Environmentally sensitive nucleic acid phase diagram analysis method based on variable temperature fluorescence resonance energy transfer

Abstract

The invention provides an environment-sensitive nucleic acid phase diagram analysis method based on variable-temperature fluorescence resonance energy transfer, and belongs to the technical field of biomedicine. According to the invention, the structure change process is disclosed through fluorescence resonance energy transfer spectrum change of fluorescently labeled DNA, and a temperature-dependent folding coefficient [phi] T is obtained by designing a series of gradient pH-ionic strength combined melting annealing experiments; different classification modes are adopted for primary data and then transposition processing is carried out to obtain a pH-dependent folding coefficient phi pH and an ionic strength-dependent folding coefficient phi S, and a fixed single variable draws a corresponding phase diagram by utilizing folding coefficients depending on other two variable conditions, so that the environmental sensitivity of a target structure can be comprehensively and intuitively reflected. The method overcomes the limitation that the existing analysis method is low in efficiency, cannot accurately reflect the whole allosteric process of the complex DNA structure and cannot comprehensively evaluate the stability of the nucleic acid structure in the complex environment, and a brand new analysis method is provided for analyzing the environment-sensitive nucleic acid structure.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to an environmentally sensitive nucleic acid phase diagram analysis method based on variable temperature fluorescence resonance energy transfer. Background Art

[0002] Fluorescence resonance energy transfer (FRET) refers to the non-radiative transfer of excited state energy from a fluorescent donor molecule to an unexcited acceptor molecule, which results in an increase in the fluorescence intensity of the acceptor molecule (it may also be transferred to a quenching group to cause fluorescence signal quenching). The energy transfer efficiency depends on the spectral properties of the donor and acceptor, the distance between the donor and acceptor molecules, etc. Since the FRET effect is extremely sensitive to changes in the distance between the donor and acceptor at the nanoscale (FRET efficiency is related to R 6 Therefore, it is widely used in studying the interaction between molecules.

[0003] Triplex DNA is an atypical nucleic acid secondary structure formed by the interaction of a single strand of a triplex oligonucleotide (triplex-forming oligonucleotide, TFO) and double-stranded DNA through the Hoogsteen bond. According to the different binding directions of the TFO chain and the double-stranded DNA, it can be divided into parallel structure and antiparallel structure. The parallel structure consists of CG*C + , CG*G and TA*T, while the antiparallel structure is usually composed of CG*C + , CG*G, TA*T and TA*A, CG*C + The formation of base pairs is accompanied by the protonation of C bases, so triplex DNA is a typical environmentally sensitive DNA secondary structure.

[0004] H-DNA formed by triplex structures in the human genome can cause genetic instability, such as double-stranded DNA breaks, which can lead to gene recombination and cancer, and can also downregulate gene transcription and replication. On the other hand, TFO is an important type of gene drug. Due to its high sequence matching and strong binding force, it can target and bind to the target double-stranded DNA in the genome to form a triplex structure, which can selectively regulate the relevant functions of the target gene and ultimately achieve the treatment of the disease. Therefore, it is of great significance to reveal in detail the conformational changes of triplex DNA in different surrounding environments.

[0005] At present, the methods used to characterize environmentally sensitive DNA structures are limited to two methods: pH titration after fixed temperature and ionic strength conditions and melting annealing experiments under a small number of fixed pH and ionic strength conditions. The former is used to explore the effect of a single environmental variable, pH, on the target structure, and the latter can only reflect the conformational changes of the target structure when the temperature changes under a small number of fixed conditions. At the same time, the existing technology mainly uses ultraviolet absorption spectroscopy as a characterization method for melting annealing experiments, but the ultraviolet absorption peaks of triplex DNA (247nm, 260nm, and 295nm) overlap seriously with the absorption peak of ordinary double-stranded DNA (260nm), and it is difficult for the ultraviolet absorption spectrum to accurately reflect the complete conformational change process of triplex DNA.

