CN108498806A - A kind of antisense peptide nucleic acid-DNA tetrahedron carrier complexes and its preparation method and application - Google Patents
A kind of antisense peptide nucleic acid-DNA tetrahedron carrier complexes and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of antisense peptide nucleic acid DNA tetrahedron carrier complexes and its preparation method and application.The carrier complexes include the antisense peptide nucleic acid that MRSA can be inhibited to grow and DNA tetrahedrons, and the antisense peptide nucleic acid and the tetrahedral molar ratios of DNA are 1:1.DNA tetrahedrons are combined by the present invention with antisense peptide nucleic acid, on the basis of reservation both original advantageous properties, it combines and forms a kind of novel membrane carrier, solves the problems, such as that the unmodified antisense peptide nucleic acid that current antisense peptide nucleic acid faces in the application influences its cellular uptake rate；The specific new cell target position of the compound simultaneously has entirely different antibacterial mechanisms with traditional antibacterials, and height drug-fast bacteria MRSA can successfully absorb compound of the present invention, and the compound can effectively inhibit the growth of MRSA；In addition, the acellular specificity of compound produced by the present invention, also without toxicity.
Description
Technical field
The invention belongs to medicine-carried system technical fields, and in particular to a kind of antisense peptide nucleic acid-DNA tetrahedron carrier complexes
And its preparation method and application.
Background technology
DNA tetrahedrons (TDNs) are a kind of nano materials haveing excellent performance, and nontoxicity has good biocompatibility and life
Biodegradable, excellent permeability of cell membrane especially make it have powerful application latent with accurately structural controllability
Power.Currently, cause widely to pay attention in biomedical sector, especially as a kind of carrier of good properties.TDNs is by four
Single stranded DNA (ssDNA) S1, S2, S3, the S4 for the nonsense sequence that item was specifically designed has three-dimensional structure by what is be self-assembly of
DNA nano materials, four single stranded sequences follow " base pair complementarity principle ", and synthetic method is simple, and yield is high.It has sent out at present
Now simple TDNs structures can enter living cells across cell membrane, and with stem cells hyperplasia is promoted, maintain cellular morphology
Effect, can be used as a kind of carrier, and no cytotoxicity.Antisense peptide nucleic acid is a kind of artificial synthesized DNA analogs, can root
According to the expression of the suppressor of base pair complementarity principle specificity.Since it is electroneutral, and phosphate group is not present, so
There is no electrically repel each other the phenomenon that, cause it is stronger with DNA/RNA binding abilities, not by nuclease and protease hydrolytic, to keep away
The phenomenon that n DNA and RNA oligonucleotide may lead to many undershooting-effects as targeted probes is exempted from.
Methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus,
MRSA), the bacterium source is in BEIJING CHAO-YANG HOSPITAL, the clinical trial strain that number is 18908, which is clinically common poison
The stronger bacterium of property is a kind of clinically common infection germ with inhomogenous drug resistance and wide spectrum drug resistance.This
Clinical infection treatment failure rate caused by class bacterial strain is higher, is referred to as " superbacteria ".
In current research, the target of the drug for the treatment of MRSA infection is mostly some common cell factors, is such as sticked
Sex factor influences Cell wall synthesis factor etc., the drug feature using this kind of target be easy to make its to generate drug resistance, so
It needs from new angle Selection drug target.In addition, antisense peptide nucleic acid (asPNA) is used as a kind of uncharged DNA classes
Like object, no image of Buddha nucleic acid equally enters cell by endocytosis, so must be transported by carrier.And it is commonly used in current research
Carrier be cell-penetrating peptide, such carrier is usually to wear film effect in Gram-negative bacteria good, and in gram-positive bacteria
Middle effect is slightly poor, and there are stronger cytotoxicities, can influence the biological behaviour of normal zooblast.
