DNA tetrahedral complex for inhibiting growth of bacterial biofilm
阅读说明：本技术 一种抑制细菌生物膜生长的dna四面体复合物 (DNA tetrahedral complex for inhibiting growth of bacterial biofilm ) 是由 林云锋 张雨欣 谢雪萍 马文娟 战雨汐 于 2020-02-19 设计创作，主要内容包括：本发明公开了一种抑制细菌生物膜生长的DNA四面体复合物,属于载药体系技术领域。本发明的DNA四面体复合物是由一种兼并寡核苷酸通过共价键连接在DNA四面体上形成的复合物。所述兼并寡核苷酸具有如下序列：DTWACANNNNNDTAACA；其中D为碱基A或G；W为碱基A或T；N为碱基A、T、C或G。本发明的DNA四面体复合物易于被细菌摄取,可有效抑制细菌胞外多糖的形成,从而显著抑制细菌生物膜的生长。本发明的复合物可应用于降低细菌抗逆性,提高抗生素灭菌的效果；还有望应用于局部慢性感染的辅助治疗,提高抗感染疗效。(The invention discloses a DNA tetrahedral complex for inhibiting growth of a bacterial biofilm, belonging to the technical field of a drug-loading system. The DNA tetrahedral complex of the invention is a complex formed by connecting a degenerate oligonucleotide to a DNA tetrahedron through a covalent bond. The degenerate oligonucleotide has the following sequence: DTWACANNNNNDTAACA, respectively; wherein D is a base A or G; w is a base A or T; n is base A, T, C or G. The DNA tetrahedral complex is easy to be absorbed by bacteria, and can effectively inhibit the formation of extracellular polysaccharide of the bacteria, thereby obviously inhibiting the growth of bacterial biofilms. The compound can be applied to reducing the stress resistance of bacteria and improving the effect of antibiotic sterilization; it is also expected to be applied to the adjuvant treatment of local chronic infection and improve the anti-infection effect.)
1. A degenerate oligonucleotide, comprising: it has the following sequence:
DTWACANNNNNDTAACA；
wherein D is a base A or G; w is a base A or T; n is base A, T, C or G.
2. The degenerate oligonucleotide of claim 1, wherein: the degenerate oligonucleotides are mixed in equimolar ratios of oligonucleotides of different sequences.
3. A DNA tetrahedral complex, characterized by: a complex comprising the degenerate oligonucleotide of claim 1 or 2 covalently linked to a DNA tetrahedron.
4. The composite of claim 3, wherein: the DNA tetrahedron is formed by complementary base pairing of DNA single chains with sequences shown as SEQIDNO.1-4.
5. The composite of claim 4, wherein: the degenerate oligonucleotide is connected to a DNA single strand with a sequence shown as SEQ ID No.2 through a covalent bond.
6. Use of the oligonucleotide of claim 1 or 2 in the manufacture of a medicament for inhibiting the growth of a bacterial biofilm; the drug is prepared by using the facultative oligonucleotide of claim 1 or 2 as an active ingredient, and adding a carrier capable of transporting the oligonucleotide into bacterial cells.
7. Use according to claim 6, characterized in that: the bacteria are streptococcus mutans.
8. Use of a complex according to any one of claims 3 to 5 in the manufacture of a medicament for inhibiting the growth of a bacterial biofilm.
9. Use according to claim 8, characterized in that: the bacteria are streptococcus mutans.
10. A medicament for inhibiting the growth of bacterial biofilms, comprising: the active ingredient of the compound is the compound as claimed in any one of claims 3 to 5.
11. The medicament of claim 10, wherein: the bacterium is streptococcus mutans.
Technical Field
The invention belongs to the technical field of drug-loaded systems, and particularly relates to a DNA tetrahedral complex for inhibiting the growth of a bacterial biofilm.
