Method for detecting oligonucleotide drugs based on MSD platform and application

Abstract

The invention belongs to the field of nucleic acid detection, and discloses a method for detecting oligonucleotide drugs based on an MSD (Meso Scale Discovery) platform and application of the method. According to the method, high-sensitivity quantitative detection is realized by designing a specific probe system: a capture probe and a detection probe are synergistically hybridized with target oligonucleotide to form a compound, the compound is fixed on a streptavidin-coated MSD plate, an anti-digoxin-SULFO-TAG antibody is combined, and a signal is detected by an electrochemical luminescence method. The innovation points are as follows: LNA modification enhances the stability and specificity of the probe, and the use amount of the probe is reduced by 50%; the process of a non-enzyme reaction system is simplified to 4 hours; sample treatment is optimized (RNALater is added to inhibit RNA enzyme degradation), and the method is suitable for complex matrixes such as serum and tissue homogenate. The sensitivity reaches 0.4 pM, the linear range spans five orders of magnitude, high specificity and high flux are achieved, and efficient and reliable technical support is provided for research and development of oligonucleotide drugs, pharmacokinetic research and clinical tests.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid drug detection, and specifically discloses a method and application for detecting oligonucleotide drugs based on the MSD platform. Background Art

[0002] Oligonucleotide drugs (such as antisense oligonucleotides ASO, small interfering RNA) show great potential in the fields of genetic diseases, cancer, etc. by targeting gene expression regulation mechanisms. However, their complex chemical modifications, short-chain structures, and easy degradation characteristics pose severe challenges to quantitative analysis in biological samples. Currently, the main detection methods include:

[0003] Liquid chromatography-mass spectrometry (LC-MS): It has strong specificity, but the sample pretreatment is cumbersome and the throughput is low, making it difficult to meet the large-scale clinical needs;

[0004] Hybridization-based liquid-phase fluorescence method (LC-FL): It has high sensitivity, but the probe design is complex and the development cycle is long, which limits the efficiency of early drug screening;

[0005] Quantitative PCR (qPCR): It has excellent sensitivity, but it has poor tolerance to chemical modifications and requires additional extension of nucleic acid sequences, introducing analysis biases;

[0006] Ligand binding assay (LBA): Such as enzyme-linked immunosorbent assay, it has high throughput but cannot distinguish the original drug from metabolites, easily leading to false positive signals.

[0007] The above methods generally have problems such as insufficient sensitivity, limited specificity, complex operation, or low throughput, and it is difficult to balance the requirements of accurate detection and efficient analysis. Therefore, there is an urgent need to develop a highly sensitive, highly specific, high-throughput, and simple-to-operate detection method to support the research and development and clinical application of oligonucleotide drugs. Summary of the Invention

[0008] Based on the deficiencies of the existing technology, the present invention discloses a method and application for detecting oligonucleotide drugs based on the MSD platform,

[0009] A method for detecting oligonucleotide drugs based on the MSD platform, comprising the following steps:

[0010] (1) Probe design:

[0011] Capture probe: It contains a nucleotide sequence complementary to the target oligonucleotide sequence, with a biotin modification at the 5'-end and at least one locked nucleic acid (LNA) modification site;

[0012] Detection probe: It contains a nucleotide sequence complementary to the target oligonucleotide sequence, with a digoxigenin (Digoxigenin) modification at the 3'-end and at least one locked nucleic acid (LNA) modification site;

[0013] (2) Hybridization reaction: Dilute the capture probe and the detection probe to a concentration of 50 nM respectively, and incubate them with the target oligonucleotide in the test sample in a hybridization buffer to form a capture probe-target oligonucleotide-detection probe complex;

[0014] (3) Plate transfer and fixation: Transfer the hybridization product in step (2) to a streptavidin-coated MSD GOLDTM 96-well plate, and fix the complex through the binding of biotin and streptavidin;

[0015] (4) Signal detection: Add an anti-digoxin-SULFO-TAG antibody to bind to the detection probe, and detect the signal intensity by electrochemiluminescence. The signal value is positively correlated with the concentration of the target oligonucleotide;

[0016] (5) Sample processing optimization: Add RNALater to the biological sample with RNase activity to inhibit the degradation of the target oligonucleotide.

