CN114317684B - Intracellular magnesium ion imaging method based on TNA molecules - Google Patents

Abstract

The invention discloses a TNA molecule-based intracellular magnesium ion imaging method. Belongs to the technical field of metal ion imaging, and comprises the following specific steps: screening magnesium ion-dependent TNA molecules with RNA cleavage activity in vitro; performing activity test and truncated analysis on the TNA molecular sequence obtained by screening; selecting TNA molecules with highest activity for biochemical characterization; constructing a TNA molecular cutting system as a magnesium ion fluorescence sensor; verifying the performance of the fluorescence sensor in an in vitro buffer; the sensor was transfected into cells for imaging of magnesium ions in vivo. The invention can effectively carry out fluorescence response on magnesium ions with different concentrations in vitro, the detection limit reaches 0.35mM, and can carry out fluorescence imaging on the magnesium ions in living cells, thus being more stable and resistant to nuclease degradation compared with a method for detecting by using natural nucleic acid, and providing a new tool for molecular biology.

Description

Intracellular magnesium ion imaging method based on TNA molecules

Technical Field

The invention belongs to the technical field of metal ion imaging, relates to a TNA molecule-based intracellular magnesium ion imaging method, and particularly relates to a method for in-vitro screening of TNA molecules capable of cutting RNA for intracellular magnesium ion imaging.

Background

Magnesium ion is the most abundant divalent cation in mammals and has wide biological effects. Magnesium ions exert important biological functions such as functional metabolism, signal transduction, and structural composition through interactions with proteins, nucleic acids, or membrane structures within cells, and thus imaging and detection of magnesium ions within cells is very important. Since natural nucleic acids have been widely studied in the field of metal ligands, there have been studies on the in vitro screening method to obtain dnase capable of specifically cleaving RNA under the activation of metal ions, and since 8-17 deoxyribose nucleic acid obtained by screening is more active than magnesium ions under the activation of zinc ions and lead ions, 8-17 is used for detecting lead ions in the environment as well as zinc ions in cells. Since natural nucleic acids are susceptible to nuclease degradation within cells, no natural ribozyme is currently available for direct detection of intracellular magnesium ions. Threose Nucleic Acid (TNA) is an RNA analogue that forms a stable duplex with DNA or RNA while being capable of storing and transmitting genetic information. Because of its simple chemical structure and different monomer connection mode from natural nucleic acid, it is more resistant to degradation of nuclease and can be used in cell environment.

Disclosure of Invention

The invention aims to: the invention aims to provide a method for imaging intracellular magnesium ions based on Threose Nucleic Acid (TNA) molecules, in particular to a method for in vitro screening of TNA enzyme capable of cutting RNA for imaging intracellular magnesium ions.

The technical scheme is as follows: the invention relates to a TNA molecule-based intracellular magnesium ion imaging method, which comprises the following steps:

(1) Screening magnesium ion-dependent TNA molecules with RNA cleavage activity in vitro;

the specific operation method is as follows:

firstly, synthesizing an initial DNA molecule random library and a primer, and then performing circular screening; in each round of screening, a TNA library is obtained by adopting a primer extension method;

then, separating by streptavidin to obtain a single-chain library containing RNA substrate and TNA molecules, incubating the library in a buffer solution containing magnesium ions, and under the catalysis of the TNA molecules, the TNA molecules with cleavage activity catalyze the substrate to break, so that the TNA molecules are eluted from the magnetic beads;

finally, the eluted TNA molecules are subjected to reverse transcription and PCR amplification to obtain an enriched DNA molecule library, and the enriched DNA molecule library is applied to the next round of screening; repeating the screening for multiple rounds, gradually increasing the screening pressure, reducing the concentration of magnesium ions from 20mM to 1mM, and reducing the incubation time from 18 hours to 1 hour to obtain TNA molecules with high activity (sequencing the enriched DNA molecules and deducing the sequence information of the TNA molecules according to the sequencing result; finally preparing single-chain TNA molecules, testing the catalytic activity of the single-chain TNA molecules in the presence of magnesium ions, wherein TNA molecules with RNA cleavage activity are threose ribozymes);

selecting TNA molecules with highest activity for biochemical characterization;

the specific contents are as follows: firstly, adding different divalent metal ions into a buffer solution, and testing the selectivity of TNA molecules to the metal ions; then adding magnesium ions with different concentrations into the buffer solution, and testing the change of the catalytic activity of TNA molecules along with the concentration of the magnesium ions; then testing the influence of different pH values on the reaction kinetic parameters; finally, the first order reaction kinetic constants, and the Mi constants, of TNA molecules were tested;

wherein, the TNA molecule with highest activity specifically refers to: the sequence of TNA molecules with highest activity obtained by the truncation experiment and treatment with snake venom phosphodiesterase is shown as follows:

