Method for analyzing and screening binding affinity of small nucleic acid drug and KRAS mutant protein

Abstract

The invention discloses a small nucleic acid drug and KRAS mutant protein binding affinity analysis screening method. A KRAS mutant, a wild type target proteome and a small nucleic acid initial library are included; performing a plurality of cycles combining positive screening and negative screening wherein the negative screening specifically uses a KRAS wild-type protein as a reverse selection target to actively enrich sequences specific for mutants; performing high-throughput sequencing on the enriched library to obtain candidate molecules; the affinity and the specificity are quantitatively verified through a biophysical technology; and the functional activity of the compound is verified in a cell model. According to the invention, through a core technology of targeting negative screening, the problem that high-specificity ligands are difficult to screen in a traditional method is effectively solved, and a rapid and efficient screening platform from combination to function integration is established.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins. Background Technology

[0002] The RAS gene is one of the most frequently mutated oncogenes in human cancers, with point mutations at codon 12 (such as G12C, G12D, and G12V) being particularly common in pancreatic cancer, colorectal cancer, and non-small cell lung cancer. Because the KRAS protein has a relatively smooth surface and binds tightly to GTP / GDP, it has long lacked effective targeted inhibitors and was considered an "undruggable" target.

[0003] Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules that can bind to target molecules (such as proteins) with high affinity and high specificity through their specific three-dimensional structure. Traditional screening methods—exponential enrichment ligand system evolution techniques—generally have the following limitations:

[0004] 1. Low screening efficiency and long cycle: It usually requires 10-15 rounds of affinity screening and amplification cycles, which is lengthy and has limited throughput.

[0005] 2. Difficulty in ensuring specificity: Traditional methods are difficult to effectively remove sequences that bind to highly homologous non-target proteins (such as KRAS-WT protein or other mutants) during the screening process, resulting in poor selectivity of the obtained aptamers and a high risk of off-target effects.

[0006] 3. Not friendly to difficult-to-prepare targets: For functional membrane proteins that are difficult to express and purify, such as full-length KRAS, traditional methods are limited in application because they require a large amount of protein.

[0007] 4. Screening and function are disconnected: Screening endpoints usually only obtain the binding sequence, and whether it has functional inhibitory activity still needs a lot of subsequent experimental verification, which prolongs the drug development process.

[0008] To this end, a method for screening small nucleic acid drugs by analyzing their binding affinity to KRAS mutant proteins is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide a method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins, so as to solve the problems mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a method for screening small nucleic acid drugs by analyzing their binding affinity to KRAS mutant proteins, comprising the following steps;

[0011] Step 1: Obtain the target proteome and a small initial nucleic acid library. The target proteome includes the target KRAS mutant protein and the control protein. The target KRAS mutant protein is a full-length protein or G domain of KRAS-G12C, KRAS-G12D, or KRAS-G12V point mutation. The control protein is the KRAS-WT protein (KRAS-WT protein is the KRAS wild-type protein).

[0012] Step 2: Perform at least two rounds of screening cycles to update the initial small nucleic acid library, obtaining an enriched small nucleic acid library, wherein at least one cycle includes:

[0013] Positive screening: The small nucleic acid library is contacted with the target KRAS mutant protein, and the first binding small nucleic acid is obtained by magnetic bead separation; the first binding small nucleic acid is a small nucleic acid sequence that binds only to the active site of the target KRAS mutant protein after specific elution (elution buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, preheated at 95°C);

[0014] Negative screening: The first binding small nucleic acid is brought into contact with the control protein, and the second binding small nucleic acid that has not bound to the control protein is collected;

[0015] Step 3: Perform high-throughput sequencing on the enriched small nucleic acid library and select candidate small nucleic acid sequences based on the sequencing results;

[0016] Step 4: Synthesize the candidate small nucleic acid sequence and determine the binding affinity of the candidate small nucleic acid sequence to the target KRAS mutant protein;

[0017] Step 5: Verify the inhibitory activity of the candidate small nucleic acid sequence on the KRAS signaling pathway at the cellular level; the functional verification is achieved by detecting the phosphorylation level of ERK1 / 2, a downstream signaling pathway molecule of KRAS, using Western blotting, or by detecting the cell proliferation inhibition rate using the CCK-8 assay.

