Method for detecting conformational change when protein recognizes target site

Abstract

The invention discloses a method for detecting conformational change when protein recognizes a target site. The Cys at the 19 / 53 / 127 / 242 / 252 / 284 / 458 sites of the CasA protein in the Casade protein is mutated into Ser amino acid, and meanwhile, the 336 / 401 sites are mutated into the Cys, so that the obtained Cys-mini CasA keeps the function, and the mutant type of the CasA protein can be subjected to FRET fluorescence pair labeling, and therefore, the conformation state of the CasA can be truly reflected on the basis of single-molecule fluorescence resonance energy transfer smFRET, and the CasA can be used for detecting the CasA conformation. An effective means is provided for in-vitro research on the structure-function relationship of a system in CRISPR / Cascade, and the method has important significance for designing Cascade protein with optimized properties and enabling the Cascade protein to exert new functions.

Description

Technical Field

[0001] This invention relates to the construction of a method for observing conformational changes of cascade protein when recognizing targets in vitro, specifically involving the application of a single-molecule fluorescence resonance energy transfer (SIR) technology system in studying conformational changes of cascade protein when recognizing targets. Background Technology

[0002] The prevalent off-target effects of CRISPR-Cas systems are a major factor limiting their application in areas requiring precise gene editing, such as the treatment of human diseases. The type I CRISPR-Cascade system is widely distributed in over 90% of bacteria and archaea. The Cascade multi-protein subunit complex structure provides a more robust target recognition mechanism during target search. Therefore, a comprehensive analysis of the CRISPR-Cascade system will provide theoretical guidance for the rational design of CRISPR-Cas systems to reduce off-target effects.

[0003] Significant progress has been made in the study of the CRISPR-Cascade system both domestically and internationally in recent years. Some studies have resolved the three-dimensional structure of Cascade and proposed potential mechanisms for Cascade protein assembly and target recognition. One article reported the use of single-molecule fluorescence energy resonance transfer (smFRET) to discover that Cascade exists in two conformational states with different functions. Other researchers have used FRET to find that Cascade has multiple molecular conformations, which interconvert during the Cascade protein's search for target sites. However, another study refuted previous reports that conformational changes in the Cascade protein on the target DNA trigger priming. While these studies have employed different techniques to elucidate the working mechanism of Cascade, whether the recognition and binding of the Cascade protein to target DNA depends on conformational changes remains controversial.

[0004] Single-molecule fluorescence resonance energy transfer (smFRET) is an imaging technique based on the energy transfer phenomenon that occurs when the emission light from one fluorescent molecule overlaps with the excitation light from another fluorescent molecule at a very close spatial distance. It can provide spatial information within or between individual biomolecules at the nanoscale and is currently one of the important single-molecule techniques for studying real-time dynamic changes in biomolecules. Although smFRET has been used by several teams to study the conformational changes of cascade proteins during target recognition, current researchers using single-molecule FRET primarily study the interaction between cascade proteins and targets by labeling fluorescent molecules on substrate DNA or by placing one fluorescent label on the substrate and another on the cascade protein. These labeling methods do not directly demonstrate that cascade proteins undergo conformational changes during target gene site recognition. Using fluorescent double labeling of different protein subunits for FRET monitoring in solution systems cannot reflect the conformational changes of single molecules when cascade proteins recognize targets. Furthermore, double labeling of different protein subunits may result in labeled proteins failing to form complete cascade complexes at low concentrations, thus lacking sufficient activity for single-molecule monitoring.

[0005] In summary, existing methods for observing conformational changes in cascade proteins still need further optimization, mainly in two aspects: 1. Selecting protein subunits that undergo conformational changes when Cas proteins recognize targets for labeling; 2. Fluorescent labeling with minimal impact on activity, ensuring that labeled Cas proteins can form Cascade complexes and interact with target sites. Summary of the Invention

[0006] The purpose of this invention is to provide a method for observing cascade single-molecule FRET conformational changes by labeling FRET fluorescent molecular pairs on CasA proteins and developing an smFRET research system that can accurately reflect cascade conformational changes.

[0007] Firstly, this invention provides a mutant CasA protein. Site-directed mutagenesis is performed on the CasA protein, mutating Cys at positions 19 / 53 / 127 / 242 / 252 / 284 / 458 of the wild-type CasA protein to Ser amino acids. Simultaneously, the 336th and 401st amino acids of CasA are mutated to Cys, resulting in the CasA mutant (C19S / C53S / C127S / C242S / C252S / C284S / N336C / E401C / C458S). It is demonstrated that this mutant does not affect its inherent functions and can avoid the possibility of unnecessary site labeling.

