RU2810742C1 - Method of determining activity of restriction endonucleases in cell extracts of microorganisms - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: reaction is started by mixing solutions of the DNA probe and cell extract in a 1:1 ratio and the change in the fluorescence intensity of the FRET pair is recorded in the time interval of 15–3,600 sec. Solutions of substrate (20 µM), buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 7% glycerol) and sample of cell extract of the microorganism (20 μg of total protein) are mixed in a 96-well fluorometer plate. A complementary oligodeoxyribonucleotide carrying the FAM/BHQ1 FRET pair at the ends of the chain and also containing a 6-mer degenerate sequence is used as a DNA probe.

EFFECT: simplifying the method and increasing the productivity of determining the activity of restriction endonucleases in cell extracts of microorganisms.

4 cl, 3 dwg, 2 ex

Description

The invention relates to the field of biochemistry and concerns a method for determining the activity of restriction enzymes (restriction endonucleases) in a cell extract obtained from a variety of microorganisms.

Restriction endonucleases (restriction enzymes), which cleave both strands of DNA in a site-specific manner, are a fundamental tool in molecular biology. The discovery of endonucleases began in the 1960s and led to their commercial availability in the early 1970s. Although restriction endonucleases are primarily found in bacterial genomes and plasmids, they also exist in archaea, viruses, and eukaryotes. It is estimated that one in four bacteria examined may contain one or more restriction enzymes (Roberts, R. J. and Halford, S. E. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1993). Neisseria and Helicobacter pylori are the richest sources of restriction enzymes. In individual strains of these microorganisms, from 7 to 14 endonuclease genes were found, although some genes are not actively expressed (Stein, D.C., Gunn, J.S., Radlinska, M., Piekarowicz, A. Gene. 1995, Lin, L-F. Posfai, J. , Roberts, R. J., and Kong, H. Nuc. Acids Res. 2001).

There are currently 3,500 known restriction enzymes (of all possible types) that recognize 259 different DNA sequences. The vast majority of them, about 3460 enzymes recognizing 234 DNA sequences, are classified as type II restriction endonucleases. In general, common type II restriction endonucleases are homodimers (in most cases with a monomeric subunit mass between 25 and 35 kDa), require a divalent metal (Mg ²⁺ ) for catalysis, and cleave DNA within palindromes, partial palindromes, or interrupted palindromes. Despite their different primary sequences, type II restriction endonucleases have a similar three-dimensional structure: a U-shaped dimeric holoenzyme in which each of the identical subunits is arranged so that on the sides there are domains that contribute to recognition and catalysis, and at the bottom bridging (connecting) ) domains. Type II restriction endonuclease monomers are not active by themselves.

Currently, only 16 site-specific DNA nickases are known that cleave one of the DNA strands at a specific site. A total of 297 restriction enzymes have been cloned to date. Restriction endonucleases are still an area of active research, including aspects such as cleavage mechanism, in vivo functions, evolutionary origins, and as the subject of a site-specific DNA recognition model. But even as the number of characterized enzymes that are a staple of molecular biology continues to grow, the number of suppliers and the variety of their product lines increase, there is still no universal approach for screening restriction enzyme activity in cell extracts of various origins.

In this regard, the development of a screening method for determining the activity of restriction endonucleases in cell extracts is an urgent task and is extremely important for the development of biotechnological and genetic technologies.

Enzyme activity is characterized by the value of the catalytic constant k _cat , equal to the number of enzyme revolutions per unit time. As is widely accepted in enzymology, the specific activity (A) of an enzyme is taken to be the number of micromoles of product formed per minute per 1 mg of enzyme (µmol × min ^-1 × mg ^-1 ), (Biochemistry, 2004, T. 69, Issue 1, pp. 134-144).

A known method for testing bacterial strains for the presence of restriction endonuclease activity involves treating the collected cells with lysozyme and Triton X-100 and, after centrifugation, the supernatant is analyzed for the presence of specific endonuclease activity using agarose gel electrophoresis. The method allows you to analyze up to 100 colonies in 3-4 hours (Belavin P.A., Dedkov V.S., Degtyarev S.Kh. Applied biochemistry and microbiology. 1988).

A few years later, this method was slightly expanded. Cells of bacterial strains treated with lysozyme and Triton X-100 were incubated for 3 hours with phage λ DNA or plasmid. The result was analyzed using agarose gel electrophoresis (Dedkov B.S., Degtyarev S.Kh. Applied biochemistry and microbiology. 1992).

The main disadvantages of the known methods are the multi-stage and multi-component nature of the approaches used.

