Fusobacterium nucleatum single-stranded DNA aptamer, screening method and kit
By screening single-stranded DNA aptamers for *Fusobacterium nucleatum* using SELEX technology, and combining this with streptavidin magnetic bead assay and marker design, the complexity and high cost of *Fusobacterium nucleatum* detection have been solved, achieving rapid, specific, and highly sensitive detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN UNIV
- Filing Date
- 2022-10-26
- Publication Date
- 2026-05-19
AI Technical Summary
Existing methods for detecting Fusobacterium nucleatum are complex to operate, time-consuming, costly, have a high false positive rate, and require strict environmental conditions, making it difficult to achieve rapid, specific, and highly sensitive detection.
The SELEX technique was used to screen for single-stranded DNA aptamers that specifically recognize *Fusobacterium nucleatum*. Probes for *Fusobacterium nucleatum* single-stranded DNA aptamers were prepared by combining forward and reverse screening with streptavidin magnetic bead method. Specific recognition elements such as FAM, FITC or biotin labels were designed for the detection of *Fusobacterium nucleatum*.
This method enables rapid, specific, and highly sensitive detection of Fusobacterium nucleatum, reducing operational complexity and cost while improving detection accuracy.
Smart Images
Figure CN115820649B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of chemical biology, specifically relating to a single-stranded DNA aptamer that specifically recognizes *Fusobacterium nucleatum*, a screening method for this single-stranded DNA aptamer, an ssDNA aptamer probe that specifically recognizes *Fusobacterium nucleatum*, and a kit for detecting *Fusobacterium nucleatum*. Background Technology
[0002] *Fusobacterium nucleatum* belongs to the genus *Fusobacterium*. Its cells are slender, typically with pinpoint ends. In liquid culture, they appear as filaments and aggregate together. *Fusobacterium nucleatum* is a Gram-negative obligate anaerobic bacterium that parasitizes the oral cavity, upper digestive tract, intestine, genitourinary tract, and soil of humans and animals. Isolated *Fusobacterium nucleatum* species require an anaerobic mixed atmosphere for culture. In research on intestinal-related diseases, scientists have found that *Fusobacterium nucleatum* can accumulate in colorectal cancer tissue and plays a role in various stages of colorectal cancer development and progression, including promoting tumor cell proliferation, tumor immune escape, cancer recurrence, and chemotherapy resistance. Extensive data indicate that *Fusobacterium nucleatum* can serve as a biomarker for colorectal cancer diagnosis and treatment. Therefore, a simple and rapid technique for detecting *Fusobacterium nucleatum* in feces can be developed for the diagnosis of colorectal cancer. The accumulation of *Fusobacterium nucleatum* in colorectal epithelial cells plays an important role in the occurrence, development, and metastasis of colorectal cancer. Gut microbiota are gradually becoming biomarkers for colorectal cancer and have been applied to the diagnosis of colorectal cancer. In the future, gut microbiota will have great applications in intestinal diseases.
[0003] The basic detection method for *Fusobacterium nucleatum* is bacterial culture and identification techniques. As the gold standard for microbial detection, these techniques are widely used clinically. However, *Fusobacterium nucleatum* is a strict anaerobe, requiring sophisticated isolation and culture techniques and environmental conditions, resulting in lengthy operation times, which limits the application of this technique in *Fusobacterium nucleatum* detection. In addition, with the development of biodetection technologies, scientists have designed various molecular biology detection techniques. Relatively well-developed techniques include immunoassay, molecular detection, and amplification product detection methods. Fecal microbiology detection methods typically involve 16S rRNA qPCR or metagenomic sequencing. Although these techniques are relatively mature, they suffer from drawbacks such as false positives, high cost, complex operation, stringent environmental requirements, and high toxicity. Therefore, there is a need to develop a novel, rapid, highly specific, and highly sensitive detection method.