[0018] Preferably, the target KRAS mutant protein is a full-length protein or G domain of KRAS-G12C, KRAS-G12D, or KRAS-G12V point mutation, and the purity of the target KRAS mutant protein is verified by SDS-PAGE to be >95%. The control protein is KRAS-WT protein.

[0019] Preferably, the separation in step two is achieved by magnetic bead method, filter membrane method or capillary electrophoresis.

[0020] Preferably, the timing of introducing negative screening is adjusted according to the enrichment efficiency of the first round of positive screening. When the binding rate of the first round is ≥30%, negative screening is introduced. The binding rate is the specific binding rate of the small nucleic acid to the target KRAS mutant protein, which is calculated by "magnetic bead method to separate the binding complex + fluorescence quantitative PCR detection of the amount of bound small nucleic acid / total amount of the initial library".

[0021] Preferably, in the negative screening round, the screening pressure is increased by gradually reducing the concentration of the target KRAS mutant protein from 200 pmol to 20-50 pmol, increasing the number of washes from 5 to 8-10, and adding 0.05-0.2 mg / mL of a competitive agent, namely yeast tRNA or salmon sperm DNA, to the washing buffer.

[0022] Preferably, the binding affinity criterion is: the dissociation constant KD of the candidate small nucleic acid with the target KRAS mutant protein is ≤100 nM, and the selectivity fold with KRAS-WT is ≥50; the selectivity fold is calculated based on the following: under the same buffer conditions (20 mM Tris-HCl, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.005% Tween-20, pH 7.4) and 25°C, the KD value is detected by a surface plasmon resonance spectrometer, and the selectivity fold is calculated as: KD value of KRAS-WT / KD value of the target KRAS mutant protein; if the KD value of KRAS-WT is >1000 nM, it is calculated as 1000 nM.

[0023] Preferably, the functional verification in step five is achieved by detecting the phosphorylation level of KRAS downstream signaling pathway molecules using Western blotting, or by detecting the cell proliferation inhibition rate using the CCK-8 assay.

[0024] Preferably, the initial small nucleic acid library is a single-stranded DNA library or a chemically modified RNA library, wherein the chemical modification is at least one of 2'-O-methyl modification, locked nucleic acid modification, or thiophosphate modification, and the library structure contains 30-50 random bases with a diversity of 10^12-10^15.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. By introducing a "negative screening" step and using KRAS wild-type protein as a control, this invention can actively and efficiently eliminate cross-reactive sequences, thereby enriching small nucleic acid sequences with extremely high selectivity for specific KRAS point mutations, solving the problem that traditional methods cannot effectively distinguish highly homologous proteins.

[0027] 2. By combining high-throughput sequencing technology, this invention can quickly identify library enrichment and accurately locate candidate sequence families, avoiding the inefficiency of blind cloning and sequencing in traditional methods and significantly shortening the screening cycle. At the same time, since the specificity is established early, the success rate of obtaining functional lead compounds is greatly improved. Attached Figure Description

[0028] Figure 1 This is an overall flowchart of the screening method described in the embodiments of the present invention.

[0029] Figure 2 This is a graph showing the change in the binding rate of the small nucleic acid library to the KRAS-G12C protein with each round during the screening process in Example 1.

[0030] Figure 3 This is an SPR sensing image of the candidate small nucleic acid molecule Apt-KC-1 binding to KRAS-G12C and KRAS-WT proteins in Example 2.

[0031] Figure 4 This is a graph showing the inhibitory effect of the candidate small nucleic acid molecule Apt-KC-1 on ERK phosphorylation in NCI-H358 cells in Example 3. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1, such as Figure 1 and Figure 2 As shown:

[0034] DNA aptamer screening for the KRAS-G12C mutant;

[0035] 1. Material preparation

[0036] a. Target proteins: Active recombinant human KRAS-G12C mutant full-length protein (with GST tag), and active recombinant human KRAS wild-type full-length protein (with GST tag). Protein purity was verified by SDS-PAGE >95%.