[0008] Furthermore, by utilizing the chemical reaction between maleimide and the thiol group (-SH) of cysteine ​​(Cys), a fluorophore is attached to the target labeling site of the protein, achieving FRET fluorescent labeling of the CasA protein. The mutant CasA protein (named Cys-minimal CasA) was induced and purified using an E. coli protein expression system. Specifically, the fluorophores Cy3-maleimide and Cy5-maleimide were mixed with the mutant CasA protein as described in claim 1 at a ratio of 7.5:10:1 and reacted at 4°C in the dark. Optionally, unreacted free fluorophores were removed using a desalting column (PD-10) to obtain the labeled mutant CasA protein.

[0009] The present invention provides a nucleic acid molecule encoding the mutant CasA protein described above.

[0010] This invention provides a method for detecting conformational changes when IE-type Cascade protein recognizes target gene sites, comprising the following steps:

[0011] (1) IE-type Cascade protein is a CasB-E&crRNA complex formed by CasA, CasB, CasC, CasD, CasE and crRNA co-expressed; crRNA is composed of a spacer sequence and a hairpin structure sequence that can complement the target gene TS.

[0012] (2) Prepare the mutant CasA protein as described in claim 1 and label it with FRET fluorescent pair;

[0013] (3) Preparation of wild-type CasB, CasC, CasD, CasE and crRNA;

[0014] (4) Prepare target TS double-stranded DNA containing a target sequence complementary to the spacer sequence, a biotinylated PAM sequence adjacent to the target sequence, and a fluorescent molecule FAM.

[0015] (5) In step (3), CasB, casC, casD, casE and crRNA can form a CasB-E&crRNA complex. CasB-E&crRNA and the mutated CasA protein labeled according to claim 2 are mixed in vitro at a molar ratio of 1:5 to obtain the Cascade complex.

[0016] (6) Biotin-PEG was modified onto the surface of a glass slide to obtain a functionalized glass slide and fabricated into a microfluidic channel. Streptavidin was then incubated in the functionalized microfluidic channel to bind to the Biotin-PEG on the surface of the glass slide. The target TS double-stranded DNA from step (5) was then incubated in the functionalized microfluidic channel modified with streptavidin. The biotin and FAM double-labeled double-stranded DNA was then linked to the glass slide through the reaction of biotin and streptavidin.

[0017] (7) Incubate the microfluidic channel obtained in step (6) with the Cascade complex from step (5);

[0018] (8) Collect the single-molecule FRET fluorescence signal on the surface of the slide after step (7) by total internal reflection fluorescence microscopy, and distinguish the conformational changes of Cascade protein between "on" and "off" by the efficiency of single-molecule FRET.

[0019] Specifically, the proteins in step (3) are obtained by recombinant expression purification using recombinant bacteria, or they are obtained by co-expression purification.

[0020] In a specific embodiment, the mutated CasA protein described in step (2) is FRET fluorescently labeled at the N336C and E401C sites.

[0021] More specifically, the FRET fluorescent pairs described in step (2) are selected from maleimide-labeled Cy3 and Cy5.

[0022] This invention provides an effective means for studying the structure-function relationship of CRISPR / Cascade systems in vitro, and is of great significance for designing Cascade proteins with optimized properties and enabling them to perform new functions. Attached Figure Description

[0023] Figure 1 The figure shows representative results of cascade protein conformational changes during substrate recognition using smFRET in an embodiment of the present invention. The top row shows the trajectory of signal intensity change over time, and the bottom row shows the trajectory of FRET efficiency (E) change over time. Detailed Implementation

[0024] The following examples will describe the present invention in more detail. These examples are for illustrative purposes only, and the scope of the invention is not limited to these examples. The following examples use the conformation of Cascade when binding to DNA as the research object to introduce the application of the present invention in investigating the conformational changes of Cascade in vitro. Specific experiments are as follows:

[0025] The plasmid, bacterial strain, and primer information used in the examples are as follows.

[0026] Table 1 Information on plasmids and bacterial strains involved in this invention.

[0027]

[0028]

[0029] Table 2 Primers used in this invention

[0030]

[0031] The amino acid sequence of the unmutated CasA is shown in Sequence 1:

[0032]

[0033] 1. Main Reagents and Materials

[0034] All plasmid constructions in this embodiment were performed in E. coli TOP10. Protein expression was performed in E. coli BL21(DE3).

