A method for screening RNA targets of a DNA-encoded compound library

Abstract

This invention provides a method for screening DNA-encoded compound libraries targeting RNA targets. The method reduces the false positive rate of DNA-encoded compound library screening by adding blocking fragments corresponding to RNA targets to the DNA-encoded compound library, thereby blocking DNA sequences that may bind to RNA targets.

Description

Technical Field

[0001] This invention belongs to the field of drug screening, specifically relating to a screening method for DNA-encoded compound libraries based on RNA targets. Background Technology

[0002] In the field of new drug development, high-throughput screening targeting biological targets is one of the main methods for rapidly obtaining lead compounds. However, traditional high-throughput screening based on single molecules is time-consuming, requires huge equipment investment, and has a limited number of compounds (millions). Furthermore, building a compound library requires decades of accumulation, limiting the efficiency and likelihood of lead compound discovery. In recent years, DNA-encoded compound library technology (WO2005058479, WO2018166532, CN103882532) has emerged, combining combinatorial chemistry and molecular biology techniques. It adds a DNA tag to each compound at the molecular level, enabling the synthesis of compound libraries with hundreds of millions of compounds in a very short time. Moreover, the compounds can be identified through gene sequencing, significantly increasing the size of the compound library and the efficiency of synthesis, becoming the trend for next-generation compound library screening technology. DNA-encoded compound library technology is beginning to be widely used in the pharmaceutical industry and has produced many positive effects (Accounts of Chemical Research, 2014, 47, 1247-1255).

[0003] However, since DNA-encoded compounds are tagged with DNA, and RNA targets may interact with these tags, false positives can occur in the screening data, interfering with the extraction and interpretation of true signals. Currently, there are methods not publicly reported in the literature to help identify such signals. One approach involves constructing a DNA library with the same DNA tag but without small molecules as a control, followed by data analysis after screening. However, this method requires library reconstruction, is time-consuming and costly, and it's difficult to ensure that the constructed DNA library perfectly matches the original library's DNA tag, thus limiting its application. Therefore, developing a rapid and economical screening method targeting RNA targets could further enhance the application value of DNA-encoded compound library screening technology. Summary of the Invention

[0004] This invention provides a method for screening RNA targets from a DNA-encoded compound library, characterized in that: before screening, a blocking fragment corresponding to the RNA target is added to the DNA-encoded compound library, and then screening is performed.

[0005] Furthermore, the blocked fragment is selected from RNA fragments, DNA fragments, and / or combinations of both.

[0006] Preferably, the design of the closed fragments corresponding to the RNA target is as follows: a 5-20 nt length sequence is truncated from the 5' end of the RNA target sequence as closed fragment sequence 1, and then a 5-20 nt length sequence is truncated from the 5' end with a shift of 1-10 nt as closed fragment sequence 2, until all are truncated, resulting in n closed fragment sequences 1 to n; preferably, the truncated length is 8-15 nt and the shift length is 2-6 nt; more preferably, the truncated length is 12 nt and the shift length is 3 nt.

[0007] More preferably, the cut of the closed fragment sequence 1 needs to cover the first unpaired circular region in the RNA target and extend beyond 3 bp.

[0008] Furthermore, the design of the closed fragment only covers the unpaired circular regions of the RNA target.

[0009] Furthermore, the design of the closed fragment covers the entire region of the RNA target.

[0010] Furthermore, the design of the closed fragment covers more than 60% of the RNA target region; preferably, it covers more than 70% of the region; more preferably, it covers more than 80% of the region.

[0011] Preferably, the step of adding the blocking fragment corresponding to the RNA target to the DNA-encoded compound library before screening is as follows: add blocking fragment sequence 1 to the DNA-encoded compound library, mix well and let stand, then add blocking fragment sequence 2, mix well and let stand, until blocking fragment sequence n is added, mix well and let stand.

[0012] Preferably, the screening method includes the following steps:

[0013] a) Before screening, add the closed fragment corresponding to the RNA target to the DNA-encoded compound library;

[0014] b) Incubate the RNA target and the sealed DNA-encoded compound library;

[0015] c) Fix the RNA target and then elute it;

[0016] d) Dissociation yields the selected DNA-encoded compounds.