[0004] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is an important in vitro screening technique and tool for nucleic acid aptamers. Its principle involves chemically synthesizing single-stranded random oligonucleotide libraries, typically 20-100 nt in length. The ssDNA library is then bound to a target under appropriate conditions. Unbound ssDNA is removed, and through positive and negative screening with staged selection pressure, amplification is performed for dozens of cycles. After 8-20 rounds of screening, oligonucleotide sequences specifically binding to the target are enriched. This screening technique has yielded many target nucleic acid aptamers, including those for bacteria, viruses, cells, and proteins. The whole-bacterial systematic evolution of ligands by exponential enrichment targets bacteria. By binding the ssDNA library to bacteria, removing unbound and negatively bound ssDNA, and performing multiple rounds of screening, aptamers specifically binding to the target bacteria are finally enriched. Summary of the Invention
[0005] The purpose of this invention is to provide a single-stranded DNA aptamer of Fusobacterium nucleatum and a screening method thereon.
[0006] A first aspect of the present invention provides a single-stranded DNA aptamer that specifically recognizes *Fusobacterium nucleatum*, the nucleotide sequence of which is shown below:
[0007] 5'-TGACTGACGACGACTCCGCX1GX2X3X4GX5AGX6X7X8X9X 10 CGTX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X19X 20 X 21 X 22 X 23 GX 24 X 25 X 26 X 27 X 28 AX 29 X 30 GX 31 X 32 X 33 X 34 X 35 X 36 AGACTGCTCGAGCTG-3'(SEQ ID NO: 1), where,
[0008] X1, X 12 X 21 X 22 X 27 X 31 X 32 X 36 Each can be independently classified as C or T;
[0009] X2, X 11 X 14 X 24 X 25 X 29 X 30 Each can be independently represented as G or T;
[0010] X3, X4, X6, X 13 X 26 X 28 X 33 Each can be independently classified as G or C;
[0011] X5, X 17 Each can be either A or C independently;
[0012] X7, X8, X9, X 10 X 18 X 19 X 34 Each can be either A or T independently;
[0013] X 15 X 16 X 20 X 23 X 35 Each can be either A or G.
[0014] According to a preferred embodiment of the present invention, X1, X4, X5, X6, X 12 X 21 X 28 X 31 X 36 For C; X7, X8, X9, X 15 X 16 X 17 X 18 X 20 X 34 X 35 For A; X2, X3, X 13 X 23 X 24 X 26 X 30 X 33 For G; X 10 X 11 X14 X 19 X 22 X 25 X 27 X 29 X 32 Let T be the value of T.
[0015] That is, the aptamer has the nucleotide sequence shown in SEQ ID NO: 2:
[0016] 5'-TGACTGACGACGACTCCGCCGGGCGCAGCAAATCGTTCGTAAAATACTGGGTGTCATGGCTGAACAGACTGCTCGAGCTG-3' (SEQ ID NO: 2);
[0017] or,
[0018] X1, X2, X7, X8, X9, X 12 X 18 X 21 X 24 X 30 X 31 X 34 X 36 For T; X3, X 13 X 17 X 22 X 26 X 27 X 32 X 33 For C; X4, X6, X 11 X 14 X 15 X 16 X 20 X 25 X 28 X 29 X 35 For G; X5, X 10 X 19 X 23 A;
[0019] That is, the aptamer has the nucleotide sequence shown in SEQ ID NO: 3:
[0020] 5'-TGACTGACGACGACTCCGCTGTCGGAAGGTTTACGTGTCGGGCTAGTCAGTGCCGAGTGTCCTGTAGACTGCTCGAGCTG-3' (SEQ ID NO: 3).
[0021] A second aspect of the present invention provides a method for screening the aforementioned Fusobacterium nucleatum single-stranded DNA aptamers, the screening principle of which is as follows: Figure 1 As shown, it includes the following steps:
[0022] (1) Culture and treatment of Fusobacterium nucleatum: Fusobacterium nucleatum was cultured overnight in a mixed gas anaerobic incubator. After the culture was completed, the supernatant was discarded by centrifugation and the bacteria were washed with buffer.
[0023] (2) Filtering, including positive filtering and optional reverse filtering:
[0024] Forward screening includes: dissolving a random DNA library in buffer, denaturing it at high temperature, and then in an ice bath. It is then mixed with the Fusobacterium nucleatum obtained in step (1) and bound at room temperature. After binding, the supernatant containing unbound ssDNA is removed by centrifugation. The supernatant is washed with buffer and sterile water and then centrifuged to remove the supernatant. The supernatant is then resuspended in sterile water, denatured at high temperature, in an ice bath, and centrifuged to retain the supernatant containing ssDNA bound to the bacteria.
