Screening and identification of a nucleic acid aptamer that specifically binds to human senescent dermal fibroblasts

Abstract

This invention proposes a nucleic acid aptamer that specifically binds to human senescent dermal fibroblasts (HDFs), along with its screening, identification, and application. Specific aptamers exhibiting high targeting specificity to human senescent HDFs were screened using Cell-SELEX technology. The three selected candidate aptamers showed binding rates of 57.5%, 86.2%, and 50.7% to senescent HDF cells within the same timeframe, respectively, demonstrating high specificity and targeted binding ability. Furthermore, the aptamer possesses potential for detecting, imaging, and regulating the function of senescent cells, successfully providing a new direction for targeted anti-aging interventions and therapeutic applications.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to the screening and identification of nucleic acid aptamers that specifically bind to human senescent dermal fibroblasts. Background Technology

[0002] Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules obtained through in vitro screening. They can recognize target molecules with high affinity and specificity and are widely used in fields such as biosensing, disease diagnosis, and treatment. Cell-SELEX (cell index enrichment ligand systematic evolution) is a common method for screening nucleic acid aptamers using intact cells as targets. It can identify membrane proteins or other biomarkers specifically expressed on the cell surface even when the target molecule is unknown. It has advantages such as short screening cycle, no need for protein purification, and preservation of the target's native conformation.

[0003] Skin aging is a significant manifestation of overall aging. Senescent cells accumulate in skin tissue and influence the tissue microenvironment by secreting aging-associated secretory phenotype (SASP) factors, thus accelerating the skin aging process. Human dermal fibroblasts (HDFs) are the main cell type in the dermis, and their aging is closely related to decreased skin elasticity and wrinkle formation. Therefore, screening for nucleic acid aptamers that can specifically recognize aging human dermal fibroblasts is of great significance for research on aging mechanisms, detection of senescent cells, and the development of anti-aging intervention strategies.

[0004] Currently, most tools for identifying senescent cells rely on antibodies, but antibodies have limitations such as large batch-to-batch variability, strong immunogenicity, and long preparation cycles. Nucleic acid aptamers, as antibody substitutes, have advantages such as small molecular weight, high stability, and ease of chemical synthesis and modification, showing promising application prospects. However, there is currently a lack of specific nucleic acid aptamers for senescent human dermal fibroblasts, limiting their application in skin aging-related research.

[0005] In summary, there is an urgent need to develop a nucleic acid aptamer that can specifically recognize dermal fibroblasts in aging humans to meet the research needs of aging cell detection, imaging, and functional regulation. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to propose a specific nucleic acid aptamer for aging human dermal fibroblasts. Through in-depth exploration of nucleic acid aptamers, new molecular tools can be provided for the bioanalysis and functional intervention of aging human dermal fibroblasts.

[0007] To achieve the above objectives, on the one hand, the present invention provides a nucleic acid aptamer that specifically binds to human senescent dermal fibroblasts, the sequence of which is as follows: 5'-AACAGCCGACTCGCATTAGGGATCCGCCTTCCTTTCTACC-3', as shown in SEQ ID NO.4; 5'-GGAGTCCGTCCCCCGTCCCCCATCGGCTTGTTGACCTAATT-3', as shown in SEQ ID NO.5; 5'-TCACGGTCTTACGTCCCAGCCGATATCCAGCAAGTTTTTT-3', as shown in SEQ ID NO.6; Or a nucleic acid sequence that has greater than 80% sequence homology with the above sequence and can specifically bind to dermal fibroblasts of aging humans.

[0008] On the other hand, the present invention provides that the nucleotide sequence of the above-mentioned nucleic acid aptamer is modified and the modified nucleic acid aptamer binds to aging human dermal fibroblasts, wherein the modification is selected from at least one of phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium and isotopization.

[0009] On the other hand, the present invention provides a conjugate of a nucleic acid aptamer from human senescent dermal fibroblasts, wherein the conjugate has a substance for labeling, detection, diagnosis or treatment attached to the nucleotide sequence of the nucleic acid aptamer, and the conjugate of the attached nucleic acid aptamer binds to senescent human dermal fibroblasts, wherein the substance is at least one of fluorescent markers such as FAM, Cy5, radioactive substances, therapeutic substances, biotin, digoxigenin, luminescent nanomaterials, small peptides, siRNA and enzyme labels.

