CN114854736A - Circular nucleic acid molecule, method for producing same, nucleic acid probe and detection method - Google Patents
Circular nucleic acid molecule, method for producing same, nucleic acid probe and detection method Download PDFInfo
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Abstract
The invention discloses a cyclic nucleic acid molecule, a preparation method thereof, a nucleic acid probe and a detection method, and relates to the technical field of molecular detection. The circular nucleic acid molecule does not contain a base C, and the linear nucleotide sequence of the circular nucleic acid molecule is (GGTTATTATT) n Wherein n is more than or equal to 3. The circular nucleic acid molecule disclosed by the invention only contains three bases, has a repeated sequence with a minimum secondary structure, is used as a template to carry out RCA, and has higher amplification efficiency and higher amplification efficiency with single-stranded DNAThe binding efficiency of the signal probe is high, so that the detection is more efficient and the sensitivity is higher. Can be widely applied to the fields of signal amplification, nano structure, biosensing, molecular diagnosis and treatment and the like.
Description
Technical Field
The invention relates to the technical field of molecular detection, in particular to a cyclic nucleic acid molecule, a preparation method thereof, a nucleic acid probe and a detection method.
Background
Rolling Circle Amplification (RCA) is a nucleic acid-based biological signal Amplification technology, and can replicate a circular nucleic acid molecule by thousands of times under the action of polymerase with the circular nucleic acid molecule as a template, so that a signal generated by low-concentration target molecules in a mediated manner can be amplified and detected. Compared to the most widely used PCR technology at present, RCA has been widely used for the detection of biomarkers at present due to its advantages of no need of a thermal cycling step and no need of special equipment. Constructing ultra-fast and highly sensitive RCA technology is especially important for Point-Of-Care Testing (POCT) and biosensing fields. However, the efficiency of the existing rolling circle amplification is low.
Disclosure of Invention
The invention aims to provide a cyclic nucleic acid molecule, a preparation method thereof, a nucleic acid probe and a detection method. The circular nucleic acid molecule provided by the invention only contains three bases, has a minimum secondary structure, is used as a template for RCA (Rolling circle amplification), has higher amplification efficiency, and can be widely applied to the fields of signal amplification, nano structures, biosensing, molecular diagnosis and treatment and the like.
The invention is realized by the following steps:
in a first aspect, the present invention provides a circular nucleic acid molecule suitable for use in an RCA reaction as a template for obtaining a long single-stranded nucleic acid molecule, which does not contain a base C and has a linear nucleotide sequence (GGTTATTATT) n Wherein n is not less than 3 and n is an integer.
Amplification efficiency of RCA is related to the DNA sequence and secondary structure of the circular template. In the patent, the invention provides a DNA circular nucleic acid molecule containing three basic groups and having a minimum secondary structure, and the novel sequence is used as a template to perform RCA, so that the amplification efficiency is higher, the DNA circular nucleic acid molecule can be widely applied to the fields of signal amplification, nano structures, biosensing, molecular diagnosis and treatment and the like, and a molecular basis is provided for a rapid and high-sensitivity RCA signal amplification technology.
Optionally, in some embodiments, n-4-6, preferably n-5.
Alternatively, in some embodiments, the linear nucleotide sequence of the circular nucleic acid molecule is as set forth in SEQ ID No. 1:
GGTTATTATTGGTTATTATTGGTTATTATTGGTTATTATTGGTTATTATT。
in a second aspect, the present invention provides a precursor nucleic acid molecule for preparing a circular nucleic acid molecule as described above, which is a linear structure that can form a circular nucleic acid molecule as described above by a cyclization reaction.
Alternatively, in some embodiments, the nucleotide sequence of the precursor nucleic acid molecule is P- (GGTTATTATT) n Wherein n is not less than 3, n is an integer, and P is a phosphate group.
Optionally, in some embodiments, n-4-6, preferably n-5.
In a third aspect, the present invention provides a method for preparing the circular nucleic acid molecule as described above, comprising: a step of ring-forming reaction,
the cyclization reaction step comprises: the precursor nucleic acid molecule as described above is placed in a loop reaction system to be reacted to obtain the circular nucleic acid molecule.
The synthesis of the circular nucleic acid molecule is carried out by ligating the precursor nucleic acid molecule, the 5' -end of which is modified with a phosphate group, by the catalytic action of a nucleic acid single-stranded ligase.