[0006] There have been related reports on using the FRET effect to reveal changes in nucleic acid structure, but triplex DNA in the molecule has three conformations: triple-stranded DNA, double-stranded DNA, and disordered coil. Using a single labeling scheme for it cannot fully characterize the allosteric process. Only conducting allosteric experiments on the target structure under a small number of conditions and simply processing the experimental folding coefficient data cannot fully and intuitively reflect the sensitivity of the target structure under complex conditions. A complete environmentally sensitive nucleic acid phase diagram analysis method has not yet been reported, and it is extremely necessary to conduct targeted research on this. Summary of the invention

[0007] The purpose of the present invention is to solve the deficiencies in the prior art and provide an environmentally sensitive nucleic acid phase diagram analysis method based on variable temperature fluorescence resonance energy transfer. First, different fluorescent labeling schemes are designed for triplex DNA sequences based on the FRET effect. Then, melting annealing experiments are performed under a series of conditions (gradient pH, ionic strength) to obtain folding coefficient data. The folding coefficient is transposed, and a phase diagram is drawn after analysis and sorting, which can be used to comprehensively and intuitively reflect the environmental sensitivity of the target structure.

[0008] The specific technical solution of the present invention is: an environmentally sensitive nucleic acid phase diagram analysis method based on variable temperature fluorescence resonance energy transfer, comprising the following steps:

[0009] 1) Fluorescently label the DNA secondary structure to obtain target DNA and reference DNA. Fluorescent labeling is mainly used to characterize the binding and dissociation of the third strand and the double strand, as well as the folding and unfolding of the double strand itself;

[0010] 2) preparing buffer solutions with different gradient pH-ionic strength combinations;

[0011] 3) mixing the target DNA with the buffer solution prepared in step 2), annealing at 95° C., performing an annealing melting procedure, and continuously collecting fluorescence emission spectra to characterize the folding changes of the target structure, and recording the FRET efficiency as the folding coefficient of the target structure;

[0012] 4) Select a temperature range that includes the complete transition range, compare the fluorescence intensity values ​​of the target DNA and the reference DNA, and obtain the temperature-dependent folding coefficient by the following formula:

[0013]

[0014] Among them, F nFIQ is the fluorescence intensity value of the target DNA, F nF is the fluorescence intensity value corresponding to the reference DNA;

[0015] The overall temperature dependence folding factor Set,consists of temperature dependence data;

[0016] 5) The temperature-dependent folding coefficients under different pH conditions were grouped according to the fixed ionic strength conditions and transposed to obtain the ionic strength-dependent folding coefficients Similarly, the temperature-dependent folding coefficients under different ionic strengths are grouped according to the fixed pH condition and the pH-dependent folding coefficients are obtained by transposition.

[0017] 6) Consider pH, ionic strength, and temperature as factors that affect the folding coefficient of the target structure For the three variables, a single variable is fixed and the corresponding phase diagram under relevant conditions is drawn using the folding coefficient that depends on the other two variables.

[0018] Furthermore, the DNA secondary structure is triplex DNA, and two schemes are used in step 1) to fluorescently label the triplex DNA to obtain the target sequence, scheme a: connecting a fluorescent molecule and a corresponding quenching group at the 3' end and the first ring region of the triplex DNA structure respectively; scheme b: connecting a fluorescent molecule and a corresponding quenching group at the 5' end and the second ring region of the triplex DNA structure respectively.

[0019] Furthermore, in step 1), a DNA sequence labeled only with a fluorescent molecule is used as a reference DNA.

[0020] Preferably, the fluorescent molecule is an Alexa series fluorescent molecule; and the quenching group is a BHQ series quenching molecule.

[0021] Furthermore, in step 2), the buffer solution is a Britton-Robinson buffer solution with broad-spectrum buffering capacity, the pH coverage range of the buffer solution is 2.0-12.0, with an interval of 0.2, and the ionic strength coverage range is 0.02 to 1.0 M, which are 0.02, 0.03, 0.05, 0.75, 0.1, 0.15, 0.2, 0.5, and 1 M, respectively. Different pH and ionic strength combinations can obtain buffer solutions with different gradient pH-ionic strength combinations.

[0022] Furthermore, in step 3), a fluorescence quantitative PCR instrument is used to measure 96 samples at one time, and the fluorescence intensity corresponding to the emission wavelength of the excited state fluorescent molecules is continuously collected during the melting and annealing process.

[0023] Furthermore, in step 3), the annealing and melting program is designed as follows: a) annealing process is performed at 95°C-4°C, with a temperature change rate of 0.9°C / min; b) 4°C is maintained for 10 minutes; c) melting process is performed at 4°C-95°C, with a temperature change rate of 0.9°C / min; d) 95°C is maintained for 10 minutes.

