阅读说明：本技术 细胞内检测生物分子间相互作用及其调控因子的方法与所用试剂 (Method for detecting interaction between biological molecules and regulating factor thereof in cell and used reagent ) 是由 李丕龙 王静 张冠伟 于 2018-08-01 设计创作，主要内容包括：本发明公开了细胞内检测生物分子间相互作用及其调控因子的方法与所用试剂。本发明的检测细胞内生物分子间相互作用的方法可用于检测细胞内的生物分子间的相互作用,并利用该方法进一步筛选影响已知具有相互作用的生物分子对间相互作用的调控因子。本发明的方法操作简便、灵敏度高、成本低廉、适用性广,适用于进行信号通路调控物的筛选,并且也可进行高通量筛选生物分子间互作的调控因子。(The invention discloses a method for detecting the interaction between biological molecules and a regulatory factor thereof in cells and a reagent used by the method. The method for detecting the interaction between biomolecules in the cell can be used for detecting the interaction between biomolecules in the cell and further screening the regulatory factors influencing the interaction between the pairs of biomolecules known to have interaction by using the method. The method has the advantages of simple operation, high sensitivity, low cost and wide applicability, is suitable for screening signal channel regulators, and can also screen the regulation factors of the interaction between biomolecules at high flux.)

1. Method for detecting the interaction between biomolecules in cells, the names of the biomolecules to be detected being X and X_LWherein X is a protein, a nucleic acid or a polysaccharide, and X is_LIs a protein, nucleic acid or polysaccharide, the method comprising U1) and U2):

u1) connecting a biomolecule named R and the X, and marking the obtained recombinant molecule as R-X; said R contains an intrinsically disordered protein/region; is connected with the X_LThe resulting recombinant molecule is designated X with the reporter group designated J_L-J；

U2) reacting said R-X with said X_L-introducing J into a biological cell, obtaining a recombinant cell, detecting in said recombinant cell whether the signal of said J is aggregated in the second phase formed by said intrinsically disordered protein/region, determining said X and said X_LWhether or not there is an interaction between: the signal of said J is concentrated in said second phase, said X and said X_LHave or are candidate for having an interaction; if the signal of J is not concentrated in the second phase, X and X_LHave no or candidate no interaction between them.

2. Method for identifying intermolecular regulation factors in cells, and biomolecule to be detected are named X and X_LAnd X is protein, nucleic acid or polySugar, said X_LIs a protein, a nucleic acid or a polysaccharide, said X and said X_LHave an interaction between them, the method comprising V1) and V2):

v1) connecting a biomolecule named R and the X, and marking the obtained recombinant molecule as R-X; said R contains an intrinsically disordered protein/region; is connected with the X_LThe resulting recombinant molecule is designated X with the reporter group designated J_L-J；

V2) reacting said R-X with said X_L-J is introduced into a biological cell to obtain a recombinant cell; culturing the recombinant cell, and adding a regulatory factor to be detected into a culture system of the recombinant cell to obtain a system to be detected; culturing the recombinant cell to obtain a control system; then detecting the signal intensity of the J in the recombinant cells in the test system and the control system in a second phase formed by the inherent disordered protein/region, and determining the X and the X of the regulatory factor to be tested_LWhether the interaction between (a) and (b) has a regulatory effect: if the signal of J is higher in the second phase of the test system than in the control system, the test regulatory factor pairs X and X_LInteraction between (a) and (b) has or is candidate to have a promoting effect; if the signal of J is equal to that of the control system in the second phase of the test system, the test regulatory factor pairs X and X_LNo or candidate for interaction between them has no regulatory effect; if the signal of J is lower in the second phase of the test system than in the control system, the test regulatory factor pairs X and X_LThe interaction between (a) and (b) has or is candidate to have an inhibitory effect.

3. The method according to claim 1 or 2, characterized in that: the R also contains a reporter group designated K, which is different from the J.