Optionally, in some embodiments, the above preparation method further comprises a purification step. For example, after the ligation product in the cyclization reaction step is subjected to PAGE electrophoresis, a nucleic acid band is stained with fluorescence by a SYBR GREEN I post-staining method, a circular nucleic acid template is cut out by a gel cutting instrument, and the circular nucleic acid molecule is purified by a PAGE purification kit to obtain a pure circular nucleic acid molecule. Of course, other purification methods may be employed by those skilled in the art to obtain the circular nucleic acid molecule, and are within the scope of the present invention.
Optionally, in some embodiments, the cyclization reaction system contains a cyclization ligase, ATP, manganese chloride, and a buffer.
Alternatively, in some embodiments, the cyclization reaction is carried out under conditions of: incubating at 58-62 deg.C for 0.5-1 hr, and incubating at 80-99 deg.C for 8-12 min to inactivate enzyme.
In a fourth aspect, the present invention provides a method of performing an RCA reaction to obtain a long single-stranded nucleic acid molecule, comprising: the RCA reaction is performed using the circular nucleic acid molecule as a template as described above.
For example, addition of a circular nucleic acid molecule to an RCA reaction mixture results in a long single-stranded nucleic acid molecule with minimal secondary structure. The RCA reaction mixture comprises polymerase buffer solution, dNTP, primer and nucleic acid polymerase. The reaction conditions were 37 ℃ incubation (the length of time depends on the requirements of the assay conditions) and inactivation by incubation at 95 ℃ for 10 minutes.
Alternatively, in some embodiments, the primer sequence used to perform the RCA reaction is shown in SEQ ID No. 4. The primer can obtain long single-stranded nucleic acid molecules through RCA reaction, and the length of the long single-stranded nucleic acid molecules can be controlled by regulating the reaction time and the like according to actual requirements by a person skilled in the art.
In a fifth aspect, the present invention provides a long single stranded nucleic acid molecule obtainable by a method as described above.
It should be noted that the length of the long single-stranded nucleic acid molecule can be controlled by controlling the time of the RCA reaction according to the needs of the art, and the length thereof is clear to those skilled in the art, and can be also adjusted and controlled.
In a sixth aspect, the present invention provides a nucleic acid probe carrying a detection signal, comprising:
at least one main nucleic acid strand, and at least one signal nucleic acid strand; wherein the primary nucleic acid strand is the long single-stranded nucleic acid molecule of claim 10.
The signal nucleic acid chain is directly or indirectly combined with the main nucleic acid chain, and is modified with a detectable signal marker;
the end of the main nucleic acid strand also has a binding site for a detection molecule that can specifically bind to a target molecule.
Alternatively, in some embodiments, the signal nucleic acid strand is in direct complementary binding with the host nucleic acid strand.
Alternatively, in some embodiments, the signal nucleic acid strand is from a long single-stranded nucleic acid molecule resulting from an RCA reaction as described above.
Alternatively, in some embodiments, the signal nucleic acid strand is indirectly joined to the main nucleic acid strand through at least one branch nucleic acid strand;
each of the nucleic acid strands includes: a head region complementarily binding to said primary nucleic acid strand and a tail region complementarily binding to at least one of said signal nucleic acid strands.
Alternatively, in some embodiments, the signal nucleic acid strand is indirectly joined to the main nucleic acid strand by at least one first branch nucleic acid strand and at least one second branch nucleic acid strand;
each of the first nucleic acid strands comprises: a first head region that complementarily binds to said primary nucleic acid strand, and a first tail region that complementarily binds to at least one of said second nucleic acid strands;
each of the second nucleic acid strands comprises: a second head region that complementarily binds to the first tail region of said first nucleic acid strand, and a second tail region that complementarily binds to at least one of said signal nucleic acid strands.
Optionally, in some embodiments, the target-binding region is located at the 5' end of the main nucleic acid strand.
Optionally, in some embodiments, the signal marker is a quantum dot. The signal label may be other labels such as a fluorescent protein, a radioisotope, or the like.
Optionally, in some embodiments, the quantum dots are selected from Q525, Q565, Q585, Q605, Q625, 655, Q705, and Q800.
Alternatively, in some embodiments, the target molecule is a protein or polypeptide and the detection molecule is an antibody that specifically binds to the protein or polypeptide.
Alternatively, in some embodiments, the target molecule is a nucleic acid fragment and the detection molecule is a nucleic acid fragment that binds complementarily to the nucleic acid fragment.
In a seventh aspect, the present invention provides a nucleic acid probe conjugate comprising a detection molecule capable of specifically binding to a target molecule, and the above nucleic acid probe linked to the detection molecule.