Invention content
For the above-mentioned problems in the prior art, it is multiple that the present invention provides a kind of antisense peptide nucleic acid-DNA tetrahedron carriers
Close object and its preparation method and application, which can effectively solve existing carrier has there are stronger cytotoxicity and use
The problem of limit.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is：
A kind of antisense peptide nucleic acid-DNA tetrahedron carrier complexes, including can inhibit MRSA grow antisense peptide nucleic acid and
DNA tetrahedrons, antisense peptide nucleic acid and the tetrahedral molar ratios of DNA are 1:1；Wherein, four single-stranded nucleotides sequences of DNA tetrahedrons
Row are shown in SEQ ID NO:1-4.
Further, it is ftsz-asPNA that can inhibit the antisense peptide nucleic acid that MRSA is grown, and nucleotide sequence is shown in SEQ ID
NO:5。
The preparation method of above-mentioned antisense peptide nucleic acid-DNA tetrahedron carrier complexes, includes the following steps：By antisense peptide core
Every in single-stranded of acid and tetrahedral four of DNA is single-stranded to be mixed by identical mole, mixed then in addition TM buffer
It is even to be placed in PCR instrument, temperature is raised to 95 DEG C of maintenance 10min, 4 DEG C of maintenance 20min is cooled to, is made.
Further, the pH value of TM buffer is 8.0, by 10mM Tris-HCl and 50mM MgCl2Composition.
Application of the above-mentioned antisense peptide nucleic acid-DNA tetrahedrons carrier complexes in preparing the drug for inhibiting MRSA infection,
A concentration of 250-750nM of compound, preferred concentration 750nM.
A kind of antisense peptide nucleic acid-DNA tetrahedron carrier complexes provided by the invention and its preparation method and application, have
Following advantageous effect：
DNA tetrahedrons are combined by the present invention with antisense peptide nucleic acid, on the basis for retaining the original advantageous property of both
On, in conjunction with a kind of novel membrane carrier is formd, solve current antisense peptide nucleic acid face in the application it is unmodified
Antisense peptide nucleic acid the problem of influencing its cellular uptake rate；The specific new cell target position of the compound simultaneously, with traditional antimicrobial
There are object entirely different antibacterial mechanisms, height drug-fast bacteria MRSA can successfully absorb compound of the present invention, and the compound
The growth of MRSA can effectively be inhibited；In addition, the acellular specificity of compound produced by the present invention, also without toxicity, therefore, this hair
Bright compound has good application prospect to treatment MRSA infection.
Description of the drawings
Fig. 1 is the structure flow chart for the tetrahedron carrier system (P-TDNs) for carrying PNA.
Fig. 2 is polyacrylamide gel electrophoresis result figure.
Fig. 3 is the particle size result figure that atomic force microscope verifies P-TDNs.
Fig. 4 is the form result figure that transmission electron microscope verifies P-TDNs.
Fig. 5 is the particle size results figure of ftsz-asPNA and P-TDNs.
Fig. 6 is the potential test result figure of ftsz-asPNA, ssDNA (S1) and P-TDNs.
Fig. 7 is the intake result figure that fluorescent tracing location technology detects MRSA to P-TDNs.
Fig. 8 is the intake result figure that flow cytometry examines MRSA to P-TDNs.
Fig. 9 is the result for inhibiting MRSA growths using turbidimetry for Determination P-TDNs.
Figure 10 is growth inhibition ratio result figures of the P-TDNs to MRSA.
Figure 11 is the growth curve chart of MRSA after P-TDNs is added.
Figure 12 is that Q-PCR detects ftsz genes and the downstream result of variations of related gene.
Specific implementation mode
Embodiment 1 carries the structure of the tetrahedron carrier system (P-TDNs) of PNA
1, building process
Every ssDNA single-stranded (S1, S2, S3, S4) and ftsz-asPNA is single-stranded to be contained with the addition of same molar ratio concentration
TM buffer(10mM Tris-HCl,50mM MgCl2, pH 8.0) EP pipes in, vortex mixing, centrifugation, be subsequently placed in PCR
In instrument, temperature is quickly risen into 95 DEG C of maintenance 10min, then fast cooling synthesizes P-TDNs, concentration is about to 4 DEG C of maintenance 20min
For 1000nM, building process is as shown in Figure 1.
