A method of optical imaging of g-quadruplex conformations
By observing the morphological differences of peptide-nucleic acid complexes using optical microscopy, the G4 conformation can be rapidly identified, solving the problems of long time consumption, equipment dependence and large sample volume of traditional methods, and realizing low-cost and efficient G4 conformation analysis.
Patent Information
- Application Number
- CN202510585546.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-01
- Publication Date
- 2026-11-03
AI Technical Summary
Existing techniques for analyzing G-quadruplex (G4) structures are time-consuming, rely on sophisticated equipment and operators, and are difficult to apply widely. Traditional methods are complex and require large sample volumes.
By using peptides that can specifically aggregate with G4, the morphological differences of peptide-nucleic acid complexes are observed through optical microscopy to achieve rapid identification of G4 conformation. The method includes preparation of peptide-nucleic acid complexes, standing, image acquisition, and data analysis.
The G4 conformation can be identified within 30 minutes using an optical microscope, with sample amounts as low as ng, making it suitable for complex samples and providing a rapid and simple method for G4 conformation analysis without inducing conformational changes.
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Figure CN123029017A_ABST
Abstract
Description
I. Technical Field
[0001] This invention belongs to the field of analytical chemistry, and specifically relates to a new method for optical imaging of G-quadruplex conformation. II. Background Technology
[0002] Currently, over 700,000 potential G-quadruplex (G4) sites are known to exist in human cells. G4 plays a crucial role in key biological functions and has been identified as a potential therapeutic target. G4 is formed by the folding of guanine-rich sequences. Its structure originates from the self-assembly of guanine bases through Hoogsteen hydrogen bonds to form G-quartets, followed by self-stacking through π-π stacking interactions to form the G4 structure. G4 structures exhibit diverse characteristics; different stem-loop arrangements, glycosidic bond angles, and chain orientations contribute to the diversity of G4 topologies, which can generally be classified into three categories: parallel, antiparallel, and hybrid. Currently, many techniques have been developed to distinguish common G4 topologies, such as circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy. However, traditional G4 structure analysis methods are time-consuming and rely on sophisticated equipment and skilled operators. In recent years, novel G4 identification methods based on nanomaterials, such as gold nanoparticles and metal-organic frameworks, have been developed. Although these methods have revolutionized the field of G4 topology identification and enabled its recognition, limitations remain, such as complex preparation processes and limited widespread application.
[0003] To overcome the limitations of existing methods in G4 topological characterization, we developed a novel method based on specific aggregation effects for identifying G-quadruplex conformations. Figure 1 This method has been successfully applied to conventional G4 topologies and extended to applications such as G4 conformational conversion and methylation modification. This method relies on peptides that specifically aggregate with G4, enabling the acquisition of G4 conformational information within 30 minutes without chemical modification. Furthermore, this method can achieve G4 conformational resolution using only a commonly used laboratory optical microscope, with sample quantities as low as ng, far lower than current common identification methods. The steps of this method include: preparation of peptide-nucleic acid complexes, incubation, image acquisition, and data processing. Figure 2 ).
[0004] In summary, this method holds promise as an alternative to G4 structural analysis, freeing us from instrument and technology limitations and expanding the available technical means for G4 research. III. Summary of the Invention
[0005] The purpose of this invention is to provide a method for optically imaging G-quadruplex conformations.
[0006] This invention is achieved through the following technical solution:
[0007] (1) Preparation of peptide-nucleic acid complexes;
[0008] (2) Let it stand;
[0009] (3) Image acquisition;
[0010] (4) Data analysis;
[0011] Compared with the prior art, the present invention has the following characteristics:
[0012] This invention utilizes peptides that specifically aggregate G4 and the morphological differences of peptide-nucleic acid aggregates to identify the G4 conformation through optical microscopy. Compared to existing G4 conformation identification methods, this method has the following advantages:
[0013] (1) Traditional G4 conformation methods rely on specialized instruments and techniques, such as nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. This method utilizes the specific aggregation effect of peptide-nucleic acid, and the G4 conformation can be identified using only an optical microscope. Furthermore, the peptide (sequence 1) used in this method can be synthesized artificially in a solid-phase environment without post-modification.
[0014] (2) Traditional G4 conformation methods rely on rigorous sample preparation and structural analysis procedures, which are quite time-consuming. This method allows for direct observation of G4 conformational differences via microscopic imaging, and results can be obtained within half an hour, greatly saving time.
[0015] (3) Traditional methods require a minimum starting amount in the μg range, while this method can achieve the same conformational identification effect with a starting amount of 1 / 1000, which can meet the testing requirements of complex samples such as small nucleic acid sample volume and low concentration.
[0016] (4) This method is applicable to the identification of various G4 conformations and can be directly applied to complex conformations.