[0024] Further, in step 6), the ionic strength dependence folding coefficient under different pH and temperature conditions is fixed at a fixed ionic strength. The pH-dependent folding coefficients under different ionic strengths and temperatures are plotted as pH-dependent phase diagrams at a fixed pH; the temperature-dependent folding coefficients under different pH and ionic strengths are plotted as pH-dependent phase diagrams at a fixed temperature. Plotted as a temperature-dependent phase diagram.

[0025] Furthermore, for the temperature dependence data, take The temperature at different ionic strength and pH conditions is used as the T 1 / 2 , T 1 / 2 The melting temperature is the temperature corresponding to 50% denaturation of the target structure, which is used to analyze the thermal stability of the target structure under complex conditions;

[0026] For pH-dependent data, take The pH at different ionic strength and temperature conditions 1 / 2 , pH 1 / 2 It is the melting pH, reflecting the pH value corresponding to 50% denaturation of the target structure, and is used to analyze the pH stability of the target structure under complex conditions.

[0027] For ionic strength-dependent data, take The ionic strength at different pH and temperature conditions is used as the S 1 / 2 , S 1 / 2 It is the melting ion strength value, reflecting the ion strength corresponding to 50% denaturation of the target structure, and is used to analyze the ion strength stability of the target structure under complex conditions. 1 / 2 , because for the triplex DNA structure given in the example, it is difficult to achieve a 10%-90% complete opening process of the structure by simply changing the ionic strength, while for other DNA secondary structures that are more sensitive to ionic strength, this method can be used to derive S 1 / 2 .

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1. This application proposes for the first time an environmentally sensitive nucleic acid phase diagram analysis method based on variable temperature fluorescence resonance energy transfer. Compared with the existing experimental methods for analyzing environmentally sensitive DNA secondary structures, this application first uses different fluorescent labeling schemes to label the selected triplex DNA before conducting subsequent experiments. The two labeling schemes can be used to characterize the binding and dissociation of the third strand and the double strand, as well as the folding and unfolding of the double strand itself, and then accurately reveal the entire conformational change process of the DNA structure;

[0030] 2. The series of melting annealing experiments, data processing and phase diagram drawing methods designed in this application overcome the limitation that existing analytical methods cannot reflect the stability of nucleic acid structure under complex environment;

[0031] 3. Although this application uses triplex DNA structure as an example to perform nucleic acid phase diagram analysis to reveal the environmental sensitivity of the target structure, this method can also be extended to analyze other environmentally sensitive DNA secondary structures (such as i-motif, G-quadruplex, etc.), providing a new experimental method and research idea for analyzing environmentally sensitive nucleic acid structures. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagrams of different fluorescent labeling schemes, wherein panel a is a schematic diagram of scheme a, and panel b is a schematic diagram of scheme b;

[0033] Figure 2 is a schematic diagram of converting fluorescence intensity into temperature-dependent folding factor;

[0034] Figure 3 It is a schematic diagram of data grouping-transposition processing;

[0035] Figure 4 The small figures a, b, and c in the middle are the temperature-dependent phase diagram, ionic strength-dependent phase diagram, and pH-dependent phase diagram, respectively;

[0036] Figure 5 Panels a and b in the middle reflect the temperature stability and pH stability of the target structure, respectively. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below in conjunction with the accompanying drawings, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0038] Experimental instruments:

[0039] Fluorescence spectrophotometer (F-4700, Hitachi), fluorescence quantitative PCR instrument (Q1plus, ABI), pH meter (PB-10, Sartorius).

[0040] Experimental reagents:

[0041] Sodium chloride (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), sodium hydroxide (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), phosphoric acid (Nanjing Chemical Reagent Co., Ltd.), boric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd.), acetic acid (Nanjing Chemical Reagent Co., Ltd.), hydrochloric acid (Nanjing Chemical Reagent Co., Ltd.), and water used were all deionized water (resistance 18.2 MΩ·cm).

[0042] Example 1

[0043] 1) Labeling triplex DNA sequences using different fluorescent labeling schemes

[0044] Plan A: See Figure 1 Panel a in the middle, fluorescent molecules and corresponding quenching groups are connected to the 3' end and the first loop region (loop 1) of the triplex DNA structure, respectively. This labeling scheme is used to characterize the binding and dissociation of the third strand with the double strand.