[0010] On the other hand, the present invention provides the application of the above-mentioned nucleic acid aptamers and their conjugates in the preparation of reagents for detecting or identifying dermal fibroblasts in aging humans.

[0011] On the other hand, the present invention provides the application of the above-mentioned nucleic acid aptamers and their conjugates in the preparation of biological probes for targeting senescent cells.

[0012] On the other hand, the present invention provides the application of the above-mentioned nucleic acid aptamers and their conjugates in the preparation of biological agents for regulating or improving the viability of dermal fibroblasts in aging humans.

[0013] On the other hand, the present invention provides a composition for improving the viability of dermal fibroblasts in aging individuals, characterized in that it comprises the above-mentioned nucleic acid aptamer and a pharmaceutically acceptable carrier.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1) This invention screens and obtains nucleic acid aptamer sequences with high affinity and targeting for senescent HDF cells, providing a novel molecular tool for the detection and treatment of skin aging.

[0015] 2) The nucleic acid aptamers obtained by the present invention have high affinity and targeting for senescent HDF cells, and have the potential for rapid detection of the degree of senescence of HDF cells, as well as precise targeting of senescent HDF cells.

[0016] 3) The nucleic acid aptamer R2 proposed in this invention has a high capture rate of senescent HDF cells, reaching 86.2%.

[0017] 4) The nucleic acid aptamer R2 proposed in this invention has the potential to improve the survival rate of senescent HDF cells. Attached Figure Description

[0018] Figure 1 The top three results of genome sequencing of aptamer libraries from senescent HDF cells.

[0019] Figure 2 The results of homology analysis of the top 20 most abundant sequences in the aptamer library of senescent HDF cells.

[0020] Figure 3 The diagram shows the secondary structure of the candidate aptamer sequences, with R1, R2, and R3 sequences from left to right.

[0021] Figure 4 To observe the affinity binding of candidate aptamers R1, R2, and R3 to senescent HDF cells using laser confocal microscopy.

[0022] Figure 5 To detect the targeted binding of different candidate aptamers to senescent HDF cells using flow cytometry.

[0023] Figure 6 To investigate the effect of CCK-8 aptamers on the relative viability of senescent HDF cells. Detailed Implementation

[0024] The following detailed embodiments further illustrate the concept and technical effects of this invention, providing a full understanding of its purpose, features, and effects. Unless otherwise specified, all methods described are conventional. All materials, unless otherwise specified, are available from publicly available commercial sources. The illustrative embodiments and descriptions of this invention are for explaining the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0025] Example 1: Cell-SELEX screening of nucleic acid aptamers targeting aging human dermal fibroblasts This example will screen for nucleic acid aptamers targeting aging human dermal fibroblasts. During the screening process, a synthetically produced random single-stranded DNA (ssDNA) library was used as the initial screening library. UVB-induced aging human dermal fibroblasts (HDF) were used as the positive screening target, and untreated raw HDF cells were used as the negative screening cells. Specific aptamers were enriched through multiple rounds of screening. The specific experimental steps are as follows: Establishment and preparation of the cell model: Human dermal fibroblasts in logarithmic growth phase were seeded in six-well plates. Senescence induction treatment was performed when the cell confluence reached approximately 70%. The cells were then irradiated under a UVB light source for 10 min at a dose of 3 J / cm². 2 After culturing for another 24 hours, observe the cell status to ensure that the confluence does not exceed 90%. These cells will be used for forward selection. Unirradiated normal HDF cells will be used as reverse selection cells.

[0026] Preparation of ssDNA library: Using a synthesized random ssDNA library (nucleotide sequence as shown in SEQ ID NO.1), centrifuge at 12,000×g for 5-10 min at 4℃, dissolve and dilute to the set concentration with ddH2O according to the instructions, and set aside for later use.

[0027] Forward selection: The ssDNA library was dissolved in binding buffer (final concentration 10-100 nM), heat-denatured in a 95°C metal bath for 5 min, and then rapidly cooled on ice. The treated library was then incubated with senescent HDF cells and virgin HDF cells on ice for 1 h.

[0028] Isolation of ssDNA: After incubation, cells were scraped off using a cell scraper and transferred to a 1.5 mL centrifuge tube. The cells were then vigorously washed four times with PBS buffer to remove unbound or non-specifically bound nucleic acids. The cell-aptamer complex was resuspended in binding buffer and heated in a 95°C water bath for 10 min to dissociate the aptamers from the cell surface. Cell debris was removed by centrifugation, and the supernatant was collected for later use.