[0017] Preferably, the screening method includes the following steps:

[0018] a) Before screening, add the closed fragment corresponding to the RNA target to the DNA-encoded compound library;

[0019] b) Fix the RNA target and incubate the RNA target with a blocked DNA-encoded compound library;

[0020] c) Elution;

[0021] d) Dissociation yields the selected DNA-encoded compounds.

[0022] More preferably, the ratio of the blocked fragment to the RNA target is 1:0.8 to 1.2.

[0023] More preferably, the incubation in step b is carried out in a screening buffer, which is 50 mM 2-aminobutyric acid, 80 mM potassium chloride, 0.3 mg / mL salmon sperm DNA, 0.01% Tween 20, pH 7.5; or the screening buffer is 50 mM 2-aminobutyric acid, 100 mM potassium chloride, 2 mM magnesium chloride, 0.3 mg / mL salmon sperm DNA, 0.05% Tween 20, pH 7.4.

[0024] More preferably, in step b or step c, the RNA target is immobilized using magnetic beads.

[0025] More preferably, in step c, elution is performed using an elution buffer, wherein the elution buffer is 50 mM 2-aminobutyric acid, 80 mM potassium chloride, 0.3 mg / mL salmon sperm DNA, 400 μM biotin, 0.01% Tween 20, pH 7.5; or the elution buffer is 50 mM 2-aminobutyric acid, 100 mM potassium chloride, 2 mM magnesium chloride, 0.3 mg / mL salmon sperm DNA, 0.05% Tween 20, pH 7.4.

[0026] More preferably, the dissociation in step d is carried out by adding a thermal dissociation buffer and incubating at 95°C for 10 minutes. The thermal dissociation buffer is 50 mM 2-aminobutyric acid, 160 mM potassium chloride, and pH 7.5.

[0027] More preferably, the dissociation in step d is performed by adding a dissociation buffer containing a positive compound that binds to the RNA target. Even more preferably, after adding the dissociation buffer containing the positive compound that binds to the RNA target, the mixture is incubated at room temperature for 15 minutes. Further, the dissociation buffer is 50 mM 2-aminobutyric acid, 100 mM potassium chloride, pH 7.4.

[0028] More preferably, the selected DNA-encoded compounds are quantified by qPCR. If the number of quantified molecules is greater than 10... 9 Then, replace the DNA-encoded compound library in step a with the selected DNA-encoded compounds, and repeat steps a to d until the number of quantified molecules is 10. 7 -10 8 .

[0029] The closed fragments mentioned in this invention refer to oligonucleotide fragments with a length of 5 to 20 nt that are consistent with the RNA target sequence.

[0030] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0031] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the TAR RNA design blocking fragment sequence 1 in Embodiment 1 of the present invention.

[0033] Figure 2 This is a schematic diagram showing different enrichment levels of degenerate sequences in the DNA tag in Example 1.

[0034] Figure 3 This is a schematic diagram showing different enrichment levels of degenerate sequences in the DNA tag in Example 2. Detailed Implementation

[0035] The DNA-encoded compound library used in this embodiment of the invention is derived from the pilot library of Chengdu Pioneer Pharmaceuticals Co., Ltd., and can be constructed according to the methods described in WO2005058479, WO2006135786, WO2018166532, or CN103882532. The DNA tag in the constructed DNA-encoded compound library incorporates degenerate sequences according to the method in WO2006135786. Other raw materials or reagents used in this embodiment of the invention can be obtained commercially.

[0036] Example 1: Screening of DNA-encoded compound libraries for TAR RNA

[0037] According to the appendix Figure 1 For example, the following 7 closed fragment sequences were designed for TAR RNA.

[0038]

[0039]

[0040] The DNA-encoded compound library for TAR RNA was screened according to the following groups;

[0041]

[0042] Follow these steps to perform the filtering operation;

[0043] Step 1: RNA blocking fragments block DNA-encoded compound libraries

[0044] Sample preparation

[0045] First, the aqueous solution of the DNA-encoded compound library was diluted with screening buffer, and then a positive control (a small peptide known to bind TAR RNA and be coupled to a DNA tag) was added. RNA blocking fragments (1-7) were added sequentially to Sample 2, mixing after each addition and incubating at room temperature for 30 minutes until all seven blocking fragments were added to the screening system. Simultaneously, Samples 1 and 3 were treated with the same procedure, adding the same volume of RNase-free deionized water as parallel controls.