The target molecule may be any polypeptide, protein, or nucleic acid fragment of interest in the art, and the like. Accordingly, the detection molecule may be an antibody capable of specifically binding to the protein or the polypeptide, or a nucleic acid fragment that complementarily binds to the nucleic acid fragment.
In an eighth aspect, the present invention provides an antibody-nucleic acid probe conjugate comprising an antibody, and the nucleic acid probe as described above linked to the antibody, wherein an end of the nucleic acid probe is linked to the antibody.
In a ninth aspect, the present invention provides a method for detecting a protein by using a single-molecule ultra-sensitive POCT, comprising: detection is carried out using a nucleic acid probe as described above, a nucleic acid probe conjugate as described above, or an antibody-nucleic acid probe conjugate as described above.
The target protein in the detection system, i.e. the target molecule, is detectable by the specific binding of a detection molecule, such as an antibody, to the target protein, and the detection of the target protein is realized by the detection of a signal marker by those skilled in the art.
Alternatively, in some embodiments, the class of antibody is selected according to the nature of the target protein, so long as it is capable of binding or specifically binding to the target protein. For example, it can be used as a primary antibody to directly detect the target protein, or as a secondary antibody, in which case the antibody is an anti-primary antibody that indirectly binds to the target protein to effect detection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a diagram illustrating the structure and thermodynamic parameters of different circular templates C1 combined with primers calculated by using MFold;
FIG. 2 is a reaction scheme of the MSS-RCA signal amplification method in example 2 with other control RCA methods;
FIG. 3 Secondary Structure prediction plots of MSS-RCA and other control RCA methods nucleic acid circular templates;
FIG. 4 is a graph of the result of PAGE for DNA circular templates of different RCAs generated by the ligase reaction in example 2;
FIG. 5 is a fluorescence standard curve diagram of the highly sensitive and accurate quantitative purification of nucleic acid according to example 2.
FIG. 6 is a graph showing agarose gel results of amplification reactions for different RCAs in example 2.
FIG. 7 is a graph showing a comparison of amplification efficiencies of amplification reactions of different RCAs in example 3.
FIG. 8 is a graph showing the comparison of the amplification efficiency of MSS-RCA (C1 circular template) and hybrid RCA (C3 circular template) with P1 as primers in example 4 at different concentrations.
FIG. 9 is an AFM image of a linear DNA molecule in example 4.
FIG. 10 is a schematic structural view of the nucleic acid probe in examples 5 to 8.
FIG. 11 is a schematic representation of the binding of the antibody-nucleic acid probe conjugate to a target protein molecule in example 9.
Reference numerals in fig. 10:
20-main nucleic acid strand, 201-the target binding region, 21-signal nucleic acid strand, 211-fluorescent quantum dot, 22-branch nucleic acid strand, 221-head region, 222-tail region, 23-first nucleic acid strand, 231-first head region, 232-first tail region, 24-second nucleic acid strand, 241-second head region, 242-second tail region.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Design of nucleic acid sequence this example designs single-stranded DNA with minimal secondary structure containing only three bases with one end labeled with phosphate group, which can be ligated into a loop to obtain circular nucleic acid molecule, which can be subjected to rapid and efficient rolling circle amplification.
To design a circular template of nucleic acid with minimal secondary structure. In this example, one base is deleted, the circular template of nucleic acid having the minimum secondary structure is limited by the characteristics of ligase, and the length of the circular template (MSS-RCA template) having the minimum secondary structure is generally designed to be several tens of bases, the base at the 5 'end is G and the phosphate group is modified, the 3' end is T, and the sequence is an integral multiple of the repeating unit having the minimum secondary structure, considering that the ligation activity of single-stranded nucleic acid ligase is related to the length of nucleic acid, to the bases at the 5 'end and the 3' end of the circular template, and also considering the cost of the circular template of nucleic acid. The primer is designed to pair with the circular template formed after the ligation reaction. The Tm value of the sequence design of the primer is determined in consideration of the temperature suitable for the amplification reaction. Considering the feature that single-stranded nucleases cannot be ligated at less than 15 bases and the problem of cost, the circular template sequence (C1) in this example was designed as a single-stranded DNA sequence having a total length of 50 bases, which comprises 5 repeated sequences with a minimum secondary structure: (GGTTATTATT) 5 The linear nucleic acid sequence is:
GGTTATTATTGGTTATTATTGGTTATTATTGGTTATTATTGGTTATTATT(SEQ ID NO.1)；
precursor structure:
in this example, a single-stranded DNA (MSS-RCA-precursor) having a minimal secondary structure and a phosphate group at the 5' end, and a Primer 1 complementary thereto;
as a control group, the SS-RCA-precursor contained four bases, including 5 repeated sequences having a secondary structure, and a Primer 2 complementary thereto.