[0029] PCR amplification and sequence enrichment: ssDNA from the supernatant was used as a template, and PCR amplification was performed using specific primers (upstream and downstream primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively) to enrich sequences that bind to target cells. The amplification products were analyzed for specificity by agarose gel electrophoresis.

[0030] Preparation of single-stranded DNA libraries: Lambda exonuclease reaction buffer (10 μL) and Lambda exonuclease (5 μL) were added to the PCR amplification products, and the mixture was reacted at 37℃ for 1 h 45 min, followed by inactivation at 75℃ for 10 min to obtain ssDNA. The digested products were verified by agarose gel electrophoresis and used as secondary libraries for the next round of screening.

[0031] Ethanol precipitation purification of ssDNA: Centrifuge the enzyme digestion product at 12,000×g for 5-10 min, collect the supernatant, add 0.1 volume of 3 mol / L sodium acetate and 2 volumes of pre-cooled anhydrous ethanol, mix thoroughly, and incubate overnight at 4℃. The next day, centrifuge at 12,000×g for 10 min, discard the supernatant, wash the precipitate with 1 mL of 70% ethanol, centrifuge again, discard the supernatant, and dry at room temperature. Finally, dissolve the DNA precipitate with 20-100 μL of ddH2O, determine the concentration using NanoDrop, and store at 4℃ for later use.

[0032] Multiple rounds of screening: The screening steps were repeated, and each round of screening used the secondary library constructed in the previous round to incubate with senescent HDF cells, for a total of 12 rounds of screening.

[0033] Reverse screening: The secondary library obtained in the last round, which binds to senescent HDF cells, is incubated with the original HDF cells, and the unbound supernatant is collected as the enriched library after reverse screening for subsequent analysis.

[0034] After screening, the enriched secondary libraries were subjected to high-throughput sequencing, followed by sequence alignment and homology analysis. Figure 2 The three candidate aptamers with the highest enrichment were selected. Figure 1 These were named R1, R2, and R3, respectively, and their nucleotide sequences are shown in SEQ ID NO:4, 5, and 6. Their secondary structures were predicted using the RNAfold web server. Figure 3 The thermodynamic free energies were calculated, and the free energies of R1 to R3 were -2.23, -2.97, and -2.52 kcal / mol, respectively. The sequence alignment results are as follows: Figure 2 As shown.

[0035] Table 1 Summary of Nucleic Acid Sequences Example 2: Identification of cell affinity binding of candidate aptamers using laser confocal microscopy Human skin fibroblasts (HDF) were seeded in six-well plates and cultured to approximately 50% confluence. The culture medium was discarded, and PBS buffer was added. Cells were irradiated with a UVB lamp for 10 min at a total dose of 3 J / cm² to induce cell senescence. After irradiation, the PBS was discarded, and the cells were cultured in fresh medium for another 24 h. Subsequently, 5 nmol / L Cy5.5 fluorescently labeled candidate aptamers R1, R2, and R3 were added, and the cells were incubated on ice for 30 min. After incubation, the cells were washed three times with PBS buffer to remove unbound aptamers, and the binding of the aptamers to the cells was observed using a laser confocal microscope.

[0036] The results showed that obvious Cy5.5 red fluorescence was visible on the cell membrane surface ( Figure 4 The results indicate that candidate aptamers R1, R2, and R3 can specifically bind to target molecules on the membrane of senescent HDF cells. These results confirm that candidate aptamers R1, R2, and R3 obtained through Cell-SELEX screening can specifically bind to senescent cells, with aptamer R2 exhibiting the highest affinity.

