissolved in the DMSO of 1.175ml and obtains DBCO- peptide solution, this reality
The concentration for applying DBCO- peptide solution in example is 10mM, and azido oligonucleotides 50mg is dissolved in the mixing of the water and DMSO of 3.63ml
Azido oligonucleotide solution is obtained in liquid, azido oligonucleotide solution concentration is 1.0mM in the present embodiment, in the present embodiment
In the mixed liquor of water and DMSO the mixed volume ratio of water and DMSO be 1:1, and respectively measurement azido oligonucleotide solution and
The high performance liquid chromatography of DBCO- peptide solution, in the present embodiment, high performance liquid chromatography uses UV detector, the ultraviolet inspection
It surveys device Detection wavelength and is set as 210nm, as shown in Figure 1, high performance liquid chromatography verifies azido oligonucleotides, this is a double-strand
Oligonucleotides, purity 85.4%, azido mark on positive-sense strand, as shown in Fig. 2, high performance liquid chromatography is verified after purification
DBCO- peptide, purity 99.40%；
C, coupling reaction: (c1), the amount for taking DBCO- peptide solution are V0It is added to reaction in azido oligonucleotide solution to obtain instead
Answer product, V in the present embodiment0=364 microlitres, wherein the molar ratio of DBCO- peptide and azido oligonucleotides is 1:1；(c2), really
Determine the position at conjugate peak: reaction product being extracted and sample and carries out efficient liquid phase chromatographic analysis, determines anti-after coupling reaction
The position at the peak of product is answered, as shown in table 1,0 corresponding diagram 3 of operating time point, 1 corresponding diagram 4 of operating time point, operating time point 2
Corresponding diagram 5,3 corresponding diagram 6 of operating time point, 4 corresponding diagram 7 of operating time point, 5 corresponding diagram 8 of operating time point, wherein Fig. 3 is gone back
Chromatogram (i.e. c1 step carries out high performance liquid chromatography measurement to azido tube nucleus thuja acid before starting) when DBCO- peptide not being added,
Fig. 3 and Fig. 4 is compared, and can learn that the peak of reaction product is P3, the area at the peak is 4700.5, unreacted azido few nucleosides
The peak of sour positive-sense strand is P2, and the peak of antisense strand is P1, in operation between when putting 0, the area A at the peak of positive-sense strand0=4807, when
Between 1 when, the area A at the peak of positive-sense strand1=2959.1, A at this time1/A0=0.6156；(c3), gradually stacking reaction: (1) folded for the first time
Add reaction: the amount that DBCO- peptide solution is added into reaction product is V1,=364 × 0.6156=224 is micro-
It rises, and 230 microlitres of actual interpolation in operating, it the results are shown in Table 1 operating time point 2, at this time the area A of the peak P2 of positive-sense strand2=
692.7, A2/A0=0.1441；(2) second of stacking reaction: the amount for continuing to add DBCO- peptide solution is V2,=(364+230) × 0.1441=85.6 microlitre, and the actual interpolation amount in operating is 80 micro-
It rises, the results are shown in Table 1 operating time point 3, at this time the area A at the peak of positive-sense strand P23=137.3, A3/A0The product peak at this time=0.0286.
P3 purity reaches 96.98%；Third time stacking reaction: the amount for continuing to add DBCO- peptide solution is V3,=(364+230+80) × 0.0286=19.3, V3Practical injected volume be 19 microlitres, knot
Fruit is shown in Table 1 operating time point 4, at this time the area A at the peak positive-sense strand P24It is 107.7, A4/A0=0.0226, this time product peak P3
Purity reaches 97.87%.Consider that the reactant concentration in previous step significantly reduces, extends total reaction time to 65 minutes, i.e. table
One operating time terminal.It extracts sample and carries out efficient liquid phase chromatographic analysis, reactant peak P3 purity slightly rises to 97.87%.
Consider that the reactant concentration of third time stacking reaction significantly reduces, and the variation of reactant residual coefficients is smaller, we will react
Time extends to 270 minutes, i.e. the operating time terminal of table one.It extracts sample and carries out efficient liquid phase chromatographic analysis, conjugate
Peak P3 purity slightly rises to 98.33%.A5/A0=0.0166.We assert that this time point is reaction end
Table 1:
Embodiment 2:
The broad applicability of gradually stacking reaction is shown in the present embodiment using a uncertain single stranded DNA of purity.The list
Chain DNA is in synthesis formula with thiophosphoric acid diester linkage instead of common phosphodiester bond.The thio chemistry for enhancing DNA molecular is steady
It is qualitative, but important consequence also is caused to DNA molecular structure simultaneously, chiral centre is introduced on all phosphorus atoms, to keep this folded
Nitrogen base oligonucleotides becomes an extremely complex racemic mixture, and it is highly difficult to detect its chromatographic purity.Although being unable to get behaviour
Make 0 data of time point, we take approximate simplified measure, still obtain relatively good result.