[0037] b. Small nucleic acid initial library: The synthesized sequence is -ATCTACGACTCACTATAGGGAGACAAGAATAAACGCCAAGA-(N40)-TTCGACAGGAGGCTCACAACGGACCCATA- A single-stranded DNA library, where N40 consists of 40 random bases. The library diversity is approximately 10^14.

[0038] c. Streptospirin magnetic beads.

[0039] d. Buffer: Binding / washing buffer: 20 mM Tris-HCl, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.005% Tween-20, pH 7.4. Elution buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

[0040] 2. Screening Process

[0041] a. First round of screening (positive screening): A 1 nmol ssDNA library was denatured at 95°C for 5 minutes and then rapidly annealed on ice. It was then incubated with 200 pmol KRAS-G12C protein in binding buffer at room temperature for 60 minutes. Subsequently, glutathione agarose beads pre-equilibrated with binding buffer were added, and the mixture was gently mixed for 30 minutes to capture the GST-tagged protein and its bound DNA complex. After centrifugation, the supernatant was carefully discarded, and the sample was washed five times rapidly with 1 mL of washing buffer. Finally, the bound DNA was eluted with 200 μL of elution buffer preheated to 95°C, purified by ethanol precipitation, and used for PCR amplification to prepare ssDNA for the next round of screening.

[0042] b. Second round of screening: The procedure is the same as the first round, but the amount of KRAS-G12C protein used is reduced to 150 pmol.

[0043] c. Third round of screening (introduction of negative screening): Since the binding rate in the first round is ≥30% (the first round binding rate refers to the percentage of small nucleic acid molecules that successfully bind to the target (e.g., KRAS-G12C mutant protein) and are recovered after the first round of positive screening, negative screening is introduced from this round onwards. First, the ssDNA enriched in the second round is incubated with 150 pmol KRAS-WT protein in binding buffer for 30 minutes. Then, this mixture is incubated with glutathione agarose beads coated with KRAS-WT protein, and the flow-through (i.e., ssDNA that has not bound to KRAS-WT protein) is collected. Subsequently, this flow-through is used for positive screening with 100 pmol KRAS-G12C protein, and the steps are the same as before.

[0044] d. Rounds 4-8 of screening: Gradually increase the screening pressure. Specifically: the amount of KRAS-G12C protein used is gradually reduced from 100 pmol to 20 pmol; the amount of KRAS-WT protein used in negative screening is kept constant or slightly higher than the amount of protein used in positive screening in the same round; the number of washes is increased from 5 to 8-10; and 0.1 mg / mL yeast tRNA is added to the wash buffer as a competitive agent starting from round 6.

[0045] In summary, the conditions for conducting positive or negative screening in the first, second, third, or fourth to eighth rounds of screening are that as long as the first round of positive screening has a binding rate of ≥30%, negative screening will be introduced in the next round. Negative screening may be in the second or third round of screening.

[0046] Example 2, as Figure 1 As shown:

[0047] Experimental Design:

[0048] Three sets of screening experiments were conducted in parallel under identical conditions (the same target KRAS-G12C protein, the same KRAS-WT control protein, the same initial small nucleic acid library, and the same buffer system). The only difference between the three sets of experiments was the timing of the introduction of negative selection:

[0049] Experimental Group A (this protocol): When the first round of positive screening binding rate is ≥30% (actual calculated value is 32%), the first round binding rate refers to the percentage of small nucleic acid molecules that successfully bind to the target (e.g., KRAS-G12C mutant protein) and are recovered after the first round of positive screening, relative to the total amount of small nucleic acid library initially invested, negative screening is introduced in the third round.

[0050] Control group B (premature introduction): Negative screening was forcibly introduced in the second round when the positive screening binding rate was only about 25% in the first round.

[0051] Control group C (introduced too late): Negative screening was introduced in the fourth round even when the positive screening binding rate in the first round had reached about 40%.