[0046] Addition of TAR RNA

[0047] Add 5 μL of 200 uM TAR RNA aqueous solution to the corresponding group's DNA-encoded compound library, mix well, and use for subsequent screening.

[0048] Magnetic bead balance

[0049] Equilibrate 120 μL of streptomycin magnetic bead suspension with 500 μL of screening buffer for several tens of seconds, then separate using a magnetic rack and discard the supernatant. Repeat the equilibration process twice more for later use.

[0050] Start filtering

[0051] Incubation of TAR RNA with DNA-encoded compound library: The DNA-encoded compound library containing TAR RNA was incubated at room temperature for 1 hour, during which time it was thoroughly mixed using a rotary mixer.

[0052] Target fixation: The incubated solution was added to the equilibrated magnetic beads, and then thoroughly mixed using a rotary mixer; fixation was carried out at room temperature for a total of 0.5 hours.

[0053] Washing: Separate using a magnetic rack and discard the supernatant. Wash the magnetic beads with 500 μL of elution buffer for 1 minute, then separate using a magnetic rack and discard the supernatant again. Repeat the washing process for a total of 5 times.

[0054] dissociation

[0055] Separate the samples using a magnetic rack and discard the supernatant. Add 55 μL of thermal dissociation buffer and incubate in a 95°C metal bath for 10 minutes. Separate the samples using a magnetic rack and collect the supernatant. Resuspend the magnetic beads in 55 μL of the corresponding dissociation buffer.

[0056] Polymerase chain reaction (qPCR): After sample dissociation, the supernatant and magnetic bead fraction were diluted 20-fold and then added to the polymerase chain reaction system for amplification (pre-denaturation at 95°C for 10 minutes; 35 cycles: 95°C, 10 seconds → 55°C, 10 seconds → 72°C, 10 seconds). The number of molecules was calculated based on the standard curve.

[0057] The number of molecules obtained after the first round of screening is as follows. If the number of molecules is greater than 10^9, the DNA-encoded compound library used for screening will be replaced with the selected DNA-encoded compound, and a second round of screening will be performed following the same procedure. After screening, the samples were sequenced and decoded to analyze the effectiveness of this closed-loop experiment in reducing false positive signals.

[0058] sample Number of library molecules put in The number of molecules obtained in the first round of screening The number of molecules obtained from the second round of screening 1 5.58E+15 3.56E+10 1.78E+08 2 5.58E+15 2.74E+10 1.35E+08 3 5.58E+15 1.35E+10 1.26E+07

[0059] Filtering results

[0060] Figure 2 This is a schematic diagram illustrating the different enrichment levels of degenerate sequences in the DNA tag in Example 1. Each point in the scatter plot represents a degenerate sequence, and the percentage of enrichment in the experimental groups (S1, S2) and the control group (S3) is marked on the X and Y axes, respectively. Figure 2 As can be seen, when comparing the unblocked S1 group with the blank group S3, data analysis shows a large number of false positive signals generated by DNA-RNA binding, as shown in the red area; when comparing the blocked S2 group with the blank group, the DNA-RNA binding signal is significantly reduced, and most of the false positive signals can be removed.

[0061] Example 2: Screening of DNA-encoded compound libraries for TAR RNA using a combination of competitive elution and blocking fragments.

[0062] The DNA-encoded compound library for TAR RNA was screened according to the following groups;

[0063]

[0064] Follow these steps to perform the filtering operation;

[0065] Step 1: RNA blocking fragments block DNA-encoded compound libraries

[0066] Sample preparation

[0067] First, the aqueous solution of the DNA-encoded compound library was diluted with screening buffer, and then a positive control (a small peptide known to bind TAR RNA and coupled with a DNA tag) was added. RNA blocking fragments (1-7) from Example 1 were added to Sample 5 sequentially. After each addition of a blocking fragment, the sample was mixed and incubated at room temperature for 30 minutes until all 7 blocking fragments were added to the screening system.