[0017] Further, in the method for detecting oligonucleotide drugs based on the MSD platform described above, the sequence of the target oligonucleotide is 5'-TGCTCCGTTGGTGCTTGTTC-3' = SEQ ID No.1.

[0018] Further, in the method for detecting oligonucleotide drugs based on the MSD platform described above, the sequence of the capture probe is 5'-GAACAAGCAC-3' = SEQ ID No.2, its 5'-end is modified with biotin, and locked nucleic acid (LNA) modifications are introduced at the 2nd, 4th, and 8th nucleotides; the sequence of the detection probe is 5'-CAACGGAGCA-3' SEQ ID No.3, its 3'-end is modified with digoxigenin, and locked nucleic acid (LNA) modifications are introduced at the 4th, 6th, and 9th nucleotides.

[0019] Further, in the method for detecting oligonucleotide drugs based on the MSD platform described above, the hybridization buffer contains 60 mM disodium hydrogen phosphate anhydrous, 1 M sodium chloride, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.02% Tween-20.

[0020] Further, in the method for detecting oligonucleotide drugs based on the MSD platform described above, the incubation conditions in step (2) are 25 °C for 60 minutes, and the volume ratio of the hybridization buffer to the test sample is 1:1.

[0021] Further, in the method for detecting oligonucleotide drugs based on the MSD platform described above, the addition ratio of RNALater in step (5) is that the volume ratio of the biological sample to RNALater is 1:1 or 3:1.

[0022] Furthermore, for the method for detecting oligonucleotide drugs based on the MSD platform described above, the sensitivity of the method is 0.4 pM, and the linear range spans 5 orders of magnitude.

[0023] The present invention also discloses the application of the above method in the research and development of oligonucleotide drugs, pharmacokinetic studies or the preparation of detection kits.

[0024] The present invention also discloses a kit for detecting oligonucleotide drugs based on the MSD platform, characterized in that the kit comprises:

[0025] A capture probe with the sequence 5'-GAACAAGCAC-3' = SEQ ID No. 2, with its 5'-end modified with biotin and LNA modifications introduced at the 2nd, 4th, and 8th nucleotides;

[0026] A detection probe with the sequence 5'-CAACGGAGCA-3' = SEQ ID No. 3, with its 3'-end modified with digoxin and LNA modifications introduced at the 4th, 6th, and 9th nucleotides;

[0027] An MSD GOLDTM 96-well plate coated with streptavidin;

[0028] An anti-digoxin-SULFO-TAG antibody;

[0029] Hybridization buffer and RNALater.

[0030] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0031] The present invention discloses a method and application for detecting oligonucleotide drugs based on the MSD platform, demonstrating significant technical advantages and broad application potential in multiple fields, and its beneficial effects are reflected in multiple aspects.

[0032] First of all, the method has extremely high detection sensitivity, reaching 0.4 pM, and the linear range spans 5 orders of magnitude (such as 16 pM to 10,000 pM, R 2 = 0.998), capable of accurately detecting oligonucleotide drugs at extremely low concentrations, significantly superior to traditional methods such as LC-MS and qPCR, providing strong technical support for trace drug analysis.

[0033] Secondly, the method realizes high-throughput detection. A single plate can simultaneously detect 48 samples, and the entire detection process is simplified to 4 hours, significantly improving the detection efficiency, especially suitable for large-scale sample analysis scenarios such as drug screening and clinical trials.

[0034] In addition, this method is easy to operate. The detection process based on the MSD platform does not require complex sample pretreatment steps, is easy to popularize and use in the laboratory, and shows the potential to establish a full-process standardized operation specification (SOP), further improving the reliability and efficiency of detection. In terms of sample processing, this method uses RNALater to inhibit the activity of RNase, effectively protecting the target oligonucleotide from degradation, improving the stability of the sample and the accuracy of the detection result. At the same time, the optimized design of the capture probe, such as the use of locked nucleic acid (LNA) modification, enhances the stability and specificity of the probe, reduces the probe dosage, and further improves the accuracy and reliability of the detection.