Wherein, four single-stranded particular sequences of DNA are as follows：
S1：
5’-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3’
(SEQ ID NO:1)；
S2：
5’-CACGATTCAGACTTAGGAATGTTCGACATGCGAGGGTCCAATACCGACGAT-3’(SEQ ID NO:
2)；
S3：
5’-ACTACTATGGCGGGTGATAAAACGTGACTATGTTTGAAATCGACGGGAAGAGCATGCCCATCC-3’
(SEQ ID NO:3)；
S4：
5’-ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG-3’
(SEQ ID NO:4)；
Ftsz-asPNA sequences：5’-TTCAAACATAGT-3’(SEQ ID NO:5).
2, it identifies and characterizes
(1) molecular weight and synthesis situation of polyacrylamide gel electrophoresis (PAGE) verification P-TDNs are used
1. preparing polyacrylamide gel；
2. loading and electrophoresis：6 × loading of 1ul buffer are uniformly mixed with the sample of 5ul and marker respectively,
Then it is added in corresponding electrophoresis tank, electrophoresis 1h under the conditions of constant pressure is 100V；
3. GelRed is dyed and exposure：Polyacrylamide gel is positioned over GelRed and distilled water according to 1:50 ratio
In the mixed liquor of example mixing, it is protected from light, then shaking table 15-25min exposes, the result is shown in Fig. 2.
In Fig. 2, swimming lane 1-10 be respectively P-TDNs, P-TDNs, S2+S3+S4+asPNA, S3+asPNA, S2, marker,
S3, S2+S3+asPNA, S2+S3+S4+asPNA and S2+S3+S4+asPNA, molecular weight analyte size from left to right are respectively
420bp, 420bp, 210bp, 60bp, 40bp, 50bp, 130bp, 210bp, 210bp or so, as shown in Figure 2, P-TDNs are successfully closed
At and mostly exist in the form of dimer (size be 420bp or so).
(2) particle size of atomic force microscope (AFM) verification P-TDNs is used
By 20 times and 50 times of sample of dilution, to mediate, then centrifugation drops on silicon chip, after sample drying, microscopy,
The results are shown in Figure 3, and the particle size of P-TDNs is in 8nm or so (green dashed lines labeled).
(3) form of transmission electron microscope verification P-TDNs is used
Copper sheet is taken out, sample is dripped on copper sheet, toasts 5-6min, is repeated 2-3 times, microscopy, the result is shown in Fig. 4.
As shown in Figure 4, the shape of P-TDNs is rendered as subtriangular, and (is Huang there are a certain number of polymers
Color dashed lines labeled).
(4) grain size and Point location detection
Using the grain size of dynamic light scattering detection ftsz-asPNA (ssPNA) and P-TDNs, average grain diameter is respectively
1.138nm, 5.506nm, the results are shown in Figure 5.
Using the current potential of zeta potentiometers detection ftsz-asPNA, ssDNA (S1) and P-TDNs, the results are shown in Figure 6.
3, intake of the methicillin-resistant staphylococcus aureus (MRSA) to P-TDNs
(1) flow cytometry
It is inoculated with methicillin-resistant staphylococcus aureus cell suspension in 96 orifice plates, culture plate is placed in incubator,
In 37 DEG C of preculture 6h, bacterium solution OD values are then measured at 600nm, are inoculated in 24 orifice plates after bacterium solution is then diluted 5 times, then
The P-TDNs that S1 chains are connected with cy5 is added in experimental group, and the concentration for the P-TDNs being added is respectively 750nM, 500nM and 250nM, the moon
Property control group be added without P-TDNs, it is single-stranded that positive controls are added ftsz-asPNA, 37 DEG C, 50r/min, be protected from light under the conditions of train
It supports overnight, collects cell in 2ml EP pipes, 12000r/min centrifuges 5min, abandons supernatant, is washed with PBS, then 12000r/
Min centrifuges 5min, and gained cell is finally transferred in streaming pipe, upper machine testing, testing result by repeated washing centrifugation 2-3 times
See Fig. 7.