[0068] Addition of TAR RNA

[0069] Add 5 μL of 200 μM TAR RNA aqueous solution to the corresponding group's DNA-encoded compound library, mix well, and use for subsequent screening.

[0070] Magnetic bead balance

[0071] Equilibrate 120 μL of streptomycin magnetic bead suspension with 500 μL of screening buffer for several tens of seconds, then separate using a magnetic rack and discard the supernatant. Repeat the equilibration process twice more for later use.

[0072] Start filtering

[0073] Incubation of TAR RNA with DNA-encoded compound library: The DNA-encoded compound library containing TAR RNA was incubated at room temperature for 1 hour, during which time it was thoroughly mixed using a rotary mixer.

[0074] Target fixation: The incubated solution was added to the equilibrated magnetic beads, and then thoroughly mixed using a rotary mixer; fixation was carried out at room temperature for a total of 0.5 hours.

[0075] Washing: Separate using a magnetic rack and discard the supernatant. Wash the magnetic beads with 500 μL of elution buffer for 1 minute, then separate using a magnetic rack and discard the supernatant again. Repeat the washing process for a total of 5 times.

[0076] dissociation

[0077] The magnetic beads were separated using a magnetic rack, and the supernatant was discarded. 55 μL of dissociation buffer containing 50 μM Tat peptides was added, and the mixture was incubated at room temperature for 15 minutes. The supernatant of each sample was then separated using a magnetic rack and collected. The supernatant samples were purified using a DNA purification and recovery kit to remove the Tat peptides for the next round of screening. Simultaneously, the magnetic beads were resuspended in 55 μL of the corresponding dissociation buffer.

[0078] Polymerase chain reaction (qPCR): After sample dissociation, the supernatant and magnetic bead fraction were diluted 20-fold and then added to the polymerase chain reaction system for amplification (pre-denaturation at 95°C for 10 minutes; 35 cycles: 95°C, 10 seconds → 55°C, 10 seconds → 72°C, 10 seconds). The number of molecules was calculated based on the standard curve.

[0079] The number of molecules obtained after the first round of screening is as follows. If the number of molecules is greater than 10^9, the DNA-encoded compound library used will be replaced with the selected DNA-encoded compound, and a second round of screening will be performed following the same procedure. After screening, the samples were sequenced and decoded to analyze the effect of this closed experiment combined with small peptide competitive elution on reducing false positive signals.

[0080] sample Number of library molecules put in The number of molecules obtained in the first round of screening The number of molecules obtained from the second round of screening 4 5.58E+15 1.07E+10 2.46E+07 5 5.58E+15 7.89E+09 2.55E+07 6 5.58E+15 5.12E+09 1.99E+07

[0081] Filtering results

[0082] Figure 3 This is a schematic diagram illustrating the different enrichment levels of degenerate sequences in the DNA tag in Example 2. Each point in the scatter plot represents a degenerate sequence, and the percentage of enrichment in the experimental group (S4, S5) and the control group (S6) is marked on the X and Y axes, respectively. Figure 3 As can be seen, when competitive elution is performed using small peptides, data analysis comparing the unblocked S4 group with the blank group S6 shows a significant number of false positive signals due to DNA-RNA binding, as indicated by the red area. However, when comparing the blocked S5 group with the blank group 6, the DNA-RNA binding signal is significantly lower. Figure 2 The S2 group was further significantly reduced, which can remove most of the false positive signals.

[0083] Example 3: Screening of DNA-encoded compound libraries from FMN Riboswitch RNA using RNA and DNA blocking fragments.