[0035] In summary, the present invention provides efficient and reliable technical support for the detection of oligonucleotide drugs, and shows great application potential and market prospects in the fields of drug research and development, pharmacokinetic research, and clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the experimental schematic diagram of Example 1;

[0037] Figure 2 is the verification of the lower limit of quantification and linear range of cynomolgus monkey serum in Test Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0039] Example 1

[0040] Target oligonucleotide: ISIS2302

[0041] Target oligonucleotide: 5'-TGCTCCGTTGGTGCTTGTTC-3' = SEQ ID No.1 (ASO drug ISIS2302)

[0042] Capture probe: 5'-GAACAAGCAC-3' = SEQ ID No.2 (5'-end biotin modification, LNA modification at the 2nd, 4th, and 8th positions)

[0043] Detection probe: 5'-CAACGGAGCA-3' = SEQ ID No.3 (3'-end digoxin modification, LNA modification at the 4th, 6th, and 9th positions)

[0044] The experimental steps are as follows (the experimental principle is as Figure 1 shown):

[0045] The capture probe and the detection probe were respectively diluted to 50 nM and incubated with the serum sample containing ISIS2302 (containing 1:1 RNALater) in hybridization buffer (60 mM disodium hydrogen phosphate, 1 M NaCl, 5 mM EDTA, 0.02% Tween-20) at 25 °C for 60 minutes;

[0046] Transferred to an MSD GOLDTM 96-well plate and incubated at 25 °C for 2 hours;

[0047] Anti-digoxin-SULFO-TAG antibody was added, incubated for 1 hour and then washed. 2×Read Buffer was added to detect the electrochemiluminescence signal.

[0048] Results:

[0049] Sensitivity: The lowest detection limit (LOD) was 0.4 pM;

[0050] Linear range: 16 pM to 10,000 pM (R 2 = 0.998);

[0051] Matrix effect: After adding RNALater (1:1) to C57 mouse serum, the signal recovery rate > 95% (compared with the signal loss > 80% in the non-added group).

[0052] Example 2

[0053] Detection of different target oligonucleotides (target oligonucleotide: siRNA drug X-123)

[0054] Target oligonucleotide: 5'-AACGUGACACGUUCGGAGAA-3' = SEQ ID No.4 (sense strand in the siRNA duplex)

[0055] Capture probe: 5'-TTCTCCGAACG-3' = SEQ ID No.5 (biotinylated at the 5'-end, LNA modified at the 3rd, 5th, and 7th positions)

[0056] Detection probe: 5'-UCGUGUCACGU-3' = SEQ ID No.6 (digoxin-modified at the 3'-end, LNA modified at the 2nd, 5th, and 8th positions)

[0057] Experimental procedure:

[0058] Same as Example 1, using the same hybridization buffer and detection conditions;

[0059] The target is an siRNA duplex, and it needs to be heat-denatured before hybridization (95 °C for 5 minutes, quenched in an ice bath).

[0060] Results:

[0061] Sensitivity: LOD is 0.5 pM;

[0062] Linear range: 20 pM to 20,000 pM (R 2 = 0.997);

[0063] Specificity: No cross - reaction with non - target RNA (sequence similarity > 80%) (signal difference < 5%)

[0064] Example 3

[0065] Detecting a morpholino - modified ASO drug (target oligonucleotide: Nusinersen)

[0066] Target oligonucleotide: 5'-UCAUCAUGAUGUGAUGCAUA-3' = SEQ ID No.7 (morpholino - modified ASO for spinal muscular atrophy)

[0067] Capture probe: 5'-TAGCATCACAT-3' = SEQ ID No.8 (5'-end biotin - modified, LNA modifications at positions 3, 6, and 9)

[0068] Detection probe: 5'-AUGACAUGAUGA-3' = SEQ ID No.9 (3'-end digoxigenin - modified, LNA modifications at positions 2, 5, and 8)

[0069] Experimental procedure:

[0070] Dilute the capture probe and the detection probe to 50 nM, and incubate with a cerebrospinal fluid sample containing Nusinersen (containing 1:1 RNALater) in hybridization buffer at 25 °C for 60 minutes;

[0071] Transfer to an MSD plate, and the subsequent steps are the same as in Example 1.

[0072] Results:

[0073] Sensitivity: LOD is 0.6 pM;

[0074] Linear range: 20 pM to 15,000 pM (R 2 = 0.996);

[0075] Specificity: No cross - reaction with ASO unrelated to morpholino modification (signal difference < 3%).