4. A method according to any one of claims 1-3, characterized in that: the J and the K are fluorescent reporter groups.

5. The method of claim 4, wherein: the fluorescent reporter group is a fluorescent protein.

6. The method according to any one of claims 1-5, wherein: the intrinsically disordered protein/region is H1) or H2) or H3):

H1) the amino acid sequence is the protein shown in the 258-772 th site of the sequence 1;

H2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the 258-772 th site of the sequence 1 in the sequence table and has the same function;

H3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of H1) or H2).

7. The method according to any one of claims 3-6, wherein: said K and said intrinsically disordered protein/region of said R are linked by a linking region or chemical bond.

8. The method according to any one of claims 1-7, wherein: said X_LX in J_LAnd said J is linked by a linking region or chemical bond;

and/or, said R and said X in said R-X are connected by said linker region or chemical bond.

9. The method according to claim 7 or 8, characterized in that: the connecting area is (Gly-Gly-Ser)_nOr contains (Gly-Gly-Ser)_nN is a natural number of 2 or more.

10. The method according to any one of claims 1-9, wherein: the R is I1) or I2) or I3) or I4):

I1) the amino acid sequence is a protein shown in 1 st-772 th position of the sequence 1;

I2) the amino acid sequence is a protein shown in the 1 st-784 th position of the sequence 1;

I3) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the 1 st to 772 th sites or the 1 st to 784 th sites of the sequence 1 in the sequence table;

I4) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of I1) or I2) or I3).

11. Use according to any one of claims 1 to 10, wherein: the biological cell is an animal cell, a plant cell or a microbial cell.

12. R according to any one of claims 1 to 10.

13. The biomaterial related to R as claimed in any one of claims 1 to 10, being any one of the following M1) to M4):

m1) a nucleic acid molecule encoding R according to any one of claims 1 to 10;

m2) an expression cassette containing the nucleic acid molecule of M1);

m3) a recombinant vector containing the nucleic acid molecule of M1) or a recombinant vector containing the expression cassette of M2);

m4) a recombinant microorganism containing M1) the nucleic acid molecule, or a recombinant microorganism containing M2) the expression cassette, or a recombinant microorganism containing M3) the recombinant vector.

14. The biomaterial of claim 13, wherein: m1) the nucleic acid molecule is any one of the following M1) -M8):

m1) the coding sequence is a cDNA molecule or DNA molecule at the 780-cozy 2324 site of the sequence 2 in the sequence table;

m2) the coding sequence is a cDNA molecule or DNA molecule at position 738-2324 of the sequence 2 in the sequence table;

m3) the coding sequence is a cDNA molecule or DNA molecule at the 9 th to 2324 th sites of the sequence 2 in the sequence table;

m4) the coding sequence is a cDNA molecule or a DNA molecule at the 780-2360 th site of the sequence 2 in the sequence table;

m5) the coding sequence is a cDNA molecule or a DNA molecule at the 738-2360 th site of the sequence 2 in the sequence table;

m6) the coding sequence is a cDNA molecule or DNA molecule at the 9 th to 2360 th site of the sequence 2 in the sequence table;

m7) has 75% or more or 75% identity to the nucleotide sequence defined by m1) or m2) or m3) or m4) or m5) or m6) and encodes the cDNA molecule or DNA molecule of R described in any one of claims 1 to 10;

m8) hybridizes under stringent conditions with a nucleotide sequence defined by m1) or m2) or m3) or m4) or m5) or m6) and encodes a cDNA molecule or DNA molecule of R as claimed in any of claims 1 to 10.

15. Use of any one of the R of claims 1-10 or the biomaterial of claim 13 or 14 for any one of the following:

x1) detecting biomolecular interactions within the cell;

x2) preparing a product for detecting the interaction between biomolecules in cells;

x3) identifying an intracellular biomolecular interaction regulator;

x4) preparing and identifying the product of the intermolecular interaction regulatory factor in the cell;

x5) screening the intermolecular interaction regulatory factor in the cell;

x6) preparing and screening the product of the intermolecular interaction regulatory factor in the cell;

x7) to detect the effect of the substance on the interaction between biomolecules in the cell.

Technical Field

The invention relates to a method for detecting the interaction between biological molecules and the regulation factor thereof in cells and a reagent used in the method, belonging to the technical field of biology.