[0084] Screening buffer: 50 mM 2-aminobutyric acid, 100 mM potassium chloride, 2 mM magnesium chloride, 0.3 mg / mL salmon sperm DNA, 0.05% Tween 20, pH 7.4; Elution buffer: 50 mM 2-aminobutyric acid, 100 mM potassium chloride, 2 mM magnesium chloride, 0.3 mg / mL salmon sperm DNA, 0.05% Tween 20, pH 7.4; Dissociation buffer: 50 mM 2-aminobutyric acid, 100 mM potassium chloride, pH 7.4

[0085] Closed segment RNA blocked fragment sequence DNA blocking fragment 1 GCUUAUUCUCAGGG GCTTATTCTCAGGG 2 GGGCGAAAUUCCC GGGCGAAATTCCC 3 ACCGGCGGUAAAU ACCGGCGGTAAAT 4 GAAAGCCCGCGAG GAAAGCCCGCGAG 5 CAGAUCCGGUGUAA CAGATCCGGTGTAA 6 AAUUCCGGGGCCGA AATTCCGGGGCCGA 7 GUUAGAGUCCGGAU GTTAGAGTCCGGAT 8 AUGGGAGAGAGUAACG ATGGGAGAGAGTAACG

[0086] The DNA-encoded compound library for FMN RNA Riboswitch was screened according to the following groups;

[0087]

[0088] Follow these steps to perform the filtering operation;

[0089] Step 1: Block the DNA-encoded compound library using RNA or DNA blocking fragments.

[0090] Sample preparation

[0091] First, dilute the aqueous solution of the DNA-encoded compound library with screening buffer. Add RNA or DNA blocking fragments (1-8) to samples 1 and 2 in sequence. After each blocking fragment is added, mix and let stand at room temperature for 30 minutes until all 8 blocking fragments are added to the screening system.

[0092] Magnetic bead balance

[0093] Equilibrate Oligo d(T)25 MagBeads with screening buffer for several tens of seconds, then separate them using a magnetic rack and discard the supernatant. Repeat the equilibration process twice more for later use.

[0094] Start filtering

[0095] Target immobilization: FMN Riboswitch RNA was added to the equilibrated magnetic beads and thoroughly mixed using a rotary mixer; immobilization was carried out at room temperature for 0.5 hours.

[0096] Library incubation: FMN Riboswitch RNA and DNA-encoded compound library were incubated: DNA-encoded compound library with added FMN Riboswitch RNA was incubated at room temperature for 1 hour, during which time it was thoroughly mixed using a rotary mixer.

[0097] Washing: Separate using a magnetic rack and discard the supernatant. Wash the magnetic beads with 500 μL of elution buffer for 1 minute, then separate using a magnetic rack and discard the supernatant again. Repeat the washing process for a total of 5 times.

[0098] dissociation

[0099] Separate the samples using a magnetic rack and discard the supernatant. Add 100 μL of dissociation buffer containing 100 mM Ribocil-C and incubate at room temperature for 10 minutes. Separate the samples using a magnetic rack and collect the supernatant. Purify the supernatant samples using a DNA purification and recovery kit to remove Ribocil-C for the next round of screening. Resuspend the magnetic beads in 55 μL of the corresponding dissociation buffer.

[0100] The number of molecules obtained after the first round of screening is as follows. If the number of molecules is greater than 10^9, the DNA-encoded compound library obtained from the screening is replaced, and the second round of screening is performed following the same procedure. After screening, the samples are sequenced and decoded.

[0101] sample Number of library molecules put in The number of molecules obtained in the first round of screening The number of molecules obtained from the second round of screening 1 4.76E+15 2.55E+11 2.82E+08 2 4.76E+15 2.24E+11 1.68E+08 3 4.76E+15 3.44E+10 2.51E+06

[0102] The method of this invention can screen DNA-encoded compound libraries for RNA targets, which can reduce the false positive rate of screening and further broaden the application scope of DNA-encoded compound libraries. It can be applied to the screening of various RNA targets.

Claims

1. A method for screening RNA targets from a DNA-encoded compound library, characterized in that: Before screening, a blocked fragment corresponding to the RNA target was added to the DNA-encoded compound library, and then screening was performed. The closed fragment is selected from RNA fragments, DNA fragments, or a combination of both; The design of the closed fragments corresponding to the RNA target is as follows: a 5-20 nt length sequence is truncated from the 5' end of the RNA target sequence as closed fragment sequence 1, and then a 5-20 nt length sequence is truncated from the 5' end with a shift of 1-10 nt as closed fragment sequence 2, until all are truncated, resulting in n kinds of closed fragment sequences 1 to n. The cut of the closed fragment sequence 1 needs to cover the first unpaired circular region in the RNA target and extend beyond 3 nt; The ratio of the blocked fragment to the RNA target is 1:0.8 ~ 1.2; The design of the closed fragment satisfies any of the following conditions: (1) the closed fragment design covers only the unpaired circular region of the RNA target; (2) the closed fragment design covers the entire region of the RNA target.