[0017] (5) This identification method does not induce G4 conformational changes and can restore the true G4 conformation. IV. Description of the attached drawings
[0018] Figure 1 Schematic diagram for identifying the G4 conformation using an optical microscope
[0019] Figure 2 Flowchart of main steps
[0020] Figure 3 Microscopic imaging image
[0021] Figure 4 Statistical chart
[0022] Figure 5 .Methylation modification detection diagram V. Detailed Implementation Methods
[0023] Example 1: Combined with Appendix Figure 2
[0024] (1) Preparation of peptide-nucleic acid complex:
[0025] Nucleic acids were pre-treated with a buffer solution containing 10 mM KCl at 95°C, slowly annealed to room temperature, and stored at 4°C for later use. Peptides were also dissolved in a buffer solution containing 10 mM KCl. The amounts of nucleic acids and peptides were calculated based on charge, and 200 μM nucleic acid and peptide samples were mixed in equal proportions, thoroughly mixed, and incubated at 25°C for ten minutes.
[0026] (2) Let it stand:
[0027] The prepared peptide-nucleic acid complex was fully resuspended, and 10 μL was transferred to a coverslip and placed in an oven at 25°C for ten minutes.
[0028] (3) Image acquisition:
[0029] Place the coverslip on the stage of the optical microscope and acquire the image using a 100x objective lens.
[0030] (4) Data processing:
[0031] The obtained images were processed using ImageJ software to obtain their feature values and perform statistical processing.
[0032] The experimental results show that:
[0033] (1) The optical identification process for the G4 conformation established in this experiment is as follows: Figure 2 As shown
[0034] (2) Microscopic imaging shows that the three conformations of G4 correspond one-to-one with the circular dichroism spectrum, such as Figure 3 As shown.
[0035] (3) Common conformations are imaged and statistically analyzed; their characteristic values can distinguish their conformations, such as... Figure 4 As shown.
[0036] (4) This method can be further extended to identify methylation modifications, such as Figure 5 As shown.
Claims
1. A method for optically imaging a G-quadruplex conformation, characterized in that, This method mainly includes the following steps: (1) Prepare peptide-nucleic acid complex and incubate at room temperature for 10 minutes; (2) Let it stand on the coverslip for 10 minutes; (3) Acquire images using an optical microscope; (4) Image J statistical analysis of particle features in images.
2. The method according to claim 1, characterized in that, The nucleic acid sequence that specifically aggregates with the peptide is rich in the base G and can form a G-quadruplex, with the following general formula: Sequence 1: H(0-10)G(1-3)H(1-10)G(1-3)H(1-10)G(1-3)H(1-10)G(1-3)H(0-10) In the above general formula, nucleic acid composition includes ribonucleic acid and deoxyribonucleic acid; H is selected from A, T, C, and U bases other than G in nucleic acids; The sequence of the peptide that specifically aggregates with nucleic acids can be any of the following general formulas: Sequence 2: ZY(1-2)-XY(1-2)-X-[X]-XY(1-2)-X(1-2)-[Y] In the above general formula, the amino acid composition includes natural and non-natural amino acids; X specifically refers to lysine (Lys) or 2,4-diaminobutyric acid (Dab); Y refers to any amino acid other than Lys and Dab; [X] and [Y] form a cyclization ring with an amide bond; Z refers to isomers such as octanoic acid, which are linked to the peptide chain by an amide bond.
3. According to claim 1, nucleic acids conforming to the general formula of sequence 1 can specifically aggregate with peptides conforming to the general formula of sequence 2, and such aggregates satisfy the following conditions: (1) Particle size is 500-2000 nm; (2) It exists stably under neutral conditions.
4. The method according to claim 1, characterized in that, Step (1) The preparation process of the peptide-nucleic acid complex is as follows: Nucleic acid and peptide samples were dissolved in buffer solution; the amount of nucleic acid and peptide substances was calculated based on charge; 200 μM nucleic acid and peptide samples were mixed in equal proportions and thoroughly mixed before incubation at 25°C for ten minutes. The buffer components are: 10mM Tris-HCl, 10mM KCl, and pH 7.
5. The method according to claim 1, characterized in that, Step (2) is as follows: after resuspending the sample from step (1), take 10 μL of the mixture and transfer it to a coverslip, and let it stand in an oven at 25°C for ten minutes.
6. The method according to claim 1, characterized in that, The image acquisition process in step (3) is as follows: place the sample from step (2) on an optical microscope, adjust to 100x objective lens for observation, and collect data.
7. The method according to claim 1, characterized in that, Step (4) Image processing process: The data obtained in step (3) is processed and analyzed by Image J software to collect the corresponding feature values.