Background

"phase transition" is a characteristic of a substance that is well known in the physical world and daily life, and in recent years, scientists have found that a phase transition (or phase separation) mechanism is also widely present in biological cells and performs an important biological function in the life activities of the cells.

Related studies have found that biological macromolecules with specific structures can be highly aggregated at a certain concentration due to interaction, and thus separated from a general solution phase to form a macromolecule-enriched independent phase (referred to as a second phase to be distinguished from the original solution phase), which is called "phase transition" (or "phase separation"). Under the microscope, a second phase can be seen to contain a large amount of aggregation products (small liquid drops, solid particles, gel substances and the like), the diameter of the second phase can reach micron level or even larger, and the second phase has higher identification degree. In biological cells, interactions between intrinsically disordered proteins/regions (IDPs/IDRs) are an important mechanism for driving phase transitions to occur. Intrinsically disordered proteins/regions are proteins/protein regions that are not stably ordered secondary and/or tertiary structures under physiological conditions, do not fold in whole or in part in the natural state, but are capable of performing biological functions normally, are widely present in organisms, and play important roles in cell signaling, protein interaction networks. The disordered structure is usually preferred in amino acid composition, and contains abundant polar amino acids such as G, P, E, S, Q, K, D, T, R and aromatic amino acids such as Y, F. It was found that the N-terminus of nucleoporin (nucleoporin) NUP98 anchored to the nuclear pore complex contains IDRs which mediate the occurrence of phase transition.

Disclosure of Invention

The technical problem to be solved by the invention is how to detect the interaction between biological molecules in cells and screen the regulatory factors influencing the interaction.

To solve the above technical problems, the present invention provides a method for detecting the interaction between biomolecules in a cell, wherein the names of the biomolecules are X and X_LWherein X is a protein, a nucleic acid or a polysaccharide, and X is_LIs a protein, nucleic acid or polysaccharide, the method comprising U1) and U2):

u1) connecting a biomolecule named R and the X, and marking the obtained recombinant molecule as R-X; the R contains inherent disordered proteins/regions (IDPs/IDRs for short); is connected with the X_LThe resulting recombinant molecule is designated X with the reporter group designated J_L-J；

U2) reacting said R-X with said X_L-introducing J into a biological cell, obtaining a recombinant cell, detecting in said recombinant cell whether the signal of said J is aggregated in the second phase formed by said intrinsically disordered protein/region, determining said X and said X_LWhether or not there is an interaction between: the signal of said J is concentrated in said second phase, said X and said X_LHave or are candidate for having an interaction; if the signal of J is not concentrated in the second phase, X and X_LHave no or candidate no interaction between them.

U2), when said X and said X are present_LAnd when said J is a protein, said R-X is reacted with said X_L-J can be introduced into a biological cell by contacting said R-X with said X_L-J encoding gene is introduced into said biological cell such that said recombinant cell obtained expresses said R-X and said X_L-J。

The invention also provides a method for identifying the intermolecular interaction regulatory factor in the cell, and the names of the biomolecules to be detected are X and X_LWherein X is a protein, a nucleic acid or a polysaccharide, and X is_LIs a protein, a nucleic acid or a polysaccharide, said X and said X_LHave an interaction between them, the method comprising V1) and V2):

v1) connecting a biomolecule named R and the X, and marking the obtained recombinant molecule as R-X; the R contains inherent disordered proteins/regions (IDPs/IDRs for short); is connected with the X_LThe resulting recombinant molecule is designated X with the reporter group designated J_L-J；

V2) reacting said R-X with said X_L-J is introduced into a biological cell to obtain a recombinant cell; culturing the recombinant cell, and adding a regulatory factor to be detected into a culture system of the recombinant cell to obtain a system to be detected; culturing the recombinant cell to obtain a control system; then detecting the signal intensity of the J in the recombinant cells in the test system and the control system in a second phase formed by the inherent disordered protein/region, and determining the X and the X of the regulatory factor to be tested_LWhether the interaction between (a) and (b) has a regulatory effect: as signal of said J in said system under testIs higher than the control system, the regulatory factor to be tested is on the X and the X_LInteraction between (a) and (b) has or is candidate to have a promoting effect; if the signal of J is equal to that of the control system in the second phase of the test system, the test regulatory factor pairs X and X_LNo or candidate for interaction between them has no regulatory effect; if the signal of J is lower in the second phase of the test system than in the control system, the test regulatory factor pairs X and X_LThe interaction between (a) and (b) has or is candidate to have an inhibitory effect.