5’-GTAGGAGAGGTTATCGTTGGAGGGAGATGAGTGTAG-2’；

as one scheme of the invention, the sequence T17-22 of the TNA enzyme is shown as SEQ ID NO:1 is shown as follows:

5’-GTAGGAGAGGTTATCGTTGGAGGGAGATGAGTGTAG-2’

(SEQ ID NO：1)

preferably, the multiple rounds of repeated screening are 16 rounds of repeated screening, for a total of 17 rounds of screening.

Specifically, the specific steps are as follows:

(A) Synthesizing a random library of initial DNA molecules and upstream and downstream primers;

(B) Taking a DNA molecule random library, and carrying out primer extension and chain separation by taking tC, tT, tG and tA nucleoside triphosphates as substrates to obtain a non-natural nucleic acid library;

(C) Incubating the non-natural nucleic acid library solution in a buffer solution containing magnesium ions at a pH of 7.5,37 ℃ and collecting an eluent;

(D) Performing reverse transcription reaction by taking the eluent as a template;

(E) The reverse transcription product is used as a template, after 19-25 cycles of PCR amplification, single chains are separated, and an enriched library obtained by the screening of the round is obtained and can be used for the next round of screening;

(F) Repeating steps (B) - (E);

(G) After 17 rounds of screening, connecting the PCR amplification products obtained in the last round of screening with the carrier, and sequencing colonies containing the successfully connected carrier;

(H) Deducing sequence information of TNA molecules according to a sequencing result, and preparing single-chain TNA molecules through polymerase extension reaction, chain separation and snake venom phosphodiesterase treatment;

(I) Testing the cleavage activity of TNA molecules on RNA substrates under the activation of magnesium ions by using an electrophoresis method to obtain a plurality of TNA molecules with catalytic activity;

(J) Carrying out secondary structure prediction and truncation optimization on TNA molecules with higher affinity to obtain the TNA enzyme;

wherein the polymerase in step (B) is selected from KOD-RI polymerases;

wherein the polymerase in step (D) is selected from Bst 2.0 polymerase;

wherein the vector in step (G) is selected from pEASY-T1;

(2) Constructing a TNA molecular cutting system as a magnesium ion fluorescence sensor;

the construction of the magnesium ion fluorescence sensor specifically comprises the following steps:

firstly, respectively marking a fluorescent group FAM and a quenching group BHQ-1 on the 5 'end and the 3' end of an RNA substrate;

then, annealing the RNA substrate and TNA molecules to form a complex; adding buffer solution into the complex for incubation, and quenching fluorescence when magnesium ions are not present; when magnesium ions exist, TNA molecules catalyze RNA substrates to break, fluorescent groups are released, and fluorescent signals are recovered;

verifying the performance of the fluorescence sensor in an in vitro buffer; the concrete steps are as follows:

when the magnesium ion concentration is less than 20mM, the fluorescence signal intensity of the system and the magnesium ion concentration show a linear relation, and the detection limit is 0.35mM;

(3) Transfecting the sensor into a cell for imaging magnesium ions in vivo; the method comprises the following steps:

firstly, transfecting a fluorescence sensor into cells by using a transfection reagent Lipofectamine 3000;

then, using a calcium ion carrier to transport magnesium ions in the culture medium into cells;

finally, imaging the cells after incubation by using a confocal microscope;

the performance of the catalyst is as follows:

the detection method can carry out fluorescence imaging on magnesium ions with the concentration of 20mM in living cells.