[0045] Option B: See Figure 1 Panel b, fluorescent molecules and corresponding quenching groups are connected to the 5' end of the triplex DNA structure and the second loop region (loop 2), respectively. This labeling scheme is used to reflect the folding and unfolding of the double helix itself;

[0046] In this embodiment, the fluorescent molecule is preferably Alex488, and the quenching group is BHQ1 corresponding to Alex488.

[0047] In order to better eliminate the influence of the surrounding environment on the fluorescence efficiency of the fluorescent molecule itself, a DNA sequence containing only a single Alex488 marker was designed as a reference.

[0048] The corresponding sequences designed based on the above scheme are as follows:

[0049] The DNA sequence was custom synthesized by Shanghai Shenggong Biotechnology Co., Ltd. and its sequence is as follows: (80TAT

[0050] The relative proportion of TA*T base pairs in the parallel triplex backbone structure is 80% (SEQ ID NO.1); F refers to the sequence labeled with only the fluorescent molecule Alex488; FIQ refers to the sequence labeled with both Alex488 and the corresponding quenching group BHQ1; among them, 80TAT-3FIQ is the sequence labeled based on scheme a, and 80TAT-5FIQ is the sequence labeled based on scheme b)

[0051]

[0052] 2) Prepare a series of Britton-Robinson buffer solutions with broad-spectrum buffering capacity

[0053] First, a Britton-Robinson buffer solution was prepared (phosphoric acid, acetic acid and boric acid were weighed and dissolved in water, phosphoric acid and acetic acid were pure products, boric acid was solid, and the three acids were weighed according to the results of molar mass and mass fraction calculation, and the final concentrations of the three acids after being dissolved in water were all 5 mM), and sodium hydroxide was added thereto to adjust the pH of the solution to obtain a series of buffer solutions with a pH coverage range of 2.0-12.0 and an interval of 0.2, for a total of 51 groups of pH gradients; then, NaCl was added to make the ionic strength of the buffer solution cover 0.02M to 1.0M (respectively 0.02, 0.03, 0.05, 0.75, 0.1, 0.15, 0.2, 0.5, 1M), for a total of 9 ionic strengths; different pH and ionic strength were combined to obtain buffer solutions with different pH-ionic strength combinations, for a total of 459 groups.

[0054] 3) Melting and annealing experiments of triplex DNA at different pH and ionic strength

[0055] The sample reaction system is 20 μL, consisting of different DNA sequences and buffer solutions with different pH-ionic strength combinations. Specifically, 20 nM DNA sequence 80TAT-3FIQ labeled with fluorescent labeling scheme a and the corresponding reference sequence 80TAT-3F are mixed with BR buffer solutions with different gradient pH-ionic strength combinations, fully shaken and centrifuged, and then placed in a fluorescent quantitative PCR instrument for annealing at 95°C. After annealing, the melting annealing program is performed, and the fluorescence changes at 520 nm are continuously collected during the entire melting annealing program.

[0056] The melting annealing program was designed as follows: a) annealing at 95°C-4°C with a temperature change rate of 0.9°C / min; b) maintaining at 4°C for 10 min; c) melting program at 4°C-95°C with a temperature change rate of 0.9°C / min; d) maintaining at 95°C for 10 min.

[0057] The DNA sequence 80TAT-5FIQ labeled with the fluorescent labeling scheme b and its corresponding reference sequence 80TAT-5F were subjected to melting annealing experiments by repeating the above operation, and the fluorescence intensity changes corresponding to the emission wavelength of the excited state fluorescent molecules were continuously collected during the melting annealing process.

[0058] 4) Temperature-dependent folding coefficient of triplex DNA at different pH and ionic strength

[0059] During the melting and annealing process, 96 samples were measured at one time using a fluorescent quantitative PCR instrument. The temperature-dependent folding coefficient was directly obtained from the melting and annealing experiment results. During the experiment, the fluorescence intensity corresponding to the emission wavelength of the excited state fluorescent molecules was continuously measured. The fluorescence emission spectrum was collected to characterize the folding changes of the target structure, that is, the FRET efficiency was recorded as the folding coefficient of the target structure.

[0060] Specifically, in the melting annealing experiment, the change in the fluorescence value of the sample nFIQ (in this embodiment, nFIQ refers to 80TAT-3FIQ and 80TAT-5FIQ) can reflect the change of the target structure with temperature (the change in the fluorescence value of the nF sequence (i.e., the reference sequence 80TAT-3F and 80TAT-5F) is used as the nFIQ sequence melting annealing curve correction).