[0037] Example 3: Flow cytometry determination of targeted binding of candidate sequences Cy5.5 fluorescently labeled nucleic acid aptamers R1, R2, and R3 were heat-denatured at 95°C for 5 minutes, followed immediately by annealing on ice for 10 minutes. Aptamers were diluted to concentration gradients of 10, 25, 50, 100, 200, and 300 nM using phosphate-buffered saline (PBS), with a blank control group included. Different concentrations of aptamers were incubated with uniformly cultured human skin fibroblasts (HDF) in six-well plates on ice in the dark for 30 minutes. After incubation, cells were gently scraped from each well and collected into corresponding centrifuge tubes. The cells were centrifuged at 300×g for 5 minutes, the supernatant was discarded, and the cells were washed three times with PBS. Finally, 200 μL of binding buffer was added to resuspend the cell pellet. Flow cytometry was used to detect the fluorescence intensity on the cell surface, and the average fluorescence intensity was quantitatively analyzed using FlowJo VX software. Data were processed using GraphPad Prism 9 software, and the results are shown below. Figure 5 As shown, the Control group was the blank control group without fluorescent aptamers, and the R1, R2, and R3 groups were the experimental groups with aptamers R1, R2, and R3, respectively. The results showed that the three candidate aptamers all exhibited different degrees of binding to the target cells. Among them, aptamer R2 showed the best affinity. Under the same incubation conditions, the proportion of positive cells binding to senescent HDF cells reached 86.2%, which was significantly higher than that of R1 (57.5%) and R3 (50.7%).

[0038] Example 4: Determination of the effect of candidate aptamers on the relative viability of senescent HDF cells using the CCK-8 assay. Human skin fibroblasts (HDF) were removed from a CO2 incubator and irradiated under a UVB light source at a dose of 3 J / cm². 2 After irradiation, cells were cultured overnight in a CO2 incubator, and then treated for 24 h with culture medium containing 1, 5, 10, 20, 40, 60, 80, and 100 μM R2 aptamers, respectively. After treatment, the culture medium was discarded, and 100 μL of CCK-8 working solution was added to each well. At the same time, 100 μL of PBS solution was added to each well in the outermost ring to maintain humidity. After incubation in the dark for 1 h, the absorbance of each well was measured at 450 nm using a microplate reader.

[0039] like Figure 6 As shown, with the increase of R2 aptamer concentration, the relative cell viability showed a recovery trend compared to the UVB group; when the concentration reached 20 μM, the cell viability tended to stabilize and no longer increased further, suggesting that this aptamer has the effect of promoting the growth of senescent HDF cells.

[0040] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer that specifically binds to senescent human dermal fibroblasts, characterized in that, The nucleic acid aptamer sequence is as follows: 5'-AACAGCCGACTCGCATTAGGGATCCGCCTTCCTTTCTACC-3', as shown in SEQ ID NO.4; 5'-GGAGTCCGTCCCCCGTCCCCCATCGGCTTGTTGACCTAATT-3', as shown in SEQ ID NO.5; 5'-TCACGGTCTTACGTCCCAGCCGATATCCAGCAAGTTTTTT-3', as shown in SEQ ID NO.6; Or a nucleic acid sequence that has greater than 80% sequence homology with the above sequence and can specifically bind to dermal fibroblasts of aging humans.

2. The nucleic acid aptamer according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid aptamer is modified and the modified nucleic acid aptamer binds to aging human dermal fibroblasts. The modification is selected from at least one of phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopization.

3. A conjugate of nucleic acid aptamers from human aging dermal fibroblasts, characterized in that, The conjugate links a substance for labeling, detection, diagnosis, or treatment to the nucleotide sequence of the nucleic acid aptamer according to claim 1 or 2, and the conjugate of the nucleic acid aptamer after linking the substance binds to aging human dermal fibroblasts, wherein the substance is at least one of fluorescent markers such as FAM, Cy5, radioactive substances, therapeutic substances, biotin, digoxigenin, luminescent nanomaterials, small peptides, siRNA, and enzyme markers.

4. The use of the nucleic acid aptamer of claim 1 or 2 or the conjugate of the nucleic acid aptamer of claim 3 in the preparation of a reagent for detecting or identifying dermal fibroblasts in aging humans.

5. The use of the nucleic acid aptamer according to any one of claims 1 or 2, or the conjugate of the nucleic acid aptamer according to claim 3, in the preparation of biological probes for targeting senescent cells.

6. The use of the nucleic acid aptamer according to any one of claims 1 or 2, or the conjugate of the nucleic acid aptamer according to claim 3, in the preparation of a biological agent for regulating or improving the viability of aging human dermal fibroblasts.

7. A composition for improving the viability of dermal fibroblasts in aging individuals, characterized in that, It contains the nucleic acid aptamer shown in SEQ ID NO. 5 and a pharmaceutically acceptable vector.

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