Background
Bacterial biofilms (or Bacterial biofilms, BF) refer to a large Bacterial aggregate membrane-like mass formed by bacteria adhering to a contact surface, secreting polysaccharide matrices, fibrin, lipoprotein, etc., and wrapping themselves around them.
Under physiological conditions, the drug resistance of BF bacteria to antibiotics can be improved by 500-fold and 5000-fold compared with planktonic bacteria because: first, the rate of metabolism is reduced due to competition for nutrients and space in the biofilm environment, bacterial growth and proliferation in the biofilm. Secondly, the protein and polysaccharide components in the EPS (extracellular polymeric substance) matrix can prevent and delay the penetration of antibiotics into the biofilm, giving the mature cells located deep in the matrix more time to develop resistance. Third, individual bacteria are resistant and produce antibiotic resistance factors, resulting in resistance of the entire biofilm community; it is called passive resistance, and resistance-associated genes can be shared between bacteria by lateral gene transfer, a mechanism by which genes are exchanged between different strains in a biofilm.
The bacteria in BF are not randomly stacked but are coordinated to each other to form a population having a highly differentiated structure. Besides the improvement of the drug resistance to antibiotics, BF has higher toxicity compared with planktonic bacteria, and the capacity of adapting to the environment (host immunity resistance, acid resistance, hunger resistance and the like) is comprehensively improved.
Microbial biofilm formation is now recognized as a major virulence factor for many local chronic infections. E.g. heart, lung tissue, skin, oral cavity, etc. Conventional therapeutic approaches (e.g., mechanical debridement, antibiotics, biofilm repellents) do not provide adequate therapeutic efficacy due to the self-protective ability and strong toxicity of biofilms, and thus inhibiting biofilm formation is critical to combating biofilm infection.
The DNA tetrahedron (TDNs or tFNAs) is a nano material with excellent performance, has no toxicity, good biocompatibility and biodegradability and excellent cell membrane permeability, and particularly has accurate structural controllability so that the DNA tetrahedron has strong application potential. Currently, the vector has attracted considerable attention in the biomedical field, and is particularly used as a vector with excellent properties. tFNAs is a DNA nano material with a three-dimensional structure formed by self-assembly of four specially designed single-stranded DNA (ssDNA) S1, S2, S3 and S4 of nonsense sequences, and the four single-stranded sequences follow the basic group complementary pairing principle, so that the synthesis method is simple and the yield is high. It has now been found that the pure tFNAs structure can penetrate the cell membrane into the living cells, and has the effects of promoting the proliferation of stem cells and maintaining the morphology of the cells, can be used as a carrier, and has no cytotoxicity.
Disclosure of Invention
The invention aims to solve the technical problem of providing a DNA tetrahedral compound for effectively inhibiting the growth of a streptococcus mutans bacterial biofilm.
In the present invention, "biofilm" is equivalent to "BF" and "bacterial biofilm".
In the present invention, a "degenerate oligonucleotide" refers to a mixture of different oligonucleotides having similar base sequences; the similar base sequence can be represented by a general formula, which is a "degenerate sequence".
The technical scheme of the invention comprises the following steps:
a degenerate oligonucleotide having a sequence of DTWACANNNNNDTAACA; wherein D is a base A or G; w is a base A or T; n is base A, T, C or G.
The degenerate oligonucleotides as described above, wherein the oligonucleotide molecules of different sequences are mixed in equimolar ratios.
A DNA tetrahedral complex, which is a complex formed by connecting the facultative oligonucleotide to a DNA tetrahedron through a covalent bond.
The DNA tetrahedron compound is formed by base complementary pairing of DNA single strands with sequences shown in SEQ ID NO. 1-4.
The facultative oligonucleotide is connected to the DNA single strand with the sequence shown in SEQ ID No.2 through a covalent bond as the DNA tetrahedral complex.
The use of an oligonucleotide as hereinbefore described in the manufacture of a medicament for inhibiting the growth of a bacterial biofilm; the medicine is prepared by taking the Facultative oligonucleotide as an active ingredient and adding a carrier capable of transporting the oligonucleotide into bacterial cells.