[0025] The sequence of the DNA random library is as follows:
[0026] 5'-TGACTGACGACGACTC-N50-GACTGCTCGAGCTG-3' (SEQ ID NO: 4), where N represents any one of the bases A, T, G, or C;
[0027] Reverse screening includes: before performing forward screening, incubating the DNA random library solution after ice bath with E. coli, centrifuging and collecting the supernatant, then incubating the supernatant with Lactobacillus rhamnosus, centrifuging and collecting the supernatant;
[0028] (3) Amplification and verification: Take the supernatant from step (2) for PCR amplification and verify it by agarose gel electrophoresis; extract DNA from the PCR system by ethanol precipitation.
[0029] (4) Next round of library preparation: Single-stranded DNA was prepared using the streptavidin magnetic bead method as the next round of library preparation;
[0030] (5) Repeated screening and sequencing: First, perform multiple rounds of forward screening, then perform multiple rounds of reverse screening + forward screening, and finally perform high-throughput sequencing on the results of the last round to obtain single-stranded DNA aptamers of Fusobacterium nucleatum.
[0031] According to a preferred embodiment of the present invention, in step (2), the high-temperature denaturation conditions are denaturation at 93-97°C for 8-12 minutes, ice bath time is 8-12 minutes, room temperature binding time is 40-60 minutes, and centrifugation conditions are centrifugation at 6000-10000 rpm / min for 8-12 minutes.
[0032] According to a preferred embodiment of the present invention, in step (3), PCR amplification is performed using biotin-labeled downstream primers to obtain biotin-labeled PCR products.
[0033] More specifically, the upstream primer used for PCR amplification was 5'-TGACTGACGACGACTC-3' (SEQ ID NO: 5); the downstream primer was 5'-CAGCTCGAGCAGTC-3' (SEQ ID NO: 6), and the 5' end was modified with biotin.
[0034] According to a preferred embodiment of the present invention, in step (4), the preparation of single-stranded DNA using the streptavidin magnetic bead method includes: taking streptavidin magnetic bead solution, washing it with PBS, adding biotin-labeled PCR product, incubating at room temperature, magnetically separating and discarding the supernatant, washing it with PBS, adding NaOH solution for incubation, and then magnetically separating and collecting the supernatant.
[0035] According to a preferred embodiment of the present invention, in step (5), rounds 1-5 use forward screening, rounds 6-13 use reverse screening + forward screening, and finally the results of round 13 are subjected to high-throughput sequencing.
[0036] A third aspect of the present invention provides an ssDNA aptamer probe that specifically recognizes Fusobacterium nucleatum, wherein the ssDNA aptamer probe is the DNA aptamer with a detection signal group or marker modified or coupled to its 5' or 3' end.
[0037] By modifying or coupling with detection signal groups or markers, the resulting ssDNA aptamers can serve as low-cost, easy-to-use, highly specific, and sensitive recognition elements for Fusobacterium nucleatum. Specifically, the detection signal groups include, but are not limited to, FAM, FITC, or biotin.
[0038] A fourth aspect of the present invention provides a kit for detecting *Fusobacterium nucleatum*, the kit comprising the aforementioned ssDNA aptamer probe specifically recognizing *Fusobacterium nucleatum*, and a 1× binding buffer, wherein the 1× binding buffer comprises: Tris: 10-30 mM, MgCl2: 4-6 mM, NaCl: 80-120 nM, KCl: 4-6 mM, pH = 7.2-7.6. Specifically, the 1× binding buffer comprises: Tris: 20 mM, MgCl2: 5 mM, NaCl: 100 nM, KCl: 5 mM, pH = 7.4.
[0039] This invention targets *Fusobacterium nucleatum*, using *Lactobacillus rhamnosus* and *Escherichia coli* as reverse screening bacteria. After 13 rounds of repeated screening, high-throughput sequencing, and affinity and specificity analyses, two aptamers, Apt-S-4 and Apt-S-5, were finally obtained. The aptamers obtained in this invention are characterized by simple operation, high sensitivity, low production cost, and strong specificity, and have broad application prospects in the detection of *Fusobacterium nucleatum*.
[0040] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description
[0041] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.