The prototype closest to the claimed method is a method for determining the activity of restriction enzyme PspXI in culture cells, which consists of the following. The reaction is started by mixing the substrate solution and cell lysate and incubating the mixture at 37°C for an hour. The reaction products are separated on an agarose gel and, after staining with ethidium bromide, the gel is visualized under UV light. As a substrate, DNA of phage λ, hydrolyzed with the restriction endonuclease HindIII or plasmid pBluescriptSK(+) (pBS), is used. The interaction of a restriction endonuclease with a DNA substrate leads to cleavage of the 2'-deoxyribose phosphate backbone at a specific site. As a result, the reaction products formed under the action of the HindIII restriction endonuclease and additional reaction products characteristic of the new restriction endonuclease are visible in the gel (Gonchar D.A., Abdurashitov M.A., Belichenko O.A., Dedkov V.S., Mezentseva N. V., Tomilova Yu.E., Degtyarev S.Kh. Bulletin of Biotechnology. 2005).

The disadvantages of the prototype are the laboriousness of the method associated with the separation of reaction products in the gel, as well as the low sensitivity of the method, due to the fact that reliable visualization of the reaction products after staining with ethidium bromide is achieved at a product concentration of at least 50 ng.

The objective of the invention is to simplify the method and increase the productivity of determining the activity of restriction endonucleases in a cell extract through the use of fluorescent DNA probes.

Technical result: simplifying the method and increasing the productivity of determining the activity of restriction endonucleases in cell extracts of microorganisms.

This task is achieved by the proposed method, which consists in recording over time changes in the fluorescence intensity of the substrate in the reaction mixture when interacting with a cell extract obtained from microorganisms of various origins. To analyze the activity of restriction enzymes, a DNA probe is used, which is a duplex:

5'-FAM - CACACANNNNNNGTAGTAGTA-3'

3'-BHQ1 - GTGTGTNNNNNNCATCATCAT-5'

where FAM is a fluorescein residue, BHQ1 is a residue of the black hole quencher 1 quencher, NNNNNN is a 6-unit fragment of a degenerate sequence (Fig. 1).

At the first stage, a cell extract is prepared. Under sterile conditions, the colony is transferred into a 1.5 ml tube containing 200 μl of lysis buffer (20 mM HEPES-KOH pH 7.5, 40 mM NaCl). For quantitative cell lysis, the resulting suspension is treated with ultrasound (10 pulses of 30 s, 22 GHz). After each pulse, the cell suspension is cooled in an ice bath for 90 s. The resulting cell extract is centrifuged for 20 minutes at 14500 rpm, 4°C.

The total protein concentration in the extract is determined based on the optical density of solutions at a wavelength of 280 nm in electronic absorption spectra. In each individual experiment, the total amount of protein in the cell extract solution was 20 μg. The cell extract is prepared each time immediately before work.

The reaction is started by mixing a solution of the substrate in the working buffer and the cell extract in a 1:1 ratio. Fluorescence is excited at a wavelength of 494 nm, registration is carried out in the spectral range of 500-550 nm. The change in the fluorescence intensity of the substrate is recorded in the time interval of 15-3600 s. Each kinetic curve is averaged over at least three experimental curves.

Kinetic curves are processed using equation (1) using the OriginPro 8.1 program (OriginLab Corp., USA). The value of the catalytic reaction rate constant k _obs obtained as a result of processing characterizes the activity of the enzyme.

where F ₀ is the amplitude, k _obs is the rate constant of the catalytic reaction.

A DNA probe is used as a substrate, which is a complementary oligonucleotide containing a 6-mer region of a degenerate sequence. At the 5' and 3' ends of this oligodeoxyribonucleotide there are fluorescent dyes, which are selected in such a way that the fluorescence emission spectrum of one dye (fluorescein, FAM) overlaps with the absorption spectrum of a second dye (fluorescence quencher, BHQ1). The formation of a duplex leads to spatial proximity of the fluorophore FAM and the fluorescence quencher BHQ1, resulting in effective quenching of fluorescence intensity (fluorescence resonance energy transfer, FRET).

The interaction of the substrate and the enzyme leads to cleavage of the 2'-deoxyribose phosphate backbone of DNA at a specific site (a specific sequence for each restriction endonuclease). As a result, the oligonucleotide chains in the reaction product diverge, which leads to spatial separation of the fluorophore and quencher. The fluorescence intensity increases as the reaction product accumulates.

The differences between the claimed method and the prototype are:

- use as a DNA probe of a duplex of oligonucleotides carrying a 6-mer region of a degenerate sequence, which allows 4096 variants of restriction endonuclease recognition sites to be realized in one sample;

- registration of the fluorescence intensity of the substrate is carried out in a tablet fluorimeter (rather than separation of reaction products in an agarose gel), which allows to significantly reduce the volume of biological material for analysis and increase the productivity of the method by reducing the analysis time by 3-4 times.