[0035] In this embodiment, the protein expression vector was either a commercially available plasmid psv272 / pCDFDuet-1 / pet52 / pACYC or a modified version thereof. All other experimental materials were commercially available. The plasmids and strains involved in this embodiment are shown in Table 1.

[0036] The reagents used for protein expression purification in this embodiment are: IPTG (isopropyl-β-d-thiogalactoside), protease inhibitor PMSF, buffer 1xNTA buffer (50mM Na2HPO4 (pH 8.0), 500mM NaCl, 5% glycerol, 1mM TCEP), buffer B1 (50mM Na2HPO4 (pH 8.0), 500mM NaCl, 5% glycerol, 1mM TCEP, 250mM imidazole), buffer C1 (20mM Tris-HCl (pH 7.5), 100mM NaCl, 5% glycerol, 1mM TCEP), 10K-MWCO centrifugation through ultrafiltration tube, 5000 molecular weight dialysis membrane, Ni-NTA affinity chromatography column, molecular sieve chromatography column, Coomassie brilliant blue rapid staining solution, and desalting column (PD-10). Except for the buffer, which was prepared in-house, all other reagents were commercially available.

[0037] The functionalized glass slides prepared in this embodiment include: adhesive tape, polytetrafluoroethylene tubing, glass slides (quartz slides), concentrated sulfuric acid, hydrogen peroxide, anhydrous ethanol, potassium hydroxide, methanol, acetic acid, APTES ((3-aminopropyl)triethoxysilane), mPEG-SVA (Laysan Bio), Biotin-PEG-SVA (Laysan Bio) (all PEG reagents were purchased from Laysan Bio), NaHCO3, and Streptavidin. Unless otherwise specified, all other reagents can be purchased from reputable reagent companies or online.

[0038] In this embodiment, binding buffer D1 (20 mM tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 5% glycerol) and imaging buffer (binding buffer supplemented with glucose oxidase, catalase (0.04 mg / mL), 0.8% glucose, 2 mM Trolox) were used. All reagents were available from Aladdin Company.

[0039] 2 Experimental Methods

[0040] 2.1 Plasmid Construction

[0041] A psv272-cfCasA-N336C / E401C plasmid (mutation sites C19S / C53S / C127S / C242S / C252S / C284S / N336C / E401C / C458S) was constructed to induce the expression of the cfCasA-N336C / E401C variant protein in *E. coli*. The construction method consisted of two steps. The psv272-cfCasA-N336C / E401C sequence fragment was obtained, specifically as follows: using the psv272-cfCasA sequence as a PCR template, primers Y-462 / 463 and Y-480 / 481 (primer sequences are shown in Table 2) were used to amplify the psv272-cfCasA-N336C / E401C fragment. The amplified product was transformed into Top10 competent cells, and single clones were selected for sequencing to obtain the plasmid.

[0042] 2.2 Protein purification

[0043] The psv272-cfCasA-N336C / E401C plasmid was transformed into Escherichia coli BL21(DE3). Single colonies were picked from the plate and cultured in LB medium containing kanamycin at 37°C until the absorbance (OD600 value) of the bacterial culture at 600 nm reached 0.6. Protein expression was then induced overnight at 16°C with 0.5 mM IPTG. The next day, the cells were collected by centrifugation and resuspended in 1xNTA buffer. 0.5 mM PMSF and 10 mM imidazole were added. The cells were lysed by sonication, and the lysate was centrifuged at 13500 rpm for 40 minutes. The supernatant was collected and filtered through a 0.2 μm filter membrane. The target protein was extracted from the clear lysate using a Ni-NTA affinity column. After loading, the protein was washed with 1xNTA buffer containing 25 mM imidazole, followed by elution with buffer B1. The protein-containing eluent was collected and mixed in one tube, and 1 / 50 of the mass fraction of rTev enzyme was added. The protein was dialyzed overnight at 4°C using a 5000 molecular weight dialysis membrane in 1xNTA buffer. The protein was loaded onto the Ni-NTA affinity column again, and the flow-through fraction was collected. The protein was concentrated to 1 mL using a 10K-MWCO centrifugal ultrafiltration tube. The protein was then purified a second time using a molecular sieve with buffer C1 at a flow rate of 0.5 mL / min. The purity of the protein in different eluent fractions was verified by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The target CasA protein was collected and concentrated to 50 μM using a 10K-MWCO centrifugal ultrafiltration tube. The CasA protein was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C for labeling.