For the aforementioned use, the bacterium is streptococcus mutans.
The use of the aforementioned DNA tetrahedral complex in the manufacture of a medicament for inhibiting the growth of bacterial biofilms.
Further, the bacterium is streptococcus mutans.
The invention also provides a medicament for inhibiting the growth of bacterial biofilms, the active ingredient of which is the DNA tetrahedral complex.
Further, the bacterium is streptococcus mutans.
The invention has the following beneficial effects:
1) the oligonucleotide provided by the invention has the capability of targeting multiple genes, and can show obvious capability of inhibiting multiple genes GtfB, GtfC, GtFD, GbpB and Ftf related to exopolysaccharide synthesis after being transported into a bacterial body by using a DNA tetrahedron.
2) The DNA tetrahedral complex of the present invention is easily taken up by bacteria. Experiments prove that compared with single oligonucleotides (ASOs) or DNA tetrahedrons (tFNAs), the capability of entering bacteria of the DNA tetrahedron compound (ASOs-tFNAs) is obviously improved; this lays a solid foundation for the oligonucleotides to play the role of inhibiting the biological membrane.
3) The DNA tetrahedral complex of the invention can effectively inhibit the formation of bacterial biofilms.
It is worth noting that in the ASOs-tFNAs of the invention, tFNAs can improve the inhibition effect on ASOs, and generate the synergistic effect: experiments show that the sum of inhibition effects of tFNAs and ASOs on biofilm formation is far lower than that of ASOs-tFNAs.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows a process for constructing DNA tetrahedral complexes (ASOs-tFNAs) according to the present invention.
FIG. 2 is a graph showing the results of polyacrylamide gel electrophoresis.
FIG. 3 is a graph showing the results of particle size and morphology of tFNAs, ASOs-tFNAs examined by atomic force microscope.
FIG. 4 is a diagram showing the results of examining the morphology of ASOs-tFNAs by transmission electron microscopy.
FIG. 5 is a graph showing the results of particle sizes of tFNAs.
FIG. 6 is a graph showing the results of the particle sizes of ASOs-tFNAs.
FIG. 7 shows the results of statistical analysis of tFNA, ASOs-tFNA particle size and potentiometric analysis.
FIG. 8 is a graph showing the results of examining the intake of ASOs-tFNAs by Streptococcus mutans by flow cytometry.
FIG. 9 is a graph showing the growth of planktonic bacteria after the addition of different concentrations of tFNAs, ASOs-tFNAs.
FIG. 10 is a graph of the crystal violet staining of biofilm and statistics of different concentrations of tFNAs, ASOs-tFNAs after 24, 48h treatment.
FIG. 11 shows the results of 3D scans of different concentrations of tFNAs, ASOs, ASOs-tFNAs treated for 24 h.
FIG. 12 is a scanning electron micrograph of biofilm after treatment with 750nmTFNAs, ASOs, ASOs-tFNAs 24.
FIG. 13 shows the results of changes in the expression of all target genes in the q-PCR detection oligonucleotides.
Detailed Description
EXAMPLE 1 Synthesis of DNA tetrahedral complexes of the invention
1. Preparation method
Each ssDNA single strand (S1, ASOs-S2, S3, S4) contained TM buffer (10mM Tris-HCl, 50mM MgCl) at the same molarity2pH 8.0), vortexing, mixing, centrifuging, placing in a PCR instrument, rapidly increasing the temperature to 95 ℃ for 10min, rapidly decreasing the temperature to 4 ℃ for 20min, and synthesizing ASOs-tFNAs with the concentration of about 1000nM, wherein the construction process is shown in FIG. 1.