As a control group of transition states, Hybrid-RCA-precursor contains 2 repeats of MSS-RCA-precursor, and 3 repeats of SS-RCA-precursor. The length of the portion where the primer and the circular template were paired was 20 bases, and the Tm value was 52 ℃.
It should be noted that the other end of the primer can be designed to be any sequence that can be paired with the target to be detected, so that the biosensor can be designed to detect the target at a later stage.
The precursor nucleic acid sequences designed in this example are shown in Table 1 below:
TABLE 1
FIG. 1 shows the structure and thermodynamic parameters of the combination of MSS-RCA circular template C1 and primer P1, SS-RCA circular template C2 and primer P2, Hybrid-RCA circular template C3 and primer P1, and C3 and primer P2 calculated by using MFold, and the results show that the Tm values are all higher than 60 ℃, and the complete complementary pairing can be realized at room temperature.
FIG. 2 shows the secondary structure formed by each single-stranded circular template predicted by the NUPACK software. The results showed that the C1 template for MSS-RCA had no secondary structure generation and had a free energy of 0kcal/mol, the C2 template for SS-RCA had a secondary structure generation and had a free energy of-9.96 kcal/mol, and the C3 template for Hybrid-RCA had less secondary structure generation than C2 and had a free energy of-4.98 kcal/mol.
Example 2
As shown in FIG. 2, the process of the circular template rolling circle amplification technology based on the nucleic acid with the minimum secondary structure is mainly realized by steps of circular template synthesis, circular template purification and RCA reaction.
FIG. 3 shows the secondary structure prediction results for MSS-RCA and other control RCA methods nucleic acid circular templates.
The specific process of each step is as follows:
(1) synthesis of circular template the ligation reaction system was 50. mu.L containing 5. mu.L of 10 Xnucleic acid single-stranded ligase buffer (ssDNA/RNA Circligase buffer), 2.5. mu.L of 50mM MnCl 2 1. mu.L of 1mM ATP, 10. mu.L of 100. mu.M cyclization precursor single-stranded template, and 2.5. mu.L of 100U/. mu.L nucleic acid single-stranded ligase (ssDNA/RNA Circligase). The nucleic acid single-stranded ligase was inactivated by incubation at 60 ℃ for 1 hour and at 95 ℃ for 10 minutes. The ligation product was stored at-20 ℃.
The product results of the ligation reactions were verified by native polyacrylamide gel electrophoresis (15% PAGE gel). As a result, as shown in FIG. 4, the 20bp DNA ladder, the MSS-RCA single-stranded C1 template without addition of nucleic acid ligase, the MSS-RCA C1 template without addition of nucleic acid ligase, the SS-RCA single-stranded C2 template without addition of nucleic acid ligase, the SS-RCA C2 template with addition of nucleic acid ligase, the Hybrid-RCA single-stranded C3 template without addition of nucleic acid ligase, and the Hybrid-RCA C3 template with addition of nucleic acid ligase were shown in the lanes from left to right. The results of denaturing PAGE electrophoresis showed that the templates all showed a linear state with only one band when no nucleic acid single-strand ligase was added to the ligation reaction. After adding nucleic acid single-stranded ligase in the ligation reaction for cyclization, new bands appear on the single-stranded template, and the bands can be determined as the nucleic acid circular template for ligation cyclization because the circular template runs slower than the linear template in electrophoresis. The results prove that the circular nucleic acid template products are generated in the system after the single-stranded nucleic acid ligase reaction.
(2) And (3) purifying the circular template, namely preparing 15% PAGE denaturing gel, adding an equal volume of formamide into a sample, incubating for 20 minutes at 95 ℃ for denaturation, and adding 1/5 loading dye of the volume of the sample. The electrophoresis was run at 100 volts until the dark blue band ran off the gel plate. Add 2 uL 10000x sybr green I into 20ml water to prepare dye liquor with 1 time sybr green I final concentration, remove the glue from the rubber plate, add into the dye liquor and incubate for 20-30 minutes. After removal of the gel, it was placed on a blue-emitting gel cutter, the circular template strip was carefully excised, collected in an EP tube, and weighed. 1-2 times the volume of the dispersion buffer (100mg of the gel was added to 150ml of the dispersion buffer), shaken at 600rpm at 50 ℃ for 2 hours, and centrifuged at 12,000rpm for 20 minutes. Collecting supernatant, adding 3 times volume of anhydrous ethanol, and standing at-80 deg.C for 1 hr. Centrifuge at 14,000rpm for 20 minutes at 4 ℃. The supernatant was discarded. 70% ethanol was added and centrifuged at 12,000rpm at 4 ℃ for 20 minutes. The supernatant was discarded. And (5) drying the clean bench. Adding a proper amount of water to dissolve. The DNA was purified again using a nucleic acid purification column. Obtaining pure circular nucleic acid template.