A kind of operating method of expansible synthetic oligonucleotide peptide conjugate comprising following steps:
A, passed through by the way that DBCO- maleimide is connected to preparation DBCO- peptide, azido on polypeptide cysteine side chain thiol
The end 5' or 3' that chemical method is covalently attached to oligonucleotides prepares azido oligonucleotides, and DBCO- peptide passes through high-efficient liquid phase color
Spectrometry is purified, and azido oligonucleotide molecules amount 11474.3 can not be purified effectively.
B, reaction prepares: DBCO- peptide 4.5mg being dissolved in the DMSO of 1.32ml and obtains DBCO- peptide solution, this implementation
The concentration of DBCO- peptide solution is 1mM in example, and azido oligonucleotides 0.69mg is dissolved in 109 microlitres of water and obtains azido
Oligonucleotide solution, azido oligonucleotide solution concentration is 0.5mM in the present embodiment, and the efficient liquid phase of DBCO- peptide solution
Chromatography, in the present embodiment, high performance liquid chromatography uses UV detector, and the UV detector Detection wavelength is set as
210nm, as shown in figure 9, the DBCO- peptide of high performance liquid chromatography verifying after purification, purity 98.99%；
C, coupling reaction: (c1), the amount for taking DBCO- peptide solution are V0It is added to reaction in azido oligonucleotide solution to obtain instead
Answer product, V in the present embodiment0=54 microlitres, wherein the molar ratio of DBCO- peptide and azido oligonucleotides is 1:1；(c2), it determines
The position at conjugate peak: extracting sample to reaction product and carry out efficient liquid phase chromatographic analysis, determines reaction after coupling reaction
The position at the peak of product, as shown in table 2,1 corresponding diagram 10 of operating time point, 2 corresponding diagram 11 of operating time point, operating time point 3
Corresponding diagram 12, the peak of reaction product are P2, and the area at the peak is 3343.5, and the peak of unreacted azido oligonucleotides is P1,
A1=4166.4, P1's and P2 and value 7509.9 are taken as A by the present embodiment0Rough approximation, A at this time1/A0=0.5548；(c3),
Gradually stacking reaction: product peak P2 purity is preset as 95% in the present embodiment, (1) first time stacking reaction: adds into reaction product
The amount for adding DBCO- peptide solution is V1,=54 × 0.5548=30 microlitres, and actual interpolation 31 in operating
Microlitre, the area A of the peak P1 of azido oligonucleotides at this time2=2647.3, A2/A0=0.3525；(2) second of stacking reaction: after
The amount of continuous addition DBCO- peptide solution is V2,=(54+31) × 0.3525=30 microlitre, in operation
Actual interpolation amount be 30 microlitres, area A3=324 of the peak P1 of azido oligonucleotides at this time, 3, A3/A0=0.0432.Product
Peak P2 purity reaches 94.68%, close to 95%, the peak shape at this time point P1 peak shape substantial deviation the first two time point, it is believed that
Its peak area value no longer has indicative significance to the stacking reaction of next step, therefore time point 3 is reaction end.
Table two
Embodiment 3:
Show that gradually stacking reaction is applicable in different from the double-stranded RNA (dsRNA) of embodiment one using another in the present embodiment
Poor in solubility and sluggish special oligonucleotides, it is molten because its antisense strand and positive-sense strand all contain the base of methylation
Xie Du test shows that it cannot form highly concentrated solution.The method according to the invention finally still obtains good reaction product and turns
Change purity.
A kind of operating method of expansible synthetic oligonucleotide peptide conjugate comprising following steps:
A, passed through by the way that DBCO- maleimide is connected to preparation DBCO- peptide, azido on polypeptide cysteine side chain thiol
The end 5' or 3' that chemical method is covalently attached to oligonucleotides prepares azido oligonucleotides, DBCO- peptide and azido few nucleosides
Acid is purified by high performance liquid chromatography.The double-strand of high performance liquid chromatography verifying azido oligonucleotides as shown in figure 13
In antisense strand purity be 91.0%, molecular weight 6457.1, high performance liquid chromatography as shown in figure 14 verifying azido oligonucleotides
Positive-sense strand both ends in double-strand are respectively coupled azido and fluorescein base, purity 87.7%, molecular weight 7444.0, as shown in figure 14
The DBCO- peptide of high performance liquid chromatography verifying after purification, purity 99.30%.