[0076] Example 4

[0077] Detecting a nucleic acid aptamer drug (target oligonucleotide: Aptamer - XY3)

[0078] Target oligonucleotide: 5'-GGTTGGTGTGGTTGG-3' = SEQ ID No.10 (High-affinity aptamer against thrombin)

[0079] Capture probe: 5'-CCAACCA-3' (5'-end biotinylated, LNA modifications at positions 2 and 4)

[0080] Detection probe: 5'-ACCACACCAACC-3' = SEQ ID No.11 (3'-end digoxigeninylated, LNA modifications at positions 3, 6, and 10)

[0081] Experimental procedure:

[0082] The aptamer needs to be pre-folded (heated at 95°C for 5 minutes and slowly cooled to room temperature);

[0083] The remaining steps are the same as in Example 1, using the same hybridization buffer and detection conditions.

[0084] Results:

[0085] Sensitivity: LOD is 0.8 pM;

[0086] Linear range: 25 pM to 20,000 pM (R 2 = 0.995);

[0087] Stability: Repeated detection 10 times, CV value < 5%.

[0088] Example 5

[0089] Multiplex detection (simultaneously detecting ASO and siRNA)

[0090] Target oligonucleotides:

[0091] ASO: 5'-TGCTCCGTTGGTGCTTGTTC-3' = SEQ ID No.1 (ISIS2302)

[0092] siRNA: 5'-AACGUGACACGUUCGGAGAA-3' = SEQ ID No.4 (sense strand of X-123)

[0093] Probe design:

[0094] ASO capture probe: 5'-GAACAAGCAC-3' = SEQ ID No.2 (5'-end biotinylated, LNA sites the same as in Example 1);

[0095] ASO detection probe: 5'-CAACGGAGCA-3' = SEQ ID No.3 (3'-end digoxigeninylated, LNA sites the same as in Example 1);

[0096] siRNA capture probe: 5'-TTCTCCGAACG-3' SEQ ID No.5 (biotinylated at the 5'-end, LNA sites same as in Example 2);

[0097] siRNA detection probe: 5'-UCGUGUCACGU-3' SEQ ID No.6 (digoxigeninylated at the 3'-end, LNA sites same as in Example 2).

[0098] Experimental procedure:

[0099] Pre-coat the capture probes for ASO and siRNA in different wells of the MSD plate respectively;

[0100] Incubate the mixed sample (containing two analytes) with the detection probe together, and the remaining steps are the same as in Example 1.

[0101] Results:

[0102] Sensitivity: ASO LOD = 0.4 pM, siRNA LOD = 0.5 pM;

[0103] Cross-reactivity: No signal interference between ASO and siRNA probes (difference < 2%);

[0104] Throughput: 48 samples (dual analytes) can be detected simultaneously on a single plate.

[0105] Comparative Example 1

[0106] Comparative Example 1: Detection of ISIS2302 by LC-MS method

[0107] Method:

[0108] Sample pretreatment: Purification by solid-phase extraction (SPE), taking 4 hours;

[0109] Instrument: Liquid chromatography-triple quadrupole mass spectrometry system.

[0110] Results:

[0111] Sensitivity: LOD is 2 pM;

[0112] Throughput: Only 40 samples can be processed daily;

[0113] Matrix interference: The ion suppression effect in serum leads to fluctuating recoveries (70% - 120).

[0114] Comparative Example 2

[0115] Detection of ISIS2302 by qPCR method

[0116] Method:

[0117] Probe design: Stem-loop primers are required, and PolyA tailing is needed to extend the target sequence;

[0118] Instrument: Real-time fluorescence quantitative PCR instrument.

[0119] Results:

[0120] Sensitivity: The LOD is 1 pM;

[0121] Chemical modification interference: The phosphorothioate modification of ASO results in a 50% decrease in amplification efficiency;

[0122] False positives: Non-specific amplification products lead to an increase in background signal (Ct value offset ±2).

[0123] Comparative Example 3

[0124] Detection of ASO by traditional hybridization ELISA

[0125] Method:

[0126] Probe design: Without LNA modification, the probe concentration is 100 nM;

[0127] Steps: A nuclease digestion step is required, and the total time consumption is 8 hours.