The beneficial effects are that: compared with the prior art, the TNA enzyme with RNA cutting activity is obtained by the in-vitro screening technology, and the TNA enzyme has higher specificity to magnesium ions. Compared with the prior screening-obtained deoxyribozyme, the deoxyribozyme has the advantages of simple structure and strong biological stability, has higher specificity on magnesium ions in buffer solution, can specifically respond to the concentration change of the magnesium ions in cells, can realize the detection of different magnesium ion concentrations in vitro after being constructed into a fluorescence sensor for magnesium ion response, can further realize the imaging of the magnesium ions in cells, and is a tool for imaging the magnesium ions in cells more stable than the deoxyribozyme.

Drawings

FIG. 1 is a screening flow chart of the method of the present invention;

FIG. 2 is a graph showing the results of the elongation reaction and the reverse transcription reaction of the present invention;

FIG. 3 shows the result of detecting TNA molecular activity of the present invention;

FIG. 4 shows the results of biochemical characterization of T17-22 molecules obtained according to the present invention

FIG. 5 is a schematic diagram of a magnesium ion fluorescence sensor according to the present invention;

FIG. 6 is a graph showing the results of imaging magnesium ions in cells using TNA enzyme-based fluorescence sensors of the present invention.

Detailed Description

The invention is further illustrated below with reference to examples.

Example 1 in vitro screening procedure for RNA-cleaving TNA enzyme of the invention:

1. synthesis of a random library of initial DNA molecules and primers:

random library nucleic acid sequences of DNA molecules are shown below:

5’-TGTCTACACTGAAGCTTAC-N40-CGTACTGCATACGAGTGTC-3’；

n40 represents a random nucleic acid sequence, and the proportions of the four deoxyribonucleotide monomers are as follows:

A:T:G:C＝2:2:2:1；

the TNA library extension reaction primer nucleic acid sequences are shown below:

5’-Biotin-AAAAA-CTCAT-r(CAUGC)-AGCTCGACACTCGTATGCAGTACG-3’；

the PCR upstream primer nucleic acid sequence is shown below:

5’-Biotin-GACACTCGTATGCAGTACG-3’；

the downstream primer nucleic acid sequence is as follows:

5’-TGTCTACACTGAAGCTTAC-3’。

2. in vitro screening procedure for TNA enzyme that cleaves RNA:

2.1, taking an initial DNA molecule random library to carry out primer extension to obtain a non-natural nucleic acid library; as shown in the left diagram of fig. 2, the specific steps are as follows:

1nmol of DNA molecule was added to 1X standard Taq enzyme reaction buffer, gradient annealed by adding 1. Mu.M downstream primer, and sequentially added with 0.1mM tNTPs,1.5mg/mL KOD-RI polymerase (1 mM MnCl in advance) ₂ Pretreatment for 15 minutes at room temperature), the reaction condition is 55 ℃ for 4 hours;

2.2, separating to obtain TNA library, and incubating in a screening buffer, wherein the method comprises the following specific steps:

1mL of streptavidin-coated magnetic beads were washed 3 times with 500. Mu.L of washing solution (50 mM Tris-HCl,200mM NaCl,1mM EDTA,pH 7.5); incubating the primer extension product and streptavidin-coated magnetic beads for 30min at room temperature, and removing the supernatant; after washing the magnetic beads 3 times with washing solution, 5 times with 500. Mu.L of cold NaOH (0.1M) containing 1mM EDTA (each washing <30 s) to remove the DNA template; the TNA library suspended on the magnetic beads was then immediately neutralized with 500. Mu.L of a neutralization buffer (50 mM Tris-HCl,1mM EDTA,pH 6.0); finally, washing with 500 mu L of enzyme-free water three times, so that the TNA single-chain library obtained by the extension reaction is immobilized on magnetic beads containing streptavidin;

to the TNA library was added 110. Mu.L of screening buffer (50 mM Tris-HCl,154mM NaCl,20mM MgCl) ₂ pH 7.5), incubating it at 37 ℃; in the first round of screening, TNA library was screened against a library containing 20mM MgCl ₂ Culturing for 18h in a screening buffer; as the screening proceeds, mgCl ₂ The concentration is gradually reduced to 1mM, and the culture time is gradually shortened to 1h;

