Raman spectrum-assisted method for rapidly screening nucleic acid aptamers and exploring combination mechanism of nucleic acid aptamers
Through the Raman spectroscopic assisted nucleic acid aptamer screening method, the Raman spectral characteristics of ssDNA and targets were analyzed in real time using deep learning models, solving the problems of low efficiency and high cost of fluorescein assisted screening, and achieving efficient and low-cost nucleic acid aptamer screening and binding mechanism exploration.
Patent Information
- Application Number
- CN202510443781.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-07-11
AI Technical Summary
The existing fluorescein-assisted nucleic acid aptamer screening methods are inefficient, costly and susceptible to environmental factors, so they cannot accurately judge the screening process and binding mechanism.
Raman spectroscopy assisted nucleic acid aptamer screening method was used, and Raman spectral characteristics of ssDNA and target were collected and analyzed in real time using the Spec-Transformer deep learning model. The screening process was judged by feature peak changes and the binding mechanism was explored.
It improves the efficiency of nucleic acid aptamer screening, reduces cost, reduces operational complexity, and accurately judges the screening process and binding mechanism.
Smart Images
Figure CN120299508A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for rapidly screening nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism, belonging to the technical field of bioinformatics engineering. Background Art
[0002] Nucleic acid aptamers are short oligonucleotide sequences obtained by in vitro screening, which can bind to corresponding aptamers with high affinity and strong specificity. Their emergence provides a new efficient and rapid recognition research platform for the chemical biology community and shows good application prospects in many aspects.
[0003] Currently, there are various methods for assisting nucleic acid screening. For example, the method of fluorescein-assisted nucleic acid aptamer screening. In this method, randomly modified ssDNA with fluorescein is used as the screening library, and the total fluorescence value at the end of incubation is calculated. If the total fluorescence value changes significantly, it enters the next round of screening; if the total fluorescence value does not change significantly, this round of screening ends. The ssDNA obtained from multiple rounds of screening is sequenced until all sequencing results no longer change significantly, and the obtained ssDNA is the required nucleic acid aptamer. This method usually requires measuring the fluorescence values of the aptamer screening process multiple times. Each round of screening and measuring fluorescence values takes a long time, with low efficiency. Moreover, multiple PCR reactions are required during the process of measuring fluorescence values, and all these steps require a large amount of reagents and equipment, resulting in high implementation costs. During the fluorescein-assisted screening process, fluorescein is easily interfered by environmental factors, which may lead to inaccurate fluorescence values and unable to judge the screening process. In addition, the fluorescence value cannot reveal the binding mechanism between the aptamer and the target. Therefore, it is necessary to develop a new method for assisting nucleic acid screening to improve screening efficiency, reduce costs, and reduce operation complexity. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for rapidly screening nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism. Before screening, record and analyze the Raman spectral characteristics of the initial state of ssDNA and the target. Real-time collect the Raman spectra of ssDNA and the target during the screening process, use the Spec-Transformer deep learning model to extract characteristic peaks, observe the peak value changes of the characteristic peaks of ssDNA and the target, so as to judge the screening process and investigate the properties of the aptamer, etc.
[0005] To achieve the above purpose, the technical solution adopted by the present invention is: a method for rapidly screening nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism, which includes the following steps:
[0006] Step 1: First, synthesize random ssDNA as the initial screening library;
[0007] Step 2: Then, the cultured highly active screening object is taken as the target, and the Raman spectral characteristics of the ssDNA and the target before screening are recorded and analyzed. The ssDNA solution and the screening object are placed on a shaker for incubation, and the free ssDNA in the solution is screened and separated;
[0008] Step 3, recording the Raman spectra of the ssDNA solution and the object to be screened obtained in step 2, placing the ssDNA solution and the object to be screened on a shaker for incubation, and screening and separating the ssDNA dissociated from the surface of the object to be screened;
[0009] Step 4, continue to operate according to step 2 and step 3 until the time error is within a certain range, and use the filter membrane to separate the ssDNA dissociated from the surface of the screened object in this round. The ssDNA is the desired aptamer, and the aptamer screening process is completed;
[0010] Step 5: By analyzing the Raman spectra of ssDNA and target before and after multiple rounds of screening, the binding mechanism of the aptamer and target is explored by comparing the Raman shift and peak characteristics.
[0011] Furthermore, the object to be screened in step 2 is a virus or a protein.
[0012] Furthermore, in steps 2 and 3, the ssDNA in the solution is separated by a filter membrane and stored at 4° C. in the dark for future use.
[0013] Furthermore, in step 2, the Spec-Transformer deep learning model is used to extract characteristic peaks, and the changes in the peak values of the characteristic peaks of ssDNA and the target are observed to judge the screening progress.
[0014] Furthermore, when the characteristic peak signal change amplitude is <5%, it indicates that the round of aptamer screening is finished, and the current time is recorded as T1.
[0015] Furthermore, in step 3, the Spec-Transformer deep learning model is used to extract characteristic peaks, and the changes in the peak values of the characteristic peaks of ssDNA and the target are observed to judge the screening progress.