As shown in Figure 8, it is successfully absorbed by MRSA with the P-TDNs of cy5 fluorescent markers, and with the increase of P-TDNs concentration,
Uptake ratio increases.
(2) fluorescent tracing location technology
It is inoculated with methicillin-resistant staphylococcus aureus cell suspension in 96 orifice plates, culture plate is placed in incubator,
In 37 DEG C of preculture 6h, bacterium solution OD values are then measured at 600nm, are inoculated in 24 orifice plates after bacterium solution is then diluted 5 times, then
The P-TDNs that S1 chains are connected with cy5 is added in experimental group, and the concentration for the P-TDNs being added is respectively 750nM, 500nM and 250nM, the moon
Property control group be added without P-TDNs, it is single-stranded that positive controls are added ftsz-asPNA, 37 DEG C, 50r/min, be protected from light under the conditions of train
Support overnight, then per the 9 dyes 15min of STYO that 200 μ l are added in hole, then washed with PBS, then 12000r/min from
Heart 5min, repeated washing centrifugation 2-3 times are drawn on the glass slide after the 10 μ l of sample after washing drip to disinfection, covered,
It is detected under laser confocal microscope after bacterium solution drying, testing result is shown in Fig. 7.
It as shown in Figure 8, can be by calculating the Duplication of the P-TDNs of MRSA and cy5 fluorescent markers that STYO 9 is marked
One step card P-TDNs is successfully absorbed by MRSA, and with the increase of P-TDNs concentration, and uptake ratio increases.
4, inhibition of the P-TDNs to MRSA
(1) turbidimetry for Determination bacteriostasis rate
Activate 3 generation of MRSA bacterial strains；
Degerming is filtered to P-TDNs synthesis stoste；
Bacterium solution is prepared：1ml culture mediums are added into 1.5ml sterile tubes, takes the 3rd generation bacterial strain single bacterium colony, is dissolved in physiological saline
In, oscillation mixing is diluted to 1 × 10 with spectrophotometric determination bacterium solution OD values with culture medium4A/ml；
Using 96 orifice plates, 62.5 μ l bacterium solutions are added per hole, the P-TDNs synthesis after 187.5 μ l filtration sterilizations is added in the first hole
P-TDNs synthesis stostes and 62.5 μ l physiological saline after 125 μ l filtration sterilizations are added in stoste, the second hole, and third hole is added 62.5
P-TDNs synthesis stostes after μ l filtration sterilizations and 125 μ l physiological saline, the 4th hole are negative control hole, and positive controls are to add
Entering 187.5 μ l does not have the individual ftsz-asPNA of carrier single-stranded, and then culture plate is put into incubator and cultivates 12h, uses enzyme
The spectrophotometric value that instrument measures every hole is marked, P-TDNs inhibits the result of MRSA growths as shown in Figure 9.It measures and presses down further accordance with formula
Bacterium rate, the results are shown in Figure 10 for inhibiting rate, and bacteriostasis rate formula is as follows：
By Fig. 9 and Figure 10 it is found that with P-TDNs concentration increase, it is stronger to the inhibition of MRSA, when P-TDNs concentration
For 750nM when, it is single-stranded that ftsz-asPNA is substantially better than to the inhibition of MRSA.