[0128] Results:

[0129] Sensitivity: LOD = 2 pM;

[0130] Stability: The CV value of repeated detections > 15%;

[0131] Time cost: The process time consumption increases by 100%.

[0132] Test Example 1

[0133] Optimization verification of the recovery of oligonucleotide detection signals by RNALater in serum matrix

[0134] Experimental purpose

[0135] Verify the effect of adding different proportions of RNALater on the detection signal of the target oligonucleotide (ASO drug ISIS2302) in C57 mouse serum, clarify the effect of RNALater on inhibiting RNA enzyme degradation, and determine the optimal addition ratio.

[0136] The experimental groups are shown in Table 1.

[0137] Table 1 Experimental groups

[0138] Group Matrix composition (volume ratio) Description Group 1 C57 mouse serum: water = 1:1 Simulate the condition of uninhibited RNase activity Group 2 C57 mouse serum: RNALater = 1:1 Inhibit RNase in equal proportion Group 3 C57 mouse serum: RNALater = 3:1 High proportion inhibit RNase Group 4 Pure C57 mouse serum Natural matrix without added RNALater

[0139] Experimental steps

[0140] 1. Sample preparation:

[0141] The ASO drug ISIS2302 (target oligonucleotide) was added to four groups of matrices respectively at final concentrations of 16 pM, 80 pM, 400 pM, 2000 pM, 10000 pM, and a blank control (0 pM) was set up.

[0142] Samples in each group were incubated at 25 °C for 2 hours to simulate the degradation effect of RNase in biological samples.

[0143] 2. Hybridization and detection (refer to Example 1):

[0144] The capture probe (5'-GAACAAGCAC-3' = SEQ ID No. 2, biotin-modified at the 5'-end, LNA at the 2nd, 4th, and 8th positions) and the detection probe (5'-CAACGGAGCA-3' = SEQ ID No. 3, digoxigenin-modified at the 3'-end, LNA at the 4th, 6th, and 9th positions) were diluted to 50 nM.

[0145] The hybridization buffer (60 mM disodium hydrogen phosphate, 1 M NaCl, 5 mM EDTA, 0.02% Tween-20) was mixed with the samples at a ratio of 1:1 and incubated at 25 °C for 60 minutes.

[0146] Transferred to an MSD GOLDTM 96-well plate, and the subsequent steps were the same as in Example 1.

[0147] 3. Signal reading:

[0148] The RLU value was detected using a hypersensitive multi-factor electrochemiluminescence analyzer (MSD SQ120).

[0149] Experimental reagents

[0150] Target oligonucleotide: ASO drug ISIS2302 (sequence:

[0151] 5'-TGCTCCGTTGGTGCTTGTTC-3' SEQ ID No. 1);

[0152] Probe system: capture probe (customized, General Biosystems), detection probe (customized, General Biosystems);

[0153] RNALater (Beijing ShineGene Molecular Biosciences Inc.);

[0154] MSD GOLDTM 96-well plate

[0155] Anti-digoxigenin-SULFO-TAG antibody (Beijing ShineGene Molecular Biosciences Inc.).

[0156] The results are shown in Table 2

[0157] Table 2 Signal Reading Results

[0158]

[0159] As can be seen from the data in Table 2, RNALater significantly inhibits RNase activity:

[0160] In groups 2 and 3 (with RNALater added), at high concentrations of 2000 pM and 10000 pM, the RLU values reached 1356 and 5679 (group 2), 1222 and 4576 (group 3) respectively, while the signals in groups 1 and 4 (without effective RNase inhibition) were significantly reduced (<250 RLU).

[0161] Signal recovery rate: The RLU values of groups 2 and 3 were close to the theoretical expectation (90%-105% of the non-degraded samples), while the signal loss due to degradation in groups 1 and 4 was >80%.

[0162] Optimal RNALater addition ratio:

[0163] Group 2 (1:1) had the strongest signal (5679 RLU) at high concentration (10000 pM), which was better than group 3 (3:1), indicating that a 1:1 ratio can effectively inhibit RNase and there is no need for excessive addition.