[0044] The PCDF-casE / pet52-CasB-D / crRNA plasmid was co-transformed into Escherichia coli BL21(DE3). Single colonies were picked from the plate and cultured in LB medium containing streptomycin, ampicillin, and chloramphenicol at 37°C until the absorbance (OD600 value) of the bacterial culture at 600 nm reached 0.6. Protein expression was then induced overnight at 18°C ​​with 0.5 mM IPTG. Subsequent steps were similar to the purification of CasA. After molecular sieving, the purity of the protein in different elution fractions was verified by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The target protein was collected and concentrated to 50 μM using centrifugal ultrafiltration tubes. The target protein was then aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0045] 2.3 Fluorescein Labeling

[0046] The protein, maleimide-Cy3, and maleimide-Cy5 were mixed thoroughly at a molar ratio of 1:7.5:10. The protein was diluted to a final concentration of 10 μM using C1 buffer and incubated at 4 °C in the dark for 2 h. Excess unreacted dye was separated from the labeled CasA protein using a desalting column. The protein concentration and labeling efficiency were measured using a Nanodrop spectrophotometer with UV-Vis absorption spectroscopy. The labeled Cys-minimal CasA-Cy3 / Cy5 protein was aliquoted and stored at -80 °C.

[0047] 2.5 Preparation of Double-Stranded DNA

[0048] The four single strands (Y-496 / 501 / 513 / 514) used for the synthesis of double-stranded DNA (see Table 2) were all ordered from Qingke Biotechnology Co., Ltd. Double-stranded DNA was prepared by mixing single strands of DNA in a molar ratio of 1:1.2:2:5, denaturing at 95°C, and then slowly cooling and annealing.

[0049] 2.6 Preparation of Functionalized Glass Slides

[0050] 1) Cleaning the slides: Drill holes 1.2 mm in diameter on both sides of the long side of the slide using a hand drill, then clean the slides with water. Immerse the slides in 1M KOH and sonicate them for 20 minutes (100% power) using an ultrasonic cleaner. Next, use tweezers to pick up each slide and rinse it with running ultrapure water, then immerse the slides in ultrapure water and sonicate for 20 minutes (100% power). Then place the slides in a glass container, pour in concentrated sulfuric acid and hydrogen peroxide in a 3:1 ratio to submerge the slides, and after 40 minutes, use corrosion-resistant tweezers to remove the slides and rinse them thoroughly with ultrapure water. Then rinse three times with anhydrous methanol and soak them in anhydrous methanol for later use. In simple terms, cleaning the coverslips involves immersing them in 1M KOH and ultrasonically cleaning them for 20 minutes (100% power). Next, each slide is picked up with tweezers and rinsed with running ultrapure water. The slides are then placed in ultrapure water and ultrasonically cleaned for 20 minutes (100% power). Finally, they are rinsed three times with anhydrous methanol and dried with nitrogen gas for later use.

[0051] 2) Use APTES to treat glass slides: Immerse the cleaned glass slides in a solution of anhydrous methanol: glacial acetic acid: APTES in a ratio of 65:3:1 and let stand for 10 minutes. After sonicating for 1 minute, let stand for another 10 minutes. Then rinse three times with anhydrous methanol, rinse with a large amount of ultrapure water, and dry with nitrogen gas for later use.

[0052] 3) Modify the surface of the glass slide with PEG: Prepare a 0.1M NaHCO3 solution (prepare fresh before use), weigh two types of PEG into an EP tube: mPEG-SVA (80mg) and Biotin-PEG-SVA (2mg), dissolve and mix with 0.64mL of 0.1M NaHCO3 until most of the solid is dissolved, centrifuge at 14000rpm for 1min; take 70μL of the supernatant onto the glass slide treated with APTES, then stack the coverslip on top, and let it stand overnight in a moist and clean box at 4 degrees Celsius in the dark, then rinse with ultrapure water and dry with nitrogen.

[0053] 4) Microfluidic channel preparation: Place the nitrogen-dried glass slide with the modified side facing up on the table. Use double-sided tape to divide the two holes into a channel parallel to the edge direction. Cover with a coverslip and seal the edge without tape with AB glue. Fix the polytetrafluoroethylene tube inserted into the hole with AB glue and let it stand for 30 minutes to allow the glue to solidify.

[0054] 2.7 Preparation of smFRET samples

[0055] 1) Add 70 μL of Streptavidin to the microfluidic channel and incubate for 1 minute to allow it to bind to the Biotin-PEG on the slide surface. Then rinse with 200 μL of buffer D1 to remove unbound Streptavidin. Add 100 μL of 20 pM substrate double-stranded DNA and ligate the double-stranded DNA onto the slide through the reaction of biotin and Streptavidin. Then rinse with 200 μL of buffer D1 to remove unbound double-stranded DNA.