B, reaction prepares: DBCO- peptide 9.4mg being dissolved in the DMSO of 0.600ml and obtains DBCO- peptide solution, this implementation
The concentration of DBCO- peptide solution is 5.70mM in example, and azido oligonucleotides 10.59mg is dissolved in the mixing of the water and DMSO of 5ml
Azido oligonucleotide solution is obtained in liquid, azido oligonucleotide solution concentration is 0.15mM, the present embodiment in the present embodiment
In the mixed liquor of middle water and DMSO the mixed volume ratio of water and DMSO be 1:5, and respectively measurement azido oligonucleotide solution and
The high performance liquid chromatography of DBCO- peptide solution, in the present embodiment, high performance liquid chromatography uses UV detector, the ultraviolet inspection
It surveys device Detection wavelength and is set as 210nm, as shown in figure 16, high performance liquid chromatography measures azido oligonucleotides, i.e. operating time
Point 0, as shown in figure 15, the DBCO- peptide of high performance liquid chromatography verifying after purification, purity 99.30%；
C, coupling reaction: (c1), the amount for taking DBCO- peptide solution are V0It is added to reaction in azido oligonucleotide solution to obtain instead
Answer product, V in the present embodiment0=100 microlitres, wherein the molar ratio of DBCO- peptide and azido oligonucleotides is 1:1.25；(c2),
It determines the position at conjugate peak: reaction product being extracted and sample and carries out efficient liquid phase chromatographic analysis, after determining coupling reaction
The position at the peak of reaction product, as shown in table 3,0 corresponding diagram 16 of operating time point, 1 corresponding diagram 17 of operating time point, the operating time
2 corresponding diagrams 18 of point, 3 corresponding diagram 19 of operating time point, 4 corresponding diagram 20 of operating time point, 5 corresponding diagram 21 of operating time point, operation
6 corresponding diagram 22 of time point, Figure 17 and Figure 16 are compared, and can learn that the peak of reaction product is P3, the area at the peak is 126.8, not
The peak of the azido oligonucleotides positive-sense strand of reaction is P2, and the peak of antisense strand is P1, in operation between when putting 0, the peak of positive-sense strand
Area A0=3099.2, in operation between when putting 1, the area A at the peak of positive-sense strand1=3618.8, as it can be seen that anti-in this example in table 3
Answer it is relatively slow, sample volume error bring influence become apparent from.We select the area 2490.3 at time point 3 as A1, this
When A1/A0=0.8035；(c3), conjugate peak purity is preset as 98% in the present embodiment, gradually stacking reaction: (1) being superimposed for the first time
Reaction: the amount that DBCO- peptide solution is added into reaction product is V1,=100 × 0.8035=80 microlitres,
And 83 microlitres of actual interpolation in operating, extracting sample simultaneously carries out efficient liquid phase chromatographic analysis, the results are shown in Table 3 operating time point
5, the peak area A of this time point P22It is 1593.4, extends reaction time to 24 hours (time point 6), the peak area A of P22It reduces
To 77.9, A2/A0=0.0251, this time point conjugate peak P3 purity reaches 98.10%, more than 95%, without further superposition
Reaction.
Table 3:
Comparative example 1:
In this comparative example, azido oligonucleotides is 0.5 milligram of a single stranded DNA (molecular weight 6460.3), is dissolved in 228 microlitres
DMSO and water, wherein the mixed volume ratio of DMSO and water is 2:1.3.2 milligrams of DBCO- peptide (molecular weight 2492.81) is molten
In 256 microlitres of 50% acetonitrile-water mixed solvents and it is added gradually in azido oligonucleotide solution, until be newly added
DBCO- peptide keeps cloudy state, and 51.6 microlitres are added altogether.Precipitating is removed with centrifugal process.
This comparative example indicates if the successive optimization operation that stacking reaction operating method not of the invention describes, DBCO-
Peptide is added when feeding intake by range estimation estimation.As shown in figure 23, conjugate molecules retain high performance liquid chromatography in chromatography as the result is shown
Time 3.458 is washed out, product purity 82.97%.Still containing the DBCO- peptide not being coupled significantly in final products, when chromatography retains
Between 5.261.