[0061] For each gradient pH-ionic strength combination, the following operations were performed on the melting annealing curve: a temperature interval covering the complete transition range was selected, and the fluorescence intensity F of the collected nFIQ and the corresponding nF sequence was calculated. nFIQ and F nF As a control group (F nFIQ and F nF The fluorescence intensities of nFIQ and nF sequences are respectively, and the fluorescence values ​​obtained under the corresponding buffer solution conditions are compared. (1-ratio) is the temperature-dependent initial folding coefficient of the target structure under different pH and ionic strength. Right now:

[0062]

[0063] Temperature-dependent folding coefficients of full gradient pH-ionic strength combinations The temperature dependence data of triplex DNA under different pH and ionic strength are constructed. The experimental process is shown in Figure 2 .

[0064] 5) Obtaining the ionic strength-dependent folding coefficient and the pH-dependent folding coefficient

[0065] Temperature-dependent folding coefficients of triplex DNA at different pH-ionic strength combinations The ion strength dependent folding coefficients were obtained by grouping in different ways and then transposing. and pH-dependent folding factor Specifically, taking a set of ionic strength and a set of pH conditions as an example, the temperature-dependent folding coefficients under different pH conditions are grouped according to the fixed ionic strength conditions (the above 9 ionic strengths can be regarded as 9 sets of ionic strength-dependent data), and one set of data is transposed to obtain the ionic strength-dependent folding coefficient (See Figure 3Similarly, the temperature-dependent folding coefficients under different ionic strengths were grouped according to the fixed pH conditions, and one group of data was transposed to obtain the pH-dependent folding coefficients. (See Figure 3 (inset in middle b).

[0066] 6) Draw a phase diagram of environmentally sensitive nucleic acids

[0067] Taking the temperature-dependent phase diagram as an example, different pH, ionic strength and corresponding temperature-dependent folding coefficients are taken. The phase diagram is drawn by the x-axis, y-axis, and z-axis data respectively. The experimental results are as follows Figure 4 As shown in the small figure in the middle (the z-axis value is represented by grayscale; the solid line in the figure represents The three conformational half-transition points of Three conformational transition points.

[0068] Similarly, using the ionic strength-dependent folding factor and pH-dependent folding factor A set of ionic strength-dependent phase diagrams (which can intuitively reflect the structural changes of DNA when pH and temperature change under fixed ionic strength conditions) and a set of pH-dependent phase diagrams (which can intuitively reflect the structural changes of DNA when ionic strength and temperature change under fixed pH conditions) can be drawn respectively. The experimental results are as follows Figure 4 As shown in panels b and c.

[0069] refer to Figure 5 In the middle panel a, for the temperature dependence data, take The temperature at different ionic strength and pH conditions is used as the T 1 / 2 (melting temperature, the temperature corresponding to 50% denaturation of the target structure), so that the thermal stability of the target structure under complex conditions can be fully analyzed.

[0070] refer to Figure 5 In panel b, for pH-dependent data, take The pH at different ionic strength and temperature conditions 1 / 2 (melting pH, the pH value corresponding to 50% denaturation of the target structure), so that the pH stability of the target structure under complex conditions can be fully analyzed.

[0071] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An environmentally sensitive nucleic acid phase diagram analysis method based on variable temperature fluorescence resonance energy transfer, characterized in that: The steps include: 1) Fluorescently label the DNA secondary structure to obtain target DNA and reference DNA; 2) preparing buffer solutions with different gradient pH-ionic strength combinations; 3) mixing the target DNA with the buffer solution prepared in step 2), annealing at 95° C., performing an annealing and melting procedure, and continuously collecting fluorescence emission spectra; 4) Select a temperature range that includes the complete transition range, compare the fluorescence intensity values ​​of the target DNA and the reference DNA, and obtain the temperature-dependent folding coefficient by the following formula: Among them, F nFIQ is the fluorescence intensity value of the target DNA, F nF is the fluorescence intensity value corresponding to the reference DNA; The overall temperature dependence folding factor Set,consists of temperature dependence data; 5) The temperature-dependent folding coefficients under different pH conditions are grouped according to the fixed ionic strength conditions and transposed to obtain the ionic strength-dependent folding coefficients Similarly, the temperature-dependent folding coefficients under different ionic strengths are grouped according to the fixed pH condition and the pH-dependent folding coefficients are obtained by transposition. 6) Consider pH, ionic strength, and temperature as factors that affect the folding coefficient of the target structure For three variables, a single variable is fixed and a phase diagram is drawn using the folding coefficient that depends on the other two variables.