[0042] Figure 1 The diagram shown illustrates the principle of screening nucleated Fusobacterium nucleic acid aptamers based on Whole-bacteria-SELEX technology.
[0043] Figure 2 The figure shows the affinity saturation binding curves of the Fusobacterium nucleatum-specific nucleic acid aptamers Apt-S-4 and Apt-S-5.
[0044] Figure 3 The image shows the Kd values and secondary structure diagrams of the Fusobacterium nucleatum-specific nucleic acid aptamers Apt-S-4 and Apt-S-5.
[0045] Figure 4 The diagram shows the specificity of the Fusobacterium nucleatum-specific nucleic acid aptamers Apt-S-4 and Apt-S-5. Detailed Implementation
[0046] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.
[0047] This invention targets *Fusobacterium nucleatum* and uses SELEX technology to screen for nucleic acid aptamers that can specifically recognize *Fusobacterium nucleatum*. After PCR and sequencing, the affinity and specificity of the nucleic acid aptamers were analyzed and verified.
[0048] Example 1
[0049] In vitro screening of nucleic acid aptamers for Fusobacterium nucleatum
[0050] Design and synthesis of random ssDNA libraries and primers: An 80 nt random ssDNA library was designed, with fixed primer sequences at both ends and a 50 nt random sequence in the middle. The library and primers were synthesized by Shanghai Sangon Biotech Co., Ltd.
[0051] Library and primer sequences:
[0052]
[0053]
[0054] (1) Culture of Fusobacterium nucleatum: Fusobacterium nucleatum was inoculated into minced meat liquid culture medium and cultured overnight in an anaerobic incubator filled with mixed gas. After thorough mixing, the OD value was measured, and the supernatant was discarded after centrifugation. The mixture was washed three times with 1× binding buffer (composition: Tris: 20mM, MgCl2: 5mM, NaCl: 100nM, KCl: 5mM, pH=7.4).
[0055] (2) In vitro screening:
[0056] Forward selection: Dissolve the ssDNA library in 1× binding buffer, denature at 95°C for 10 minutes, and immediately incubate on ice for 10 minutes. Resuspend the ssDNA solution with the bacteria (washed three times), and bring the volume to 500 μL. Incubate at room temperature for 45 minutes using a four-way spiral mixer. After incubation, centrifuge at 4500 rpm for 5 minutes, remove the supernatant containing unbound ssDNA, wash three times, and resuspend in 200 μL of sterile water. Denature the bacterial culture at 95°C for 10 minutes, immediately incubate on ice for 10 minutes, centrifuge at 8000 rpm for 10 minutes, and retain the supernatant containing ssDNA bound to the bacteria.
[0057] Reverse screening: In the initial stage of this round, the ssDNA solution is incubated with E. coli, centrifuged at 4500 rpm / min to collect the supernatant, and then incubated with Lactobacillus rhamnosus, centrifuged to collect the supernatant and then performed forward screening.
[0058] (3) PCR amplification: The ssDNA obtained in each round was subjected to conventional PCR. The PCR system is shown in Table 1. Amplification conditions: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 s, 56.4℃ annealing for 30 s, 72℃ extension for 45 s, for 12 cycles, 72℃ extension for 10 min, and finally maintained at 4℃.
[0059] Table 1 PCR system
[0060]
[0061]
[0062] DNA was precipitated from the PCR product using ethanol precipitation.
[0063] a. Add 1 / 10 volume of 3M sodium acetate solution (pH 5.2) to the PCR product and mix thoroughly.
[0064] b. Add 4 μL of Dr. GenTLE Precipitation Carrier and mix thoroughly.
[0065] c. Add 2.5 times the volume of pre-cooled anhydrous ethanol and mix thoroughly.
[0066] Centrifuge at 15,000 rpm at 4°C for 15 minutes. Discard the supernatant and retain the white precipitate.
[0067] e. Add 70% pre-cooled ethanol and centrifuge at 15,000 rpm at 4°C for 5 minutes.
[0068] f. Discard the supernatant, allow the precipitate to air dry for 10 minutes, then dissolve it in sterile water and use it directly as the library for the next round of screening.