The related sequences are shown in Table 1, S1-S4 are ssDNA which is the most basic of DNA tetrahedron, ASOs are degenerate sequences of the antisense oligonucleotide with BF inhibitory activity in the complex of the present invention, and the ASOs are connected with any single strand of S1-S4 (in this example, connected with S2 to form ASOs-S2) through phosphodiester bonds, and can be fixed on the finally synthesized DNA tetrahedron.
TABLE 1 related sequences in the Complex
Note: d is A or G; w is A or T; n is any base; s1, S2, S3, S4 and ASOs respectively correspond to SEQ ID NO. 1-5 in the nucleotide sequence table; in actual experimental procedures, although ASOs are obtained by mixing various sequences (subsequences) having sequences shown as the degenerate sequence DTWACANNNNNDTAACA in Table 1 at equimolar ratios, the ASOs are not necessarily required to be at equimolar ratios in theory and the amounts of ASOs present may be adjusted.
2. Identification
(1) Polyacrylamide gel electrophoresis (PAGE) is adopted to verify the molecular weight and the synthesis condition of ASOs-tFNAs
Preparing polyacrylamide gel;
sample loading and electrophoresis: respectively mixing 1 mu of 16 × loading buffer with 5 mu of sample and marker uniformly, then adding into a corresponding electrophoresis tank, and carrying out electrophoresis for 90min under the condition of constant voltage of 80V;
③ GelRed dyeing and exposure: the polyacrylamide gel was placed in a 1: 50, shaking the mixture for 15-25min in a dark place, and then exposing, the results are shown in FIG. 2.
In FIG. 2, lanes 2-6 are marker, S1, S2, S3, S4, ASOs-S2, and lanes 10-18 are marker, S1, S1+ S2, S1+ ASOs-S2, S1+ S2+ S3, S1+ S3+ ASOs-S2, tFNA, and ASOs-tFNA, the molecular weights of the samples from left to right are 60bp, 40bp, 40bp, 40bp, 80bp, 40bp, 120bp, 180bp, 240bp, 300bp, 450bp, and about 500bp, and the ASOs-tFNA is successfully synthesized and exists in the form of a dimer (about 500bp in size).
(2) Atomic Force Microscope (AFM) was used to verify the particle size of ASOs-tFNAs
Samples diluted 20 and 50 times were vortexed, centrifuged, and dropped onto silicon wafers, and after drying the samples, they were examined under a microscope, as shown in FIG. 3, with particle sizes of tFNAs around 10nm and ASOs-tFNAs around 15nm (circle marks).
(3) Transmission electron microscope is adopted to verify the morphology of ASOs-tFNAs
Taking out the copper sheet, dripping the sample on the copper sheet, baking for 5-6min, repeating for 2-3 times, and performing microscopic examination, wherein the result is shown in FIG. 4.
As can be seen from FIG. 4, the ASOs-tFNAs appear approximately triangular in shape and a certain number of multimers (all marked with dashed lines) are present.
(4) Particle size, zeta potential
The particle sizes of tFNAs and ASOs-tFNA were measured by dynamic light scattering, and the average particle sizes were about 10.58nm and 16.66nm, respectively, as shown in FIGS. 5 and 6.
The potentials of tFNAs, ASOs-tFNA were measured by a zeta-potentiometer, and the results are shown in FIG. 7.
The above results indicate the success of the preparation of ASOs-tFNA.
The ability of the complexes ASOs-tFNAs of the present invention to inhibit biofilm formation is further illustrated below by way of experimental examples. When tFNAs, ASOs-tFNAs and the like are required to be traced in an experimental example, a fluorophore Cy5 is added into any single chain of S1-S4 in advance, and the Cy5-tFNAs and the Cy5-ASOs-tFNAs are obtained after synthesis by the method of the example 1; ASOs can also be directly labeled with Cy5 fluorophore to obtain Cy 5-ASOs.