2.3, after incubation, reversely transcribing the supernatant serving as a template into cDNA (complementary deoxyribonucleic acid), wherein the right graph of the graph is shown in FIG. 3; the reaction contained 0.1. Mu.M primer, 1 XThermoPol buffer, 0.5mM dNTPs and 0.4U/mL Bst 2.0 polymerase, incubated at 55℃for 4h;

2.4, PCR amplification is carried out by using a PCR primer pair and Taq DNA polymerase with a reverse transcription product as a template: 95 ℃,5min, N cycles (95 ℃,15s;49 ℃,15s;72 ℃,10 s), 72 ℃,5min; determining the cycle times (N) by adopting a small-system PCR gradient experiment;

2.5, carrying out chain separation on the PCR product, wherein the specific steps are as follows:

washing resin loaded with streptavidin twice by using PBS, adding an extension reaction product solution for incubation for 30min, washing once by using PBS, eluting 3 times by using 200mM NaOH, collecting 3 eluents, adding a sodium acetate solution for regulating pH to be neutral, desalting by using a desalting column, and using the collected DNA sequence for templates for the next round of screening;

2.6, repeating the steps 2.1-2.5;

2.7, after 17 rounds of screening, connecting the PCR amplification product obtained after the last round of screening to a pEASY-T1 vector, and sequencing a colony containing a successfully connected vector;

2.8, deducing TNA molecular sequence information according to a sequencing result, synthesizing a primer extension template, performing primer extension reaction, and separating single chains to obtain TNA molecules;

2.9, incubating TNA molecules and fluorescent-labeled RNA substrates in a screening buffer solution, and testing the catalytic activity of the TNA molecules by an electrophoresis method;

2.10, carrying out secondary structure prediction and truncation optimization on TNA enzyme with highest activity to obtain TNA enzyme T17-22 with high activity and strong specificity. The predicted result of the secondary structure of T17-22 is shown in FIG. 5;

the sequence of TNA enzyme T17-22 is shown in SEQ ID NO:1 is shown as follows:

5’-GTAGGAGAGGTTATCGTTGGAGGGAGATGAGTGTAG-2’(SEQ ID NO：1)

wherein, the monomers ATCG are TNA monomers;

further, the polymerase used for primer extension in the step 2.1 is KOD-RI reported in the literature;

further, the polymerase used in the reverse transcription reaction in the step 2.3 is Bst 2.0 reported in the literature;

further, the fluorescent marker modified at the 5' end of the RNA substrate in the step 2.9 is Cy5.5;

example 2 the procedure of the present invention for constructing a fluorescence sensor for magnesium ions for intracellular magnesium ion imaging:

1. the initial sequence was synthesized as follows:

the DNA template sequence of biotin-labeled T17-22 is shown in the following figure:

5’-Biotin-AACTCATCTCCCTCCAACGATAACCTCTCCTACCGTACTGCATACGAGTGTC-3’；

2. preparation of TNA enzyme T17-22 molecule:

2.1, obtaining a hybridization double chain of TNA and DNA through a primer extension reaction, wherein the specific steps are as follows:

first, the primer and the biotin-labeled template were heated in 1×thermo pol buffer at 90 ℃ for 5 minutes, then gradient cooled to 4 ℃ (10 ℃/min) for annealing; then 1mM MnCl is added ₂ KOD-RI polymerase (1 mg/mL) after 15min pretreatment, 0.1mM tNTPs reacted at 55℃for 4h;

2.2, after primer extension, the double-stranded product was immobilized on streptavidin resin, and TNA single strands were eluted with 200mM NaOH; then desalting by a desalting column;

2.3, removing the DNA primer part of the TNA enzyme single chain, which comprises the following specific steps:

the reaction comprises 1 mu M double-chain product and 1U/mL snake venom phosphodiesterase I, wherein the double-chain product and the snake venom phosphodiesterase I are incubated in a reaction buffer solution of the enzyme for 16 hours at 37 ℃, and enzymolysis products are purified by a denaturing PAGE gel and then desalted by a desalting column and then quantified by ultraviolet;

3. construction of magnesium ion dependent fluorescence sensor:

3.1, synthesizing a substrate sequence labeled with a fluorescent group and a quenching group, as follows:

5’-FAM-AACTCAT-r(CAUGC)-AGCTCGA-BHQ-1-3’；

3.2, T17-22 molecules and substrate (E: s=2:1 (200 nM)) were heated in water at 90 ℃ for 5 minutes and then annealed on ice for 10 minutes to form a fluorescent sensor complex;