[0164] Background signal control:

[0165] The RLU values of the blank control group (0 pM) were all <250, indicating good method specificity and no significant background interference.

[0166] Experimental conclusion:

[0167] This test example directly verified the core innovation point of "sample processing optimization" in the claims (adding RNALater to inhibit degradation) and quantified the effects of different addition ratios. The data show that the addition of RNALater can significantly improve the detection sensitivity and accuracy, especially in complex biological matrices (such as serum), providing reliable technical support for the preclinical research of oligonucleotide drugs.

[0168] Test Example 2

[0169] Verification of Cross-Matrix Applicability (Including Cynomolgus Monkey Serum)

[0170] Experimental purpose

[0171] Verify the applicability of the detection method based on the MSD platform in different biological matrices (including mouse serum, rat tissue homogenate, human plasma and cynomolgus monkey serum), and evaluate its lower limit of quantification (LLOQ = 40 pM) and linear range (40 - 2560 pM) in cynomolgus monkey serum to ensure the stability and reliability of the method in complex biological samples.

[0172] The experimental groups are shown in Table 3.

[0173] Table 3 Experimental Groups

[0174]

[0175] Experimental Procedures

[0176] 1. Sample Preparation:

[0177] Conventional Matrix Group (mouse serum, rat tissue homogenate, human plasma): Add the ASO drug ISIS2302 to a final concentration of 400 pM, mix well, then add RNALater (1:1), and incubate at 25 °C for 2 hours.

[0178] Cynomolgus Monkey Serum Group: Add ISIS2302 to 40, 80, 160, 320, 640, 1280, 2560 pM respectively, repeat each concentration 3 times, add RNALater (1:1), and incubate at 25 °C for 2 hours.

[0179] 2. Hybridization and Detection (refer to Example 1):

[0180] Use the capture probe (5'-GAACAAGCAC-3' = SEQ ID No.2, LNA modified) and the detection probe (5'-CAACGGAGCA-3' = SEQ ID No.3, LNA modified), and dilute them to 50 nM.

[0181] Mix the hybridization buffer and the sample at a ratio of 1:1, incubate at 25 °C for 60 minutes, and transfer to the MSD plate for subsequent detection.

[0182] 3. Signal Reading:

[0183] Use the MSD SQ120 analyzer to detect the RLU value and calculate the recovery rate through the standard curve.

[0184] Experimental Reagents

[0185] Target Oligonucleotide: The ASO drug ISIS2302 (sequence:

[0186] 5'-TGCTCCGTTGGTGCTTGTTC-3' = SEQ ID No.1);

[0187] Probe System: Capture Probe (General Biosystems), Detection Probe (General Biosystems);

[0188] Biological Matrix: C57 mouse serum, SD rat liver tissue homogenate, healthy human plasma, cynomolgus monkey serum (all containing RNALater 1:1);

[0189] MSD GOLDTM 96-well plate, anti-digoxin-SULFO-TAG antibody, hybridization buffer.

[0190] Analysis of experimental results

[0191] 1. Verification of cross-matrix recovery rate (400 pM)

[0192] The results are shown in Table 4

[0193] Table 4 Verification of cross-matrix recovery rate

[0194] Matrix type Detection concentration (pM) Recovery rate (%) Mouse serum 400 98±3 Rat tissue homogenate 400 95±5 Human plasma 400 102±4 Cynomolgus monkey serum 400 96±4

[0195] Conclusion: The recovery rates in all matrices meet the requirements of bioanalysis (85%-115%), indicating that the method has good tolerance to complex matrices.

[0196] 2. Verification of lower limit of quantitation and linear range in cynomolgus monkey serum

[0197] The results are shown in Table 5 and Figure 2 as follows

[0198] Table 5 Verification of lower limit of quantitation and linear range in cynomolgus monkey serum

[0199]

[0200]

[0201] The following conclusions can be drawn from the data in Table 5 above:

[0202] Lower limit of quantitation (LLOQ): When the concentration is 40 pM, the recovery rate is 95±12%, CV<15%, meeting the LLOQ standard;

[0203] Linear range: In the range of 40-2560 pM, R 2 =0.999, indicating a good linear relationship;

[0204] Precision and accuracy: The recovery rates at all concentration points are between 85%-115%, and the intra-day / inter-day CV<10%.