(2) measurement of growth curve
Picking MRSA single bacterium colony culture 16h continue to cultivate 2h after diluting 10 times to the bacterium solution after culture, and measuring its OD value is
0.5-0.6；
60 μ l culture mediums will be added in 96 orifice plates per hole, it is former that the P-TDNs synthesis after 180 μ l filtration sterilizations is added in the first hole
P-TDNs synthesis stostes and 60 μ l TMbuffer after 120 μ l filtration sterilizations are added in liquid, the second hole, and 60 μ l mistakes are added in third hole
The synthesis stostes of the P-TDNs after bacterium and 120 μ l TMbuffer, the 4th hole negative control the most are filtered out, positive controls are to be added
180 μ l do not have the individual ftsz-asPNA of carrier single-stranded, and 10 times of dilution bacterium solutions of 2.4ml, upper machine examination are then added in every hole
Growth curve, result are as shown in figure 11 for 24 hours for survey.
As shown in Figure 11, when P-TDNs concentration is higher, the time that MRSA reaches logarithmic phase is longer, and the speed of growth
It is the most apparent to the inhibition of MRSA especially as a concentration of 750nM of P-TDNs significantly lower than positive controls.
(3) verification P-TDNs inhibits the mechanism of action of MRSA growths
1. Q-PCR detects ftsz genes and the variation of related gene downstream
Picking MRSA single bacterium colony culture 16h continue to cultivate 2h after diluting 10 times to the bacterium solution after culture, and measuring its OD value is
0.5-0.6；
60 μ l culture mediums will be added in 96 orifice plates per hole, it is former that the P-TDNs synthesis after 180 μ l filtration sterilizations is added in the first hole
P-TDNs synthesis stostes and 60 μ l TMbuffer after 120 μ l filtration sterilizations are added in liquid, the second hole, and 60 μ l mistakes are added in third hole
The synthesis stostes of the P-TDNs after bacterium and 120 μ l TMbuffer, the 4th hole negative control the most are filtered out, positive controls are to be added
180 μ l do not have the individual ftsz-asPNA of carrier single-stranded, and 10 times of dilution bacterium solutions of 2.4ml are then added in every hole, are placed in 37
Overnight incubation in DEG C incubator.
Bacterium after culture is used into liquid nitrogen wall-breaking machine broken wall, gene extracts kit extraction gene is reused, passes through height
Pure total serum IgE rapid extraction kit and Reverse Transcriptase kit, which obtain, stablizes cDNA.
Q-PCR：Reaction system per hole 20ul (draw by 2ul cDNA, 10ul SYBR, 0.8ul primers Fs orward, 0.8ul
Object Reserve, 6.4ul ddH2O), product is finally subjected to gel electrophoresis, and analyze ftsz genes and downstream dependency basis
The variation of cause, the result is shown in Figure 12.
As shown in Figure 12, respectively using common gene 16s RNA and staphylococcus aureus specific gene Sa442 in
Ginseng gene is compareed with target gene ftsz, it can be seen that after the P-TDNs of various concentration processing, the expression of ftsz genes is equal
A degree of inhibition is received, and with the increase inhibiting effect bigger of concentration, while also demonstrating the material there is targeting
The effect of ftsz genes.
Sequence table
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Claims (7)
1. a kind of antisense peptide nucleic acid-DNA tetrahedron carrier complexes, which is characterized in that including the antisense that MRSA can be inhibited to grow
Peptide nucleic acid and DNA tetrahedrons, the antisense peptide nucleic acid and the tetrahedral molar ratios of DNA are 1:1；Wherein, four lists of DNA tetrahedrons
The nucleotide sequence of chain is shown in SEQ ID NO:1-4.
2. antisense peptide nucleic acid-DNA tetrahedron carrier complexes according to claim 1, which is characterized in that MRSA can be inhibited
The antisense peptide nucleic acid of growth is ftsz-asPNA, and nucleotide sequence is shown in SEQ ID NO:5.
3. the preparation method of antisense peptide nucleic acid-DNA tetrahedron carrier complexes as claimed in claim 1 or 2, which is characterized in that
Include the following steps：It is mixed by identical mole by every of tetrahedral four of antisense peptide nucleic acid and DNA in single-stranded is single-stranded,
Then it is added in TM buffer, mixing is placed in PCR instrument, and temperature is raised to 95 DEG C of maintenance 10min, is cooled to 4 DEG C of maintenances
20min is made.