- kinetic curves are processed using the OriginPro8.1 program (OriginLab Corp., USA), which allows you to minimize the processing time of kinetic curves and reliably calculate V ₀ - the initial rate of product accumulation.

The invention is illustrated by the following examples of specific implementation.

Example 1

Determination of restriction endonuclease activity in a cell extract obtained from cells of the microorganism Priestia aryabhattai.

To determine the activity of restriction endonucleases, prepare a buffer with the following composition: 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, 5 mM MgCl ₂ , 1 mM DTT, 7% glycerol. A 20 μM substrate solution is prepared in a buffer, which is a duplex of oligonucleotides carrying a 6-unit region of a degenerate sequence (Fig. 1).

Lysis of Priestia aryabhattai cells is carried out in a buffer composition of 10 mM Tris-HCl, pH 7.5, 1 mM MgCl ₂ , 1 mM EDTA, 0.5% CHAPS, 10% glycerol, 0.1 mM PMSF, 0.5 mM β-mercaptoethanol. 150 μl of lysis buffer is added to the cell pellet, kept on ice for 30 minutes, then centrifuged (14500 rpm, 10 min). The total protein concentration in the resulting supernatant is determined using the Bradford method (20 μg of total protein is taken for analysis in each sample).

The reaction is started by mixing solutions of the substrate and extract in a 1:1 ratio and the change in fluorescence intensity of the substrate is recorded in the time interval of 15-3600 s at a temperature of 37°C. Fluorescence is excited at a wavelength of 485 nm, and fluorescence is recorded at a wavelength of 520 nm.

Figure 2 shows the results of recording the FRET signal for a cell extract from Priestia aryabhattai.

The kinetic curve is analyzed using the OriginPro8.1 program (OriginLab Corp., USA), which allows you to calculate the parameters of eq. 1. As a result of processing, the value k _obs = 0.0003±0.00002 s ^-1 is obtained, characterizing the activity of restriction endonucleases in the cell extract.

Example 2

Determination of the activity of recombinant preparations of restriction endonucleases using the example of commercially available restriction endonucleases NheI and HpaII using the claimed method.

To determine the activity of recombinant preparations of restriction endonucleases NheI and HpaII, prepare a buffer of the following composition: 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, 5 mM MgCl ₂ , 1 mM DTT, 7% glycerol. A 20 μM substrate solution is prepared in a buffer, which is a duplex of oligonucleotides carrying a 6-unit region of a degenerate sequence (Fig. 1).

A recombinant enzyme concentration of 20 μM is used. The reaction is started by mixing solutions of the substrate and extract in a 1:1 ratio and the change in fluorescence intensity of the substrate is recorded in the time interval of 15-3600 s at a temperature of 37°C. Fluorescence is excited at a wavelength of 485 nm, and fluorescence is recorded at a wavelength of 520 nm.

Figure 3 shows the results of recording the FRET signal obtained from the interaction of restriction endonucleases NheI and HpaII with a DNA probe containing a degenerate 6-mer sequence.

Kinetic curves are analyzed using the OriginPro8.1 program (OriginLab Corp., USA), which allows you to calculate the parameters of eq. 1. As a result of processing, the values of k _obs = 0.0009±0.0002 s ^-1 and k _obs = 0.0012±0.0004 s ^-1 are obtained for NheI and HpaII, respectively, which characterize the activity of recombinant restriction endonucleases.

Using the method makes it possible to simplify the determination of the activity of restriction enzymes in a cell extract, and, at the same time, speed up the analysis, which is necessary to increase the productivity of the search for new restriction enzymes.

Claims (7)

1. A method for determining the activity of restriction endonucleases in cell extracts of microorganisms, including mixing the substrate in a working buffer and the cell extract in a 1:1 ratio and recording changes in the fluorescence intensity of the substrate using a fluorimeter, followed by processing the resulting kinetic curves using a program, characterized in that A DNA probe, which is a duplex, is used as a substrate: 5’-FAM-CACACANNNNNNGTAGTAGTA-3’ 3’-BHQ1-GTGTGTNNNNNNCATCATCAT-5’ Where FAM is a fluorescein residue, BHQ1 is a residue of the black hole quencher 1 quencher, NNNNNN is a 6-mer fragment of a degenerate sequence. 2. The method according to claim 1, characterized in that the buffer contains 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, 5 mM MgCl ₂ , 1 mM DTT, 7% glycerol. 3. The method according to claim 1, characterized in that the recording of the fluorescence intensity of the substrate is carried out in a tablet fluorimeter. 4. The method according to claim 1, characterized in that the kinetic curves are processed using the OriginPro8.1 program.