[0205] This test example further verifies the detection performance of the method in cynomolgus monkey serum, demonstrating that it is not only applicable to conventional biological matrices (such as serum, tissue homogenate, plasma), but also can achieve highly sensitive (LLOQ = 40 pM) and wide linear (40-2560 pM) quantitative analysis in the serum of non-human primate models (cynomolgus monkeys). This result provides key technical support for preclinical pharmacokinetic studies of oligonucleotide drugs (especially non-human primate experiments), further highlighting the broad application potential of the present invention.

[0206] The above are only a few limited preferred embodiments of the present invention, and the description thereof is relatively specific and detailed. However, it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for detecting oligonucleotide drugs based on the MSD platform, characterized in that, It includes the following steps: (1) Probe design: Capture probe: It contains a nucleotide sequence complementary to the target oligonucleotide sequence, with a biotin modification at the 5'-end and at least one locked nucleic acid (LNA) modification site; Detection probe: It contains a nucleotide sequence complementary to the target oligonucleotide sequence, with a digoxigenin modification at the 3'-end and at least one locked nucleic acid (LNA) modification site; (2) Hybridization reaction: Dilute the capture probe and the detection probe to a concentration of 50 nM respectively, and incubate with the target oligonucleotide in the test sample in a hybridization buffer to form a capture probe-target oligonucleotide-detection probe complex; (3) Transfer and fixation: Transfer the hybridization product in step (2) to a streptavidin-coated MSD GOLDTM 96-well plate, and fix the complex through the binding of biotin and streptavidin; (4) Signal detection: Add an anti-digoxigenin-SULFO-TAG antibody to bind to the detection probe, and detect the signal intensity by electrochemiluminescence. The signal value is positively correlated with the concentration of the target oligonucleotide; (5) Sample processing optimization: Add RNALater to the biological sample with RNase activity to inhibit the degradation of the target oligonucleotide.

2. The method according to claim 1, wherein The sequence of the target oligonucleotide is 5'-TGCTCCGTTGGTGCTTGTTC-3'=SEQ ID No.

1.

3. The method according to claim 2, wherein The sequence of the capture probe is 5'-GAACAAGCAC-3'=SEQID No.2, with a biotin modification at the 5'-end and locked nucleic acid (LNA) modifications introduced at the 2nd, 4th, and 8th nucleotides; the sequence of the detection probe is 5'-CAACGGAGCA-3'=SEQ ID No.3, with a digoxigenin modification at the 3'-end and locked nucleic acid (LNA) modifications introduced at the 4th, 6th, and 9th nucleotides.

4. The method according to claim 1, wherein The hybridization buffer contains 60 mM disodium hydrogen phosphate anhydrous, 1 M sodium chloride, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.02% Tween-20.

5. The method according to claim 1, wherein The incubation conditions in step (2) are 25°C for 60 minutes, and the volume ratio of the hybridization buffer to the test sample is 1:

1.

6. The method according to claim 1, wherein In step (5), the addition ratio of RNALater is that the volume ratio of the biological sample to RNALater is 1:1 or 3:

1.

7. The method according to claim 1, characterized in that, The target oligonucleotide is an antisense oligonucleotide (ASO) or a small interfering RNA (siRNA).

8. The method according to claim 1, characterized in that, The sensitivity of the method is 0.4 pM, and the linear range spans 5 orders of magnitude.

9. The application of the method according to any one of claims 1-8 in the research and development of oligonucleotide drugs, pharmacokinetic studies, or the preparation of detection kits.

10. A kit for detecting oligonucleotide drugs based on the MSD platform, characterized in that, The kit contains: A capture probe with the sequence 5'-GAACAAGCAC-3' =SEQ ID No.2, with a biotin modification at the 5'-end and LNA modifications introduced at the 2nd, 4th, and 8th nucleotides; A detection probe with the sequence 5'-CAACGGAGCA-3' = SEQ ID No.3, whose 3'-end is modified with digoxigenin and LNA modifications are introduced at the 4th, 6th, and 9th nucleotides; Streptavidin-coated MSD GOLDTM 96-well plate; Anti-digoxigenin-SULFO-TAG antibody; Hybridization buffer and RNALater.

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