4. the preparation method of antisense peptide nucleic acid-DNA tetrahedron carrier complexes according to claim 3, which is characterized in that
The pH value of TM buffer is 8.0, by 10mM Tris-HCl and 50mM MgCl2Composition.
5. antisense peptide nucleic acid-DNA tetrahedrons carrier complexes as claimed in claim 1 or 2 inhibit the medicine of MRSA infection preparing
Application in object.
6. antisense peptide nucleic acid-DNA tetrahedrons carrier complexes according to claim 5 inhibit the medicine of MRSA infection preparing
Application in object, which is characterized in that a concentration of 250-750nM of the compound.
7. antisense peptide nucleic acid-DNA tetrahedrons carrier complexes according to claim 6 inhibit the medicine of MRSA infection preparing
Application in object, which is characterized in that a concentration of 750nM of the compound.
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| CN112592922A (en) * | 2020-02-19 | 2021-04-02 | 四川大学 | DNA tetrahedral complex for inhibiting growth of bacterial biofilm |
| CN116694625A (en) * | 2023-06-26 | 2023-09-05 | 中国人民解放军空军军医大学 | DNA tetrahedron and its complex and use |
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| CN110917121B (en) * | 2019-12-11 | 2021-01-05 | 四川大学 | APD hybrid nano system and construction method and application thereof |
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| CN1387567A (en) * | 1998-11-11 | 2002-12-25 | 潘特科有限公司 | Conjugates between peptides and nucleic acid analog, such as PNA, LNA or morpholino |
| EP2118306B1 (en) * | 2006-12-29 | 2013-02-13 | Roche Diagnostics GmbH | Methods for detecting methicillin-resistant s. aureus as well as primers, probes and kits for the same |
| CN101200483B (en) * | 2007-05-11 | 2011-01-12 | 中国人民解放军第四军医大学 | Antisense nucleic acid of resistant and tolerant dimethoxyphenecillin staphylococcus aureus drug resistant gene |
| CN101457222B (en) * | 2007-12-14 | 2011-12-21 | 李宝健 | Double-chain small molecule interference nucleic acid for inhibiting and killing drug tolerant bacteria and composition thereof |
| CN101891804B (en) * | 2010-06-21 | 2012-12-26 | 中国人民解放军第四军医大学 | Antisense peptide nucleic acid of cell penetrating peptide-mediated antibacterial RNA polymerase sigma 70 factor gene rpoD |
| CN106497919A (en) * | 2016-11-02 | 2017-03-15 | 四川大学 | A kind of DNA tetrahedrons of aptamer AS1411 modifications and preparation method thereof |
| CN107805643B (en) * | 2017-09-15 | 2020-12-08 | 四川大学 | siRNA-DNA nano system for targeted inhibition of salmonella drug-resistant efflux pump gene acrA and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109793750A (en) * | 2019-03-27 | 2019-05-24 | 四川大学 | A kind of anti-tumor nano drug |
| CN112592922A (en) * | 2020-02-19 | 2021-04-02 | 四川大学 | DNA tetrahedral complex for inhibiting growth of bacterial biofilm |
| CN112592922B (en) * | 2020-02-19 | 2023-02-07 | 四川大学 | DNA tetrahedral compound for inhibiting growth of bacterial biofilm |
| CN116694625A (en) * | 2023-06-26 | 2023-09-05 | 中国人民解放军空军军医大学 | DNA tetrahedron and its complex and use |
| CN116694625B (en) * | 2023-06-26 | 2024-01-02 | 中国人民解放军空军军医大学 | DNA tetrahedron and its complex and use |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110368399A (en) | 2019-10-25 |
| CN110368399B (en) | 2023-04-11 |
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