Since the detection sensitivity of the nanodrop is generally ng/. mu.L, and the concentration of the circular nucleic acid template obtained by the purification method of this example is generally ng/. mu.L or even lower, the circular nucleic acid template purified by the nanodrop cannot be accurately quantified. Failure to accurately quantify the circular template will affect subsequent comparison and determination of RCA efficiency. Thus, in this example, a more sensitive sybr gold nucleic acid dye was used to perform highly sensitive and accurate quantification of purified circular nucleic acid templates. The quantitative standard curve system is 50. mu.L, and the system contains 10. mu.L of single-stranded nucleic acid template with different concentrations, 1. mu.L of 100x sybr gold. As shown in FIG. 5, the results of the calibration curve show that the fluorescence intensity of the MSS-RCA single-stranded C1 template bound to sybr gold is the highest as a whole, and the fluorescence intensity is consistent with the property 3 that the fluorescence intensity is high when the sybr gold is bound to a single strand. The single stranded C1 template, also validated for MSS-RCA, had minimal secondary structure. The single-stranded C3 template of Hybrid-RCA combines with the C2 template of sybr gold with higher fluorescence intensity than that of SS-RCA, and proves that the single-stranded C3 template of Hybrid-RCA has more secondary structures than the C2 template of SS-RCA. The results also show that the fluorescence signals of the three nucleic acid templates after being combined with sybr gold are linear in the range of 0.25nM to 256nM, and the purified nucleic acid circular templates with low concentration levels can be accurately quantified with high sensitivity.
(3) RCA reaction the reaction system contained 2. mu.L 10 × Phi29 DNA polymerase buffer, 1. mu.L 10mM dNTP, 5U Phi29 DNA polymerase, 2. mu.L 100nM purified circular template of nucleic acid, primers of different concentrations. Reactions were carried out at 37 ℃ for 10 min (time required) and incubations were carried out at 95 ℃.
The combination of the circular template C1 and the primer P1 initiates MSS-RCA reaction, the combination of the circular template C2 and the primer P2 initiates SS-RCA reaction, the combination of the circular template C3 and the primer P1 initiates Hybrid-RCA-1 reaction, and the combination of the C3 and the primer P2 initiates Hybrid-RCA-2 reaction, so that the feasibility of the RCA reaction is verified. The reaction system was 20. mu.L, reacted at 37 ℃ for 1 hour, and incubated at 95 ℃ for 10 minutes. The remaining components of the RCA reaction were identical to those of example 2 with the primers containing the circular template at a final concentration of 20nM MSS-RCA and a final concentration of 200 nM. The result shows that after 2x sybr gold is added to 10 μ LRCA DNA product, fluorescence signal is generated under blue light irradiation, and it is evident that the fluorescence signal emitted by the SS-RCA reaction triggered by the combination of the circular template C2 and the primer P2 is the lowest (as shown in FIG. 6).
The above results can be said to be: 1. the SS-RCA reaction produces a DNA product with a lot of secondary structures and thus results in low fluorescence intensity after binding to sybr gold; 2. and the C2 template contains more secondary structures, thus resulting in insufficient DNA product generated by RCA amplification.
The amplification results from the above procedure were verified by electrophoresis on 0.6% agarose gel and the agarose gel was irradiated with blue light by sybr gold spot-staining. The results are shown in FIG. 6. From left to right, the lanes are DNA ladder, MSS-RCA reaction triggered by the combination of the circular template C1 and the primer P1, SS-RCA reaction triggered by the combination of the circular template C2 and the primer P2, Hybrid-RCA-1 reaction triggered by the combination of the circular template C3 and the primer P1, and Hybrid-RCA-2 reaction triggered by the combination of the C3 and the primer P2, respectively. From the results of agarose electrophoresis, it was revealed that long-chain DNA products were produced after 1 hour of the RCA reaction using the circular templates designed and synthesized in this example and the primers corresponding thereto.
Example 3
In order to compare the amplification efficiency of different RCA reaction systems, the combination of the circular