EXAMPLE 1 intake of ASOs-TDNs by Streptococcus mutans
The 96-well plate was inoculated with a cell suspension of Streptococcus mutans, and the plate was placed in 5% CO2The cells were pre-incubated at 37 ℃ for 6 hours in an incubator, then the OD value of the cells was measured at 595nM, then the cells were diluted 5-fold and inoculated into 24-well plates, and the cells were treated with tFNAs (Cy5-tFNAs, 500,750nM), ASOs (Cy5-ASOs, 500,750nM) and ASOs-tFNAs (Cy5-ASOs-tFNAs, 500,750nM) for 12 hours (5% CO)2Culturing overnight at 37 ℃ in the dark), collecting cells in a 2ml EP tube, centrifuging at 12000r/min for 5min, discarding the supernatant, washing with PBS, centrifuging at 12000r/min for 5min, repeatedly washing and centrifuging for 2-3 times, transferring the obtained cells to a flow tube, and detecting on a computer. The results are shown in FIG. 8.
As can be seen from FIG. 8, tFNAs, ASOs-tFNAs fluorescently labeled with cy5 were successfully ingested by Streptococcus mutans, and ASOs promoted the ingestion of tFNAs.
Experimental example 2 growth test of planktonic bacteria
A single colony of Streptococcus mutans UA159 was picked and grown until early log phase was reached (OD595nm ═ 0.2-0.3). Adjusting the OD value of the bacteria to be consistent. They were co-cultured with different concentrations of tFNAs and ASOs-tFNAs (500nM, 750nM) and incubated with BHI (sucrose-free) in cell culture plates. The growth of planktonic bacteria was measured using an automated spectrophotometer (BioTek, Winooski, VT, usa) and corresponding optical density readings (595nm) were taken within 24 hours; OD595nm values were measured every 2 hours, and the plates were shaken every 30 minutes.
As can be seen from FIG. 9, the addition of tFNAs and ASOs-tFNAs did not significantly differ from each other in the growth of suspended bacteria in the absence of sucrose.
And (4) conclusion: under the condition of no sugar, the ASOs-tFNAs can not influence the growth of bacteria.
Experimental example 3 analysis of biofilm formation
(1) Crystal violet staining of biofilms
To test the inhibitory effect of ASOs-tFNAs on biofilm formation, the experimental strain was inoculated into the wells of a 96-well plate containing BHIS (containing 1% sucrose). Treatment was carried out for 24h and 48h with tFNAs, ASOs, ASO-tFNAs (500, 750nM), respectively. Then, the culture broth was aspirated and washed again with PBS. And 100ul of methanol was added to each well to fix for 15 minutes, and excess liquid was aspirated and naturally dried. 0.1% crystal violet staining solution was added to each well and stained at 20 ℃ for 5 minutes, and examined under a microscope. Then, the staining solution in the wells was sucked up and dried in a drying oven. Finally, 100. mu.l of acetic acid (33%) was added to each well to dissolve the crystal violet staining solution for 30 minutes at 37 ℃. The OD of the eluate sample (OD595nm) was measured with an ultraviolet spectrophotometer.
The results are shown in FIG. 10, and FIGS. 10a and 10b are the results of treatments 24 and 48h, respectively, and it can be seen that pure tFNAs does not affect the formation of bacterial biofilm, and pure ASOs has a certain inhibiting effect but is not obvious; the inhibition effect of ASOs-tFNAs on the biological membrane is very obvious and is several times higher than that of simple ASOs.
(2) Laser confocal microscopy analysis of bacterial biofilm and extracellular polysaccharide distribution
Overnight cultures of the experimental strains were inoculated into BHIS with tFNAs, ASOs and ASOs-tFNA (500, 750nM) for 24 hours. By in situ labelling of S.mutans and EPS, we observed the structure of the bacterial biofilm. Alexa Fluor 647-labeled dextran conjugate (1. mu.M; Thermo Fisher Scientific, MA, USA) was added to BHIS medium prior to inoculation of the strain. After incubation in the cell incubator for 24h, the medium was aspirated and each sample was washed twice with sterile PBS to remove planktonic and loosely bound cells. Then adding SYTOTMThe biofilm was labeled for 15 minutes with a ratio of 100: 1 for 9 dyes (Saimer Fei technology). The structure of bacterial biofilms was examined by confocal laser microscopy (Nikon A1R MP +, japan). We used the Z-section to record the thickness of the biofilm, each layer being 1 μ M thick. Each sample was scanned at five randomly selected positions. Finally, the confocal images are three-dimensional structural images of the biofilm, we used COMSTAT image processing software to analyze the confocal image stack and calculate EPS and fineBiomass of bacterial cells (s.mua 159).