Although the result indicates that common operating method is easy, error is larger.
Comparative example 2:
In this comparative example, azido oligonucleotides is the single stranded RNA (molecular weight without high-efficient liquid phase chromatogram purification
21571) 556 microgram of azido oligonucleotides, is taken to be dissolved in 52 microlitres of DMSO.By 5.6 milligrams of DBCO- peptide (molecular weight 2661.66)
210 microlitres of DMSO are dissolved in, take 4.6 microlitres to be added in azido oligonucleotide solution.
RNA molecule in this comparative example for synthesis is not purified before the coupling reaction, is directly thrown with DBCO- peptide
Material operation, discovery can find product peak, highest peak as shown in figure 24 be exactly be product peak, purity 55.9%.But due to raw material
RNA is impure, causes other lesser peaks that can not determine type, and this impure product must be further purified after having reacted.
Therefore, reactant is before coupling, it is necessary to raising purity as far as possible.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Claims (8)
1. a kind of operating method of expansible synthetic oligonucleotide peptide conjugate, which comprises the following steps:
Prepare azido oligonucleotides and DBCO- peptide and highly purified；
Reaction prepares: DBCO- peptide being dissolved in DMSO and obtains DBCO- peptide solution, azido oligonucleotides is dissolved in water or water
With azido oligonucleotide solution is obtained in the mixed liquor of DMSO；
Coupling reaction: (c1), efficient liquid phase chromatographic analysis is carried out to azido oligonucleotides, the area at starting peak is A0, take
The amount of DBCO- peptide solution is V0It is added in azido oligonucleotide solution reaction and obtains reaction product, wherein DBCO- peptide and folded
The molar ratio of nitrogen base oligonucleotides is 1:1.00-1.25；(c2), it determines the position at conjugate peak: sample is extracted to reaction product
And efficient liquid phase chromatographic analysis is carried out, determine the position at the peak of reaction product after coupling reaction, unreacted azido widow core
The area at the peak of thuja acid is A1；(c3), DBCO- first time stacking reaction: gradually stacking reaction: (1) is added into reaction product
The amount of peptide solution is V1,；(2) second of stacking reaction: the amount for continuing to add DBCO- peptide solution is V2,；(3) n-th stacking reaction: the amount for continuing to add DBCO- peptide solution is Vn,, wherein after each stacking reaction, sample is extracted to product and carries out high performance liquid chromatography
Analysis, obtains the area A at the peak of unreacted azido oligonucleotidesn+1, until An+1No longer reduce, terminates stacking reaction and obtain
Final product.
2. the operating method of the expansible synthetic oligonucleotide peptide conjugate of one kind according to claim 1, feature exist
In: the concentration of the DBCO- peptide solution is in 1-10mM, and the concentration of the azido oligonucleotide solution is in 0.1-1.0mM.
3. the operating method of the expansible synthetic oligonucleotide peptide conjugate of one kind according to claim 1, feature exist
In: the high performance liquid chromatography uses UV detector.
4. the operating method of the expansible synthetic oligonucleotide peptide conjugate of one kind according to claim 3, feature exist
In: the UV detector Detection wavelength is set as 210nm.
5. the operating method of the expansible synthetic oligonucleotide peptide conjugate of one kind according to claim 1, feature exist
In: after step e, carrying out vacuum freeze drying i.e. to final product can be directly used for testing in next step.
6. the operating method of the expansible synthetic oligonucleotide peptide conjugate of one kind according to claim 1, feature exist
In: in stepb, azido oligonucleotides is dissolved in the mixed liquor of water or water and DMSO and obtains concentration in 0.1mM-100mM
Azido oligonucleotide solution, wherein in water and DMSO mixed liquor, the mixed volume ratio of water and DMSO are 1:1-5.
7. the operating method of the expansible synthetic oligonucleotide peptide conjugate of one kind according to claim 6, feature exist
In: in water and DMSO mixed liquor, the mixed volume ratio of water and DMSO are 1:1.
8. according to the operating method of the expansible synthetic oligonucleotide peptide conjugate of one kind described in claim 1, it is characterised in that:
The molar ratio of DBCO- peptide and azido oligonucleotides is 1:1 in step (c1).
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| ANUP M.JAWALEKAR等: "Oligonucleotide Tagging for Copper-Free Click Conjugation", 《MOLECULES》 * |
| 卢奎等: "肽与寡核苷酸缀合物的合成及应用研究进展", 《化学研究》 * |
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