[0056] 2) Prepare a dual-labeled cascade complex by incubating Cys-minimal CasA-Cy3 / Cy5 at a final concentration of 500 nM and CasB-E (crRNA) at 2.5 μM in binding buffer for 10 minutes; then add 100 μL of imaging buffer containing 1.5 nM of the cascade complex to the channel to enable the cascade complex to recognize the target DNA.

[0057] 2.8smFRET data acquisition

[0058] The sample was observed using a prism-type TIRFM in the imaging buffer; fluorescence imaging was performed using a Nikon inverted fluorescence microscope; images were acquired using a 532nm laser excitation, a 60× / 1.2 objective lens, an EMCCD camera (Andor), and Nikon imaging software (parameter settings: exposure time set to 100ms, electron multiplication gain (EM gain) set to 300).

[0059] 2.9 Analysis of smFRET data

[0060] Signal point locations were extracted using ImageJ and a written Python code. Potential single-molecule signal points were then filtered out, and the fluorescence intensity changes of the extracted Cy3 / Cy5 signal points over time were obtained. Signals exhibiting single-molecule characteristics in both Cy3 and Cy5 intensity changes were used for subsequent analysis. FRET efficiency was calculated using the formula: E FRET = A / (A+D), where A represents the signal intensity of the acceptor fluorophore (Cy5) and D represents the signal intensity of the donor fluorophore (Cy3). The smFRET method described above was used to conduct experiments, and the representative signal point information obtained was analyzed as follows: Figure 1 As shown.

Claims

1. A mutant CasA protein, characterized in that, It was obtained by mutating Cys to Ser amino acids at positions 19 / 53 / 127 / 242 / 252 / 284 / 458 of the wild-type CasA protein, and mutating Cys to Cys at positions 336 / 401.

2. The mutant CasA protein as described in claim 1, characterized in that, It also includes fluorescent labeling, specifically by mixing the fluorophores Cy3-maleimide and Cy5-maleimide with the mutated CasA protein as described in claim 1 in a ratio of 7.5:10:1 and reacting at 4°C in the dark. Optionally, unreacted free fluorophores are removed using a desalting column (PD-10) to obtain the labeled mutated CasA protein.

3. A nucleic acid molecule encoding the mutant CasA protein as described in claim 1.

4. A method for detecting conformational changes when IE-type Cascade protein recognizes a target gene site, characterized in that, Includes the following steps: (1) IE-type Cascade protein is a CasB-E&crRNA complex formed by CasA, CasB, CasC, CasD, CasE and crRNA co-expressed; crRNA is composed of a spacer sequence and a hairpin structure sequence that can complement the target gene TS. (2) Prepare the mutant CasA protein as described in claim 1, and label it with FRET fluorescent pair; (3) Preparation of wild-type CasB, CasC, CasD, CasE and crRNA; (4) Prepare target TS double-stranded DNA containing a target sequence complementary to the spacer sequence, a biotinylated PAM sequence adjacent to the target sequence, and a fluorescent molecule FAM. (5) In step (3), CasB, casC, casD, casE and crRNA can form a CasB-E&crRNA complex. CasB-E&crRNA and the mutated CasA protein labeled according to claim 2 are mixed in vitro at a molar ratio of 1:5 to obtain the Cascade complex. (6) Biotin-PEG was modified onto the surface of a glass slide to obtain a functionalized glass slide and fabricated into a microfluidic channel. Streptavidin was then incubated in the functionalized microfluidic channel to bind to the Biotin-PEG on the surface of the glass slide. The target TS double-stranded DNA from step (5) was then incubated in the functionalized microfluidic channel modified with streptavidin. The biotin and FAM double-labeled double-stranded DNA was then linked to the glass slide through the reaction of biotin and streptavidin. (7) Incubate the microfluidic channel obtained in step (6) with the Cascade complex from step (5); (8) Collect the single-molecule FRET fluorescence signal on the surface of the slide after step (7) by total internal reflection fluorescence microscopy, and distinguish the conformational changes of Cascade protein between "on" and "off" by the efficiency of single-molecule FRET.

5. The method as described in claim 4, characterized in that, The proteins from step (3) are obtained by recombinant expression purification using recombinant bacteria, or by co-expression purification.

6. The method as described in claim 5, characterized in that, The mutated CasA protein described in step (2) is FRET fluorescently labeled at the N336C and E401C sites.

7. The method as described in claim 5, characterized in that, The FRET fluorescent pairs mentioned in step (2) are selected from maleimide-labeled Cy3 and Cy5.

Images