Results as shown in FIG. 11, FIGS. 11a and 11b are the results of 3d layer sweeps after 24 hours of treatment with 500,750 nMASOs-tFNAs, respectively. At a concentration of 500nM, the EPS and Bacteria coverage Ratio (EPS/Bacteria Ratio) was significantly lower in the ASOs-tFNAs-treated group than in the ASOs group; at a concentration of 750nM, the ratio of EPS and bacterial coverage was more than ten times lower in the ASOs-tFNAs treated group than in the ASOs group (area under the curve).
The experiment shows that ASOs-tFNAs have more remarkable biofilm inhibiting ability than ASOs.
(3) Scanning electron microscope for observing form of biological membrane
The influence of ASOs-tFNAs on biofilm structure and EPS content was observed by scanning electron microscopy (FEI, Hillsboro, OR, USA), and Streptococcus mutans was inoculated overnight into cell culture plates with glass coverslips and BHIS medium. After one day of incubation, additional washes with sterile PBS were performed to remove planktonic bacteria and loose cells, each sample was fixed with 2.5% glutaraldehyde overnight at 4 ℃ and then washed once per sample. The bacterial cells were maintained in morphology by washing with sterile PBS and dehydration in a series of absolute ethanol, each sample was coated with gold and observed under SEM.
As shown in FIG. 12, which is the result of scanning electron microscope of the biofilm of Streptococcus mutans treated with 750 nMSOSs-tFNAs for 24h, it is evident that pure tFNAs exists, extracellular polysaccharides (white arrows, spongy pores) exist in the ASOs group, the structure of the biofilm is complete, but the complete biofilm structure cannot be seen in the ASOs-tFNAs experimental group.
And (4) conclusion: the DNA tetrahedral complex of the invention can achieve the effect of inhibiting the formation of the biofilm by inhibiting the production of exopolysaccharides.
Experimental example 4 mechanism for inhibiting exopolysaccharide production
The inventors verified the multiple targeting of ASOs-tFNAs by quantitative RT-PCR, and involved target genes GtfB, GtfC, GtFD, GbpB and Ftf, which are genes related to extracellular polysaccharide synthesis.
The expression of the target gene was quantified using 16S rRNA as a control gene. The strains grew to late log phase in BHIS with tFNA and ASOs-tFNA (750 nM). The strains were then harvested by centrifugation (4000g, 4 ℃, 10 min) and snap frozen in TRIzol reagent (Thermo Fisher Scientific) until needed. Total RNA was extracted from each sample and purified by RNeasy Mini Kit (Hiden, Germany) with genomic DNA remover. The extracted sample was dissolved in RNase-free water. Preparation of cDNA, cDNA Synthesis kit (TaKaRa, China) was used. Amplification of all target mrnas was performed by quantitative RT-PCR. In this experiment, tFNAs was used as a control group.
It can be seen from FIG. 13 that the expression of the target genes was all reduced.
And (4) conclusion: this experiment demonstrates the effectiveness of multiple targeting and the inhibition of biofilm formation by inhibiting the expression of exopolysaccharide-associated genes.
In conclusion, the complexes ASOs-tFNAs of the present invention can be absorbed by Streptococcus mutans, which inhibits the formation of exopolysaccharides in bacteria by inhibiting multiple genes in the latter, thereby inhibiting biofilm formation.
SEQUENCE LISTING
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