[0016] Furthermore, when the characteristic peak signal change amplitude is less than 5%, indicating that the round of aptamer screening is finished, the current time is recorded as T2.
[0017] Furthermore, in step 4, the operation is continued according to step 2 and step 3, and the time is recorded in sequence as T3...Tn, until the time error between Tn-1 and Tn is within a certain range.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: Traditional SELEX methods require fluorescent labeling or immobilized targets, etc. The method of the present invention does not require fluorescent labeling and immobilization. The present invention uses random ssDNA as the screening library, records and analyzes the Raman spectral characteristics of ssDNA and the target before screening, and uses the real-time acquisition of the Raman spectra of ssDNA and the target as a control during the screening process. It is possible to screen for aptamer Raman spectra corresponding to different stages of the same batch of samples in one round of operation, so as to judge the screening progress, investigate the properties of the aptamer, infer the binding mechanism, save costs, and greatly improve the screening efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 It is a schematic flow chart of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0020] The present invention will be described in detail below with reference to the drawings and specific embodiments.
[0021] Example 1
[0022] Raman-assisted rapid screening of ssDNA aptamers against HCoV-OC43 virus.
[0023] (a) First, synthesize random ssDNA as the initial screening library;
[0024] (b) Then, take the well-cultured highly active HCoV-OC43 virus and record and analyze the Raman spectral characteristics of ssDNA and HCoV-OC43 virus before screening. The ssDNA solution and HCoV-OC43 virus are incubated on a shaker, and the Raman spectral characteristics of ssDNA and HCoV-OC43 virus during this screening process are recorded and analyzed in real time. The Spec-Transformer deep learning model is used to extract the characteristic peaks to observe the peak value changes of the characteristic peaks during the binding process of ssDNA and HCoV-OC43 virus. If the change amplitude of the characteristic peak signals of ssDNA and HCoV-OC43 virus < 5%, then this round of screening is completed, record the current time as T1 (Tn is adjusted according to the screening results, and n is the number of screening rounds). The free ssDNA in the solution is separated by a filter membrane and stored in the dark at 4 °C for later use.
[0025] (c) Record the Raman spectra of the ssDNA solution and HCoV-OC43 virus obtained in (b), incubate the ssDNA solution and HCoV-OC43 virus on a shaker, and record and analyze the Raman spectra of the ssDNA and HCoV-OC43 virus in real time during the screening process. Use the Spec-Transformer deep learning model to extract characteristic peaks and observe the changes in the characteristic peaks during the binding process of ssDNA and HCoV-OC43 virus. If the change amplitude of the characteristic peak signal of ssDNA and HCoV-OC43 virus is less than 5%, the current time is recorded as T2, and then the ssDNA dissociated from the surface of HCoV-OC43 virus is separated by a filter membrane and stored at 4°C in the dark.
[0026] (d) Continue to operate according to steps (b) and (c), and record the time as T3...Tn in sequence, until the time error between Tn-1 and Tn is within a certain range, and use the filter membrane to separate the ssDNA dissociated from the surface of the HCoV-OC43 virus. The ssDNA is the desired aptamer, and the aptamer screening process is completed.
[0027] (e) By analyzing the Raman spectra of ssDNA and HCoV-OC43 virus before and after multiple rounds of screening, the binding mechanism between the aptamer and the target was explored by comparing the Raman shift and peak characteristics.
[0028] Example 2
[0029] Raman-assisted rapid screening of aptamers for exploring drug resistance in Escherichia coli.
[0030] (a) First, random ssDNA is synthesized as the initial library for screening;
[0031] (b) Then take the cultured highly active E. coli and put it into a certain concentration of antibiotic solution, record and analyze the Raman spectral characteristics of ssDNA and E. coli before screening, incubate the ssDNA solution and E. coli on a shaker, and record and analyze the Raman spectral characteristics of ssDNA and E. coli during the screening process in real time. Use the Spec-Transformer deep learning model to extract characteristic peaks and observe the changes in the peak values of characteristic peaks during the binding process of ssDNA and E. coli. If the change amplitude of the characteristic peak signal of ssDNA and E. coli is <5%, the current round of screening is completed. Record the current time as T1, (Tn is adjusted as the screening results change, and n is the number of screening rounds). Separate the free ssDNA in the solution through a filter membrane and store it at 4°C in the dark for later use.
[0032] (c) Record the Raman spectra of the ssDNA solution and E. coli obtained in (b), incubate the ssDNA solution and E. coli on a shaker, and record and analyze the Raman spectra of ssDNA and E. coli during the screening process in real time. Use the Spec-Transformer deep learning model to extract characteristic peaks and observe the changes in the peak values of characteristic peaks during the binding process of ssDNA and E. coli. If the change amplitude of the characteristic peak signal of ssDNA and E. coli is <5%, the current round of screening is completed, and the current time is recorded as T2. Then, use a filter membrane to separate the ssDNA dissociated from the surface of E. coli and store it at 4°C in the dark.