[0052] All groups underwent a total of 8 rounds of screening. The final enriched libraries were then subjected to high-throughput sequencing. The candidate sequences with the highest abundance in each group were selected for synthesis and their affinity and specificity were uniformly verified.

[0053] Results and Data Analysis

[0054] 1. Comparison of enrichment efficiency during the screening process, as shown in Table 1.

[0055] Table 1

[0056] Screening rounds Experimental Group A Control group B Control group C First round of binding rate 32% 26% 38% Introducing negative screening rounds Round 3 Round 2 Round 4 4th round of binding rate 65% 28% 70% 8th round of union rate 88% 45% 75% Document Diversity (Round 8) Significantly reduced, but sequence families are concentrated. Excessive reduction, single sequence Still remains high, sequence noise

[0057] Control Group B: Due to the premature introduction of negative screening, excessive sequences were removed from the already scarce library, severely impairing library diversity. Subsequent screening and enrichment were inefficient, resulting in a final binding rate of only 45%, and the candidate sequence library was small with limited potential.

[0058] Control group C: Due to the late introduction of negative screening, a large number of non-specific binding sequences (which can bind both G12C and WT) had accumulated in the library. These "miscellaneous sequences" dominated, making it difficult for subsequent negative screening to completely remove them. As a result, although the binding rate of the final library was acceptable (75%), the specific background was high.

[0059] Experimental Group A: By introducing negative screening while ensuring initial enrichment, sequence diversity was effectively protected and specificity was promptly enhanced. Ultimately, the desired results of high binding rate (88%) and high specificity were achieved.

[0060] Example 3, as Figure 1 and Figure 3 As shown:

[0061] Analysis and affinity determination of candidate sequences

[0062] 1. The ssDNA libraries enriched in the 8th round of screening in the three control experiments of Example 2 were subjected to high-throughput sequencing using the Illumina NovaSeq platform: tens of millions of sequences were quality filtered, and then sequence clustering, abundance statistics, and common sequence deduction were performed using professional software. One representative sequence that was significantly enriched (abundance in the top 0.1%) and had a large sequence family was selected and named Apt-A1 (experimental group A), Apt-B1 (control group B), and Apt-C1 (control group C), respectively, and then chemically synthesized and purified by HPLC.

[0063] 2. Kinetic analysis using a surface plasmon resonance (SPR) spectrometer: Biotinylated KRAS-G12C protein was immobilized on the test channel of the SA chip, and KRAS-WT protein was immobilized on the reference channel. A series of concentrations (0.78 nM to 100 nM) of Apt-KC-1 were flowed through the chip at a constant flow rate, and the binding and dissociation processes were recorded. The sensor maps were globally fitted using the instrument's accompanying kinetic evaluation software to obtain the binding kinetic parameters.

[0064] 4. Results are as follows Figure 3 As shown in Table 2, Apt-A1 has a nanomolar affinity for KRAS-G12C protein and a very weak affinity for KRAS-WT protein, demonstrating excellent selectivity.

[0065] Table 2

[0066] Candidate molecules target protein Dissociation constant KD(nM) Selectivity factor relative to KRAS-G12C Apt-A1 KRAS-G12C 8.2 - KRAS-WT >1000 >120 KRAS-G12D 650 79 Apt-B1 KRAS-G12C 105 - KRAS-WT 580 5.5 Apt-C1 KRAS-G12C 25 - KRAS-WT 150 6

[0067] Among them, the smaller the value of the dissociation constant KD, the stronger the binding affinity; the selectivity factor is calculated as KD(WT) / KD(G12C); >1000 indicates that no obvious binding was detected in this concentration range; >120 indicates the minimum selectivity factor calculated based on the detection limit; - indicates that it is used as a reference and the selectivity factor is not calculated.

[0068] 5. Conclusion

[0069] a. Control group B: The generated candidate molecule Apt-B1 has weak affinity (KD=105nM) and extremely poor specificity (selectivity fold of only 5.5), making it almost impossible to distinguish between mutants and wild types, and it has no potential for drug development.