2. The method according to claim 1, characterized in that: The design of the closed fragments corresponding to the RNA target is as follows: starting from the 5' end of the RNA target sequence, a sequence of 8-15 nt in length is extracted as closed fragment sequence 1. Then, a sequence of 8-15 nt in length is extracted from the 5' end with a shift of 2-6 nt as closed fragment sequence 2, until all are extracted, resulting in n kinds of closed fragment sequences 1 to n.

3. The method according to claim 1, characterized in that: The design of the closed fragments corresponding to the RNA target is as follows: a 12nt sequence is truncated from the 5' end of the RNA target sequence as closed fragment sequence 1, and then a 12nt sequence is truncated from the 5' end with a 3nt shift as closed fragment sequence 2, until all are truncated, resulting in n closed fragment sequences 1 to n.

4. The method according to claim 1, characterized in that: The steps for adding the blocking fragment corresponding to the RNA target to the DNA-encoded compound library before screening are as follows: add blocking fragment sequence 1 to the DNA-encoded compound library, mix well and let stand, then add blocking fragment sequence 2, mix well and let stand, until blocking fragment sequence n is added, mix well and let stand.

5. The method according to claim 1, characterized in that: The screening method includes the following steps: a) Before screening, add the closed fragment corresponding to the RNA target to the DNA-encoded compound library; b) Incubate the RNA target and the sealed DNA-encoded compound library; c) Fix the RNA target and then elute it; d) Dissociation yields the selected DNA-encoded compounds.

6. The method according to claim 1, characterized in that: The screening method includes the following steps: a) Before screening, add the closed fragment corresponding to the RNA target to the DNA-encoded compound library; b) Fix the RNA target and incubate the RNA target with a sealed DNA-encoded compound library; c) Elution; d) Dissociation yields the selected DNA-encoded compounds.

7. The method according to claim 5 or 6, characterized in that: The incubation in step b is performed in a selection buffer; the selection buffer is 50 mM glycerol, 80 mM potassium chloride, 0.3 mg / mL salmon sperm DNA, 0.01% Tween 20, pH 7.5; or the selection buffer is 50 mM glycerol, 100 mM potassium chloride, 2 mM magnesium chloride, 0.3 mg / mL salmon sperm DNA, 0.05% Tween 20, pH 7.

4.

8. The method according to claim 5, characterized in that: In step c, the RNA target is immobilized using magnetic beads.

9. The method according to claim 6, characterized in that: In step b, the RNA target is immobilized using magnetic beads.

10. The method according to claim 5 or 6, characterized in that: In step c, elution is performed using an elution buffer; the elution buffer is 50 mM glycerol, 80 mM potassium chloride, 0.3 mg / mL salmon sperm DNA, 400 uM biotin, 0.01% Tween 20, pH 7.5; or the elution buffer is 50 mM glycerol, 100 mM potassium chloride, 2 mM magnesium chloride, 0.3 mg / mL salmon sperm DNA, 0.05% Tween 20, pH 7.

4.

11. The method according to claim 5 or 6, characterized in that: In step d, dissociation is carried out by adding a thermal dissociation buffer and incubating at 95°C for 10 minutes. The thermal dissociation buffer consists of 50 mM aminobutanetriol, 160 mM potassium chloride, and pH 7.

5.

12. The method according to claim 5 or 6, characterized in that: In step d, dissociation is performed by adding a dissociation buffer containing a positive compound that has the ability to bind to RNA targets.

13. The method according to claim 5 or 6, characterized in that: The DNA-encoded compounds screened are quantified by qPCR, and if the quantified molecules are greater than 10 9 , the DNA-encoded compounds screened are replaced with the DNA-encoded compound library in step a, and then the operations of steps a to d are repeated until the quantified molecules are between 10 7 -10 8 .

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