[0033] (d) Continue to operate according to steps (b) and (c), and record the time as T3..., Tn in sequence until the time error between Tn-1 and Tn is within a certain range. Use the filter membrane to separate the ssDNA dissociated from the surface of E. coli. The ssDNA is the desired aptamer, and the aptamer screening process is completed.
[0034] (e) By analyzing the Raman spectra of ssDNA and E. coli before and after multiple rounds of screening, the binding mechanism of the aptamer and the target was explored by comparing the Raman shift and peak characteristics.
[0035] Aptamers bind to target molecules through spatial conformational complementarity to form stable complexes. Their ssDNA forms a specific three-dimensional structure by folding, and matches and binds to target molecules in terms of physical and chemical properties. Raman spectroscopy can directly reflect the formation process of aptamer-target complexes by analyzing the changes in molecular vibration energy levels and capturing the characteristics of chemical bond vibration and conformational changes when aptamers bind to targets. The real-time detection capability of Raman spectroscopy can directly observe the binding dynamics of aptamers and targets during the screening process.
[0036] By comparing the Raman shift and peak characteristics of the binding objects, the binding mechanism between the aptamer and the target is explored, thereby achieving continuous and dynamic screening, greatly improving screening efficiency, reducing costs, reducing operational complexity, and shortening screening time.
[0037] In summary, this method is based on real-time collection of Raman spectra of ssDNA and target during the screening process, using the Spec-Transformer deep learning model to extract characteristic peaks, observe the characteristic peaks of ssDNA and target and their signal change amplitudes, so as to judge the screening process and examine the properties of aptamers. By comparing the Raman shift and peak characteristics of the aptamer-target conjugate, the binding mechanism of the aptamer and the target is explored.
[0038] The method for rapid screening of aptamers assisted by Raman spectroscopy and exploring their binding mechanism can be applied to multiple fields, including but not limited to biomedical research, molecular recognition and targeted detection, bioanalysis and detection, etc. Compared with other aptamer screening methods, the Raman spectroscopy-assisted screening method constructed in the present invention can judge the screening progress in real time based on the changes of aptamers, targets, aptamer-target conjugates, etc., greatly shortening the incubation time.
[0039] The above shows and describes the basic principles, main features and advantages of the present invention. Those of ordinary skill in the art should understand that the above embodiments do not limit the protection scope of the present invention in any form. Any technical solutions obtained by means of equivalent replacement and the like fall within the protection scope of the present invention. The parts not involved in the present invention are the same as or can be implemented by the prior art.
Claims
1. A method for rapid screening of nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism, characterized in that, The steps include: Step 1: First, random ssDNA is synthesized as the initial library for screening; Step 2: Then, the cultured highly active screening object is taken as the target, and the Raman spectral characteristics of the ssDNA and the target before screening are recorded and analyzed. The ssDNA solution and the screening object are placed on a shaker for incubation, and the free ssDNA in the solution is screened and separated; Step 3, recording the Raman spectra of the ssDNA solution and the object to be screened obtained in step 2, placing the ssDNA solution and the object to be screened on a shaker for incubation, and screening and separating the ssDNA dissociated from the surface of the object to be screened; Step 4, continue to operate according to step 2 and step 3 until the time error is within a certain range, and use the filter membrane to separate the ssDNA dissociated from the surface of the screened object in this round. The ssDNA is the desired aptamer, and the aptamer screening process is completed; Step 5: By analyzing the Raman spectra of ssDNA and target before and after multiple rounds of screening, the binding mechanism of the aptamer and target is explored by comparing the Raman shift and peak characteristics.
2. The method for rapid screening of nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism according to claim 1, wherein, The object to be screened in step 2 is a virus or a protein.
3. A method for rapid screening of nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism according to claim 1, characterized in that, In the steps 2 and 3, the ssDNA in the solution is separated by a filter membrane and stored at 4° C. in the dark for future use.
4. A method for rapid screening of aptamers assisted by Raman spectroscopy and exploring their binding mechanism according to claim 1, characterized in that, In step 2, the Spec-Transformer deep learning model is used to extract characteristic peaks, and the changes in the peak values of the characteristic peaks of ssDNA and the target are observed to judge the screening progress.
5. A method for rapid screening of nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism according to claim 4, characterized in that, When the characteristic peak signal change amplitude is less than 5%, it indicates that the round of aptamer screening is over, and the current time is recorded as T1.
6. The method for rapid screening of nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism according to claim 5, characterized in that, In step 3, the Spec-Transformer deep learning model is used to extract characteristic peaks, and the changes in the peak values of the characteristic peaks of ssDNA and the target are observed to judge the screening progress.
7. A method for rapid screening of nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism according to claim 6, characterized in that When the characteristic peak signal change amplitude is less than 5%, it indicates that the round of aptamer screening is over, and the current time is recorded as T2.
8. A method for rapid screening of nucleic acid aptamers assisted by Raman spectroscopy and exploring their binding mechanism according to claim 7, characterized in that, In step 4, continue to operate according to step 2 and step 3, and record the time as T3...Tn in sequence until the time error between Tn-1 and Tn is within a certain range.