2. The method for analyzing environmentally sensitive nucleic acid phase diagram based on variable temperature fluorescence resonance energy transfer according to claim 1, characterized in that: The DNA secondary structure is triplex DNA. In step 1), two schemes are used to fluorescently label the triplex DNA to obtain the target sequence. Scheme a: a fluorescent molecule and a corresponding quenching group are connected to the 3' end of the triplex DNA structure and the first ring region respectively; scheme b: a fluorescent molecule and a corresponding quenching group are connected to the 5' end of the triplex DNA structure and the second ring region respectively.

3. The method for analyzing environmentally sensitive nucleic acid phase diagram based on variable temperature fluorescence resonance energy transfer according to claim 2, characterized in that: In step 1), a DNA sequence labeled only with a fluorescent molecule is used as a reference DNA.

4. The method for analyzing environmentally sensitive nucleic acid phase diagram based on variable temperature fluorescence resonance energy transfer according to any one of claims 2 to 3, characterized in that: The fluorescent molecules are Alexa series fluorescent molecules; the quenching groups are BHQ series quenching molecules.

5. The method for analyzing environmentally sensitive nucleic acid phase diagram based on variable temperature fluorescence resonance energy transfer according to claim 1, characterized in that: In step 2), the buffer solution is a Britton-Robinson buffer solution with broad-spectrum buffering capacity, the pH coverage range of the buffer solution is 2.0-12.0, with an interval of 0.2, and the ionic strength coverage range is 0.02 to 1.0 M, which are 0.02, 0.03, 0.05, 0.75, 0.1, 0.15, 0.2, 0.5, and 1 M respectively. Different pH and ionic strength combinations can obtain buffer solutions with different gradient pH-ionic strength combinations.

6. The method for analyzing environmentally sensitive nucleic acid phase diagram based on variable temperature fluorescence resonance energy transfer according to claim 2, characterized in that: In step 3), a fluorescence quantitative PCR instrument is used to measure 96 samples at one time, and the fluorescence intensity corresponding to the emission wavelength of the excited state fluorescent molecules is continuously collected during the melting and annealing process.

7. The method for analyzing environmentally sensitive nucleic acid phase diagram based on variable temperature fluorescence resonance energy transfer according to claim 1, characterized in that: In step 3), the melting annealing program is designed as follows: a) annealing process is performed at 95°C-4°C with a temperature change rate of 0.9°C / min; b) 4°C is maintained for 10 minutes; c) melting program is performed at 4°C-95°C with a temperature change rate of 0.9°C / min; d) 95°C is maintained for 10 minutes.

8. The method for analyzing environmentally sensitive nucleic acid phase diagram based on variable temperature fluorescence resonance energy transfer according to claim 1, characterized in that: In step 6), the ionic strength dependence folding coefficient under different pH and temperature conditions is calculated under fixed ionic strength. The pH-dependent folding coefficients under different ionic strengths and temperatures are plotted as pH-dependent phase diagrams at a fixed pH; the temperature-dependent folding coefficients under different pH and ionic strengths are plotted as pH-dependent phase diagrams at a fixed temperature. Plotted as a temperature-dependent phase diagram.

9. The method for analyzing environmentally sensitive nucleic acid phase diagram based on variable temperature fluorescence resonance energy transfer according to claim 7, characterized in that: For temperature dependence data, take The temperature at different ionic strength and pH conditions is used as the T 1 / 2 , T 1 / 2 The melting temperature is the temperature corresponding to 50% denaturation of the target structure, which is used to analyze the thermal stability of the target structure under complex conditions; For pH-dependent data, take The pH at different ionic strength and temperature conditions 1 / 2 , pH 1 / 2 It is the melting pH, reflecting the pH value corresponding to 50% denaturation of the target structure, and is used to analyze the pH stability of the target structure under complex conditions; For ionic strength-dependent data, take The ionic strength at different pH and temperature conditions is used as the S 1 / 2 , S 1 / 2 It is the melting ion intensity value, reflecting the ion intensity corresponding to 50% denaturation of the target structure, and is used to analyze the ion intensity stability of the target structure under complex conditions.

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