[0070] b. Control group C: The generated candidate molecule Apt-C1 has acceptable affinity (KD=25nM), but its specificity is still insufficient (selectivity fold of only 6), and the off-target risk is high. It requires a lot of resources for subsequent optimization or re-screening.

[0071] c. Experimental Group A: The generated candidate molecule Apt-A1 possesses both high affinity (KD=8.2nM) and high specificity (selectivity fold >120), fully meeting the preset screening criteria (KD≤100nM, selectivity≥50-fold), making it an ideal lead compound.

[0072] Example 4, as Figure 4 As shown,

[0073] Cellular level functional verification

[0074] The human non-small cell lung cancer cell line NCI-H358 (endogenously expressing the KRAS-G12C mutation) was used. Chemically synthesized Apt-KC-1 and a random sequence negative control were introduced into the cells via electroporation. Total cell protein was collected 24 hours after transfection. The protein levels of phosphorylated ERK1 / 2 and total ERK1 / 2 were detected by Western blotting. Figure 4 As shown, compared with the negative control group, the p-ERK1 / 2 level in the Apt-KC-1 treatment group was significantly reduced, while the total ERK1 / 2 level remained unchanged, indicating that Apt-KC-1 can effectively invade cells and specifically inhibit the MAPK signaling pathway downstream of KRAS-G12C, thus possessing the expected biological function.

[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for screening small nucleic acid drugs by analyzing their binding affinity to KRAS mutant proteins, characterized in that: Includes the following steps; Step 1: Obtain the target proteome and a small initial nucleic acid library, wherein the target proteome includes the target KRAS mutant protein and the control protein; Step 2: Perform at least two rounds of screening cycles to update the initial small nucleic acid library, obtaining an enriched small nucleic acid library, wherein at least one cycle includes: Positive screening: The small nucleic acid library is brought into contact with the target KRAS mutant protein to separate and obtain the first binding small nucleic acid; Negative screening: The first binding small nucleic acid is brought into contact with the control protein, and the second binding small nucleic acid that has not bound to the control protein is collected; Step 3: Perform high-throughput sequencing on the enriched small nucleic acid library and select candidate small nucleic acid sequences based on the sequencing results; Step 4: Synthesize the candidate small nucleic acid sequence and determine the binding affinity of the candidate small nucleic acid sequence to the target KRAS mutant protein; Step 5: Verify the inhibitory activity of the candidate small nucleic acid sequence on the KRAS signaling pathway at the cellular level.

2. The method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins according to claim 1, characterized in that: The target KRAS mutant protein is a full-length protein or G domain of KRAS-G12C, KRAS-G12D, or KRAS-G12V point mutation, and the control protein is KRAS-WT protein.

3. The method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins according to claim 1, characterized in that: The separation in step two is achieved by magnetic bead method, filter membrane method or capillary electrophoresis method.

4. The method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins according to claim 1, characterized in that: The timing of introducing negative screening is adjusted based on the enrichment efficiency of the first round of positive screening. When the binding rate of the first round is ≥30%, negative screening is introduced.

5. The method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins according to claim 1, characterized in that: In the negative screening round of step two, the screening pressure is increased by gradually reducing the concentration of the target KRAS mutant protein from 200 pmol to 10-50 pmol, increasing the number of washes from 5 to 5-12, and adding 0.05-0.2 mg / mL of a competing agent to the wash buffer.

6. The method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins according to claim 1, characterized in that: The binding affinity criteria for step four are: the dissociation constant KD of the candidate small nucleic acid with the target KRAS mutant protein is ≤100 nM, and the selectivity fold with KRAS-WT is ≥50.

7. The method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins according to claim 1, characterized in that: The functional verification in step five is achieved by detecting the phosphorylation level of downstream signaling pathway molecules of KRAS using Western blotting, or by detecting the cell proliferation inhibition rate using the CCK-8 assay.

8. The method for analyzing and screening the binding affinity of small nucleic acid drugs to KRAS mutant proteins according to claim 1, characterized in that: The initial small nucleic acid library is a single-stranded DNA library or a chemically modified RNA library, with a library structure containing 30-50 random bases and a diversity of 10^12-10^15.

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