4. adding reaction buffers with different magnesium ion concentrations into the compound, and reacting for 1h at 37 ℃; under the excitation of 460nm, scanning and collecting fluorescence spectrum by using an enzyme-labeled instrument, wherein the emission wavelength is 505-650nm;

5. intracellular magnesium ion imaging:

5.1, hela cell culture, the steps are as follows:

HeLa cells were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS), 100U/mL penicillin, 100mg/mL streptomycin and 2.5. Mu.g/mL tetracycline, and placed at 37℃with 5% CO ₂ Is cultured in a humidifying incubator; before confocal imaging, cells were plated in 35mm glass bottom dishes for 24 hours to reach 60-70% confluence;

5.2, carrying out transfection of the fluorescence sensor by adopting a transfection reagent, wherein the method comprises the following steps:

100pmol of sensor and 4. Mu.L of Lipofectamine 3000 were incubated in Opti-MEM medium at room temperature for 10 min each, followed by mixed incubation for 10 min; adding the mixture to a culture medium and incubating the mixture with cells for 12 hours;

5.3, confocal imaging, comprising the following steps:

after 12h of transfection, the cells were transfected with a solution containing 2. Mu.M calcium ionophore and 20mM Mg ²⁺ Instead of the medium used for transfection, and then incubated with cells for 12h; after the incubation, the cells were stained with 2.5ng/mL Hoechst 33258 nuclear stain for 15min at 37℃and carefully washed with PBS, and finally fixed with 4% paraformaldehyde for 15min at 37 ℃; confocal imaging was taken at 60 x resolution using an olympus laser scanning confocal microscope; fluorescence emission of Hoechst 33258 was measured in the range of 450-520nm under 401nm excitation; FAM was excited at 488nm to obtain fluorescence and collected at 493-563 nm.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the invention without departing from the principles thereof are intended to be within the scope of the invention as set forth in the following claims.

Claims (5)

1. A method for imaging intracellular magnesium ions based on TNA molecules, which is characterized by comprising the following specific operation steps:

(1) Screening magnesium ion-dependent TNA molecules with RNA cleavage activity in vitro; constructing a TNA molecular cutting system as a magnesium ion fluorescence sensor;

the TNA molecule specifically refers to: the sequence of TNA molecules with highest activity obtained by the truncation experiment and treatment with snake venom phosphodiesterase is shown as follows:

5’-GTAGGAGAGGTTATCGTTGGAGGGAGATGAGTGTAG-2’；

(2) Verifying the performance of the fluorescence sensor in an in vitro buffer;

(3) The sensor is transfected into cells for imaging of magnesium ions in vivo.

2. The method for intracellular magnesium ion imaging based on TNA molecules according to claim 1, wherein,

in the step (1), the construction of the magnesium ion fluorescence sensor specifically comprises the following steps:

marking a fluorescent group FAM and a quenching group BHQ-1 at the 5 'end and the 3' end of the RNA substrate respectively, and quenching fluorescence when magnesium ions are not present; when magnesium ions exist, TNA molecules catalyze RNA substrates to break, fluorescent groups are released, and fluorescent signals are recovered.

3. The method for intracellular magnesium ion imaging based on TNA molecules according to claim 1, wherein,

in step (2), the performance of the fluorescent sensor in the in vitro buffer is demonstrated as follows:

when the magnesium ion concentration is less than 20mM, the fluorescence signal intensity of the system and the magnesium ion concentration show a linear relationship, and the detection limit is 0.35mM.

4. The method for intracellular magnesium ion imaging based on TNA molecules according to claim 1, wherein,

in step (3), the imaging step of the sensor into cells for in vivo magnesium ions is as follows:

firstly, transfecting a fluorescence sensor into cells by using a transfection reagent Lipofectamine 3000;

then, using a calcium ion carrier to transport magnesium ions in the culture medium into cells;

finally, cells were imaged with confocal microscopy after incubation.

5. The method of claim 4, wherein in step (3), the imaging of magnesium ions in the sensor body is performed as follows: the method performs fluorescence imaging of magnesium ions at a concentration of 20mM in living cells.