In the above method, the R may further comprise a reporter group having the name K, the K being different from the J.

In the above method, both J and K may be a fluorescent reporter group.

In the above method, the fluorescent reporter group may be a fluorescent protein.

Further, J may specifically be a red fluorescent protein mCherry, and K may specifically be a green fluorescent protein GFP.

In the above method, the intrinsically disordered protein/region may be H1) or H2) or H3):

H1) the amino acid sequence is the protein shown in the 258-772 th site of the sequence 1;

H2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the 258-772 th site of the sequence 1 in the sequence table and has the same function;

H3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of H1) or H2).

To facilitate purification of the protein in H1), the amino-or carboxy-terminus of H1) may be attached a tag as shown in table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of H2) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of H2) above may be artificially synthesized, or may be obtained by synthesizing the encoding gene and then performing biological expression.

The gene encoding the protein in H2) above can be obtained by deleting one or several amino acid residues of codons in the DNA sequence encoding the inherently disordered protein/region, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end thereof.

In the above method, said K and said intrinsically disordered protein/region of said R may be connected by a linking region or a chemical bond.

In the above process, said X_LX in J_LAnd said J may be connected by said connecting region or chemical bond.

Said R and said X in said R-X may be connected by said connecting region or a chemical bond.

In the above method, the linking region may be (Gly-Gly-Ser)_nOr contains (Gly-Gly-Ser)_nN is a natural number of 2 or more.

n may be specifically 4 or 2.

In the above method, the R may be I1) or I2) or I3) or I4):

I1) the amino acid sequence is a protein shown in 1 st-772 th position of the sequence 1;

I2) the amino acid sequence is a protein shown in the 1 st-784 th position of the sequence 1;

I3) the protein which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the 1 st to 772 th sites or the 1 st to 784 th sites of the sequence 1 in the sequence table;

I4) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of I1) or I2) or I3).

To facilitate purification of the protein of I1), the amino-or carboxy-terminus of I1) was attached a tag as shown in Table 1.

The protein of I2) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in I2) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of I2) above can be obtained by deleting one or several amino acid residues from the DNA sequence encoding said R, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching to its 5 'end and/or 3' end the coding sequence of the tag shown in Table 1 above.

In the above method, the biological cell may be an animal cell, a plant cell or a microbial cell. In one embodiment of the invention, the animal cell is a HEK293 cell.

In one embodiment of the present invention, said X is p53 and said X_LIs MDM 2.

The invention also provides the R.

The invention also provides a biological material related to the R, wherein the biological material is any one of the following M1) to M4):

m1) a nucleic acid molecule encoding R according to any one of claims 1 to 10;

m2) an expression cassette containing the nucleic acid molecule of M1);

m3) a recombinant vector containing the nucleic acid molecule of M1) or a recombinant vector containing the expression cassette of M2);

m4) a recombinant microorganism containing M1) the nucleic acid molecule, or a recombinant microorganism containing M2) the expression cassette, or a recombinant microorganism containing M3) the recombinant vector.

In the above-mentioned biomaterial, M1) the nucleic acid molecule may be any one of the following M1) -M8):

m1) the coding sequence is a cDNA molecule or DNA molecule at the 780-cozy 2324 site of the sequence 2 in the sequence table;

m2) the coding sequence is a cDNA molecule or DNA molecule at position 738-2324 of the sequence 2 in the sequence table;

m3) the coding sequence is a cDNA molecule or DNA molecule at the 9 th to 2324 th sites of the sequence 2 in the sequence table;

m4) the coding sequence is a cDNA molecule or a DNA molecule at the 780-2360 th site of the sequence 2 in the sequence table;

m5) the coding sequence is a cDNA molecule or a DNA molecule at the 738-2360 th site of the sequence 2 in the sequence table;