[0069] (4) Next round of library preparation: The complementary strand of the desired sequence was removed using streptavidin magnetic beads. 200 μL of streptavidin magnetic bead solution was washed twice with 1×PBS (pH 8.0), and 200 pmol of biotin-labeled PCR product was added. The mixture was incubated at room temperature for 1 minute. The supernatant was discarded after magnetic separation. The mixture was washed three times with PBS, and 200 μL of freshly prepared 0.15 M NaOH solution was added. The mixture was incubated at 37°C for 15 minutes, and the supernatant was collected after magnetic separation. The prepared single-stranded DNA was used as the library for the next round of screening.
[0070] (5) Repeated screening and sequencing: First, perform multiple rounds of forward screening, then perform multiple rounds of reverse screening + forward screening, and perform high-throughput sequencing on the results of the last round to obtain single-stranded DNA aptamers of Fusobacterium nucleatum. The screening conditions for each round are shown in Table 2.
[0071] Table 2
[0072]
[0073] The DNA obtained from the 13th round of screening was amplified by PCR and sent to Shanghai Sangon Biotech Co., Ltd. for high-throughput sequencing. Based on the sequencing results, sequences with higher abundance were selected for further verification.
[0074] Example 2
[0075] Fluorescence analysis was used to determine the dissociation constant Kd of the aptamer, and software was used to predict the secondary structure and ΔG of candidate aptamers.
[0076] FAM-labeled aptamers with different concentration gradients were set up and washed with 2×10⁻⁶ phosphate groups. 8 CFU was incubated at room temperature for 45 minutes, washed three times, and 200 μL of 1× binding buffer was added. The mixture was thoroughly mixed, and the entire liquid was transferred to a black 96-well plate. Fluorescence intensity was measured using a multi-mode microplate reader, and the relative fluorescence intensity F was calculated (F = F1 - F0, where F1 is the fluorescence signal output value of the test sample, and F0 is the fluorescence signal output value of the blank control). Nonlinear regression analysis of the measured relative fluorescence intensity was performed using GraphPad 9 software, and the results were plotted as shown below. Figure 2 The saturation binding curves were obtained, and the Kd value of the aptamer was calculated using the formula Y = Bmax × (Kd + X) (where Y is the fluorescence intensity, Bmax is the maximum relative fluorescence intensity of the system, and X is the aptamer concentration). The measured Kd values for Apt-S-4 and Apt-S-5 were 37.97 ± 0.5206 nM and 23.25 ± 0.3572 nM, respectively. Figure 3 The secondary structure and ΔG of the aptamers were predicted using Mfold software. The ΔG values for aptamers Apt-S-4 and Apt-S-5 were -3.05 kcal·mol⁻¹. -1 -6.89 kcal·mol -1 .
[0077] Example 3
[0078] Aptamer specificity
[0079] FAM-aptamers at a final concentration of 100 nM were incubated with *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, *Escherichia coli*, *Lactobacillus mucosae*, *C. moraceae*, *Lactobacillus rhamnosus*, and a mixed bacterial strain washed with binding buffer at room temperature for 45 minutes. After three washes, fluorescence intensity was measured using a multi-functional microplate reader, and relative fluorescence intensity was calculated and plotted. The results are shown below. Figure 4 As shown, the aptamers obtained by screening in this invention have high specificity for Fusobacterium nucleatum.
[0080] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.
Claims
1. A single-stranded DNA aptamer that specifically recognizes Fusobacterium nucleatum, wherein the nucleotide sequence of the aptamer is the nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of the aptamer is the nucleotide sequence shown in SEQ ID NO:
3.
2. An ssDNA aptamer probe that specifically recognizes Fusobacterium nucleatum, wherein the ssDNA aptamer probe is the DNA aptamer of claim 1 that is modified or coupled with a detection signal group or marker at its 5' or 3' end.
3. The ssDNA aptamer probe for specifically recognizing *Fusobacterium nucleatum* according to claim 2, wherein, The detection signal group is FAM, FITC or biotin.
4. A kit for detecting Fusobacterium nucleatum, characterized in that, The kit comprises: the ssDNA aptamer probe for specifically recognizing Fusobacterium nucleatum as described in claim 2 or 3, and a 1× binding buffer, wherein the 1× binding buffer comprises: Tris: 10-30 mM, MgCl2: 4-6 mM, NaCl: 80-120 nM, KCl: 4-6 mM, pH=7.2-7.6.