m6) the coding sequence is a cDNA molecule or DNA molecule at the 9 th to 2360 th site of the sequence 2 in the sequence table;

m7) has 75% or more or 75% identity with the nucleotide sequence defined by m1) or m2) or m3) or m4) or m5) or m6), and encodes the cDNA molecule or DNA molecule of the R;

m8) hybridizes with the nucleotide sequence defined by m1) or m2) or m3) or m4) or m5) or m6) under strict conditions, and codes the cDNA molecule or DNA molecule of the R.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identical to the nucleotide sequence of the present invention that encodes said R. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

M2) the expression cassette containing a nucleic acid molecule encoding said R (R gene expression cassette) refers to a DNA capable of expressing said R in a host cell, which DNA may include not only a promoter for initiating transcription of said R gene but also a terminator for terminating transcription of said R gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the R gene expression cassette can be constructed using an existing vector. The vector may be a plasmid, cosmid, phage or viral vector. The plasmid can be pcDNA3.1 vector.

X13) the recombinant vector can be pcDNA3.1-GFP-NUPN, the pcDNA3.1-GFP-NUPN is a recombinant vector obtained by replacing the DNA fragment (containing the recognition sequences of NotI and XbaI) between the NotI and XbaI recognition sequences of the pcDNA3.1 vector with the DNA molecule shown in the sequence 2 in the sequence table. The pcDNA3.1-GFP-NUPN can express a fusion protein GFP-NUPN of GFP fusion NUPN shown in a sequence 1.

The microorganism may be a yeast, bacterium, algae or fungus.

The invention also provides any one of the following applications of the R or the biomaterial:

x1) detecting biomolecular interactions within the cell;

x2) preparing a product for detecting the interaction between biomolecules in cells;

x3) identifying an intracellular biomolecular interaction regulator;

x4) preparing and identifying the product of the intermolecular interaction regulatory factor in the cell;

x5) screening the intermolecular interaction regulatory factor in the cell;

x6) preparing and screening the product of the intermolecular interaction regulatory factor in the cell;

x7) to detect the effect of the substance on the interaction between biomolecules in the cell.

In the above application, the cell may be an animal cell, a plant cell or a microbial cell. In one embodiment of the invention, the animal cell is a HEK293 cell.

In the above application, the product may be a kit.

The screening of the intermolecular interaction-regulating factor can be carried out by high-throughput screening, and the identification of the intermolecular interaction-regulating factor can also be carried out by high-throughput identification.

Experiments prove that the method for detecting the interaction between the biomolecules in the cells can be used for detecting the interaction between the biomolecules in the cells, and the method is utilized to further screen the regulatory factors influencing the interaction between the pairs of biomolecules known to have interaction. The method has the advantages of simple operation, high sensitivity, low cost and wide applicability, is suitable for screening signal channel regulators, and can also screen the regulation factors of the interaction between biomolecules at high flux.

Drawings

FIG. 1 shows the detection of the interaction between P53 and MDM 2. A is the observation of the second phase morphology generated by the system 1, and the right image is an enlarged image of a frame selection area of the left image; and B is laser confocal scanning microscopic imaging analysis of the systems 1-8. Scale 20 μm.

Figure 2 is a confocal scanning laser microscopy imaging analysis of the effect of inhibitors on the interaction between P53 and MDM 2. Scale 20 μm.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

The pcDNA3.1 vector (Yoo et al, A new strain for assessing the selectivity of protein function: siRNA knock down/knock-in targeting the 3' -UTR, RNA (2007),13: 921-929.) in the following examples was publicly available from the applicant, and this biomaterial was only used for repeating the experiments related to the present invention, and was not used for other purposes.

The HEK293 cells (SHIN et al, Overexpression OF PGC-1 α enhanced cell proliferation and modelling OF HEK293 cells through the regulation OF Sp1 and Acyl-CoA binding protein, INTERNATIONAL JOURNAL ONCOLOGY 46:1328-1342,2015) in the examples described below were publicly available from the Applicant only for the purpose OF repeating the experiments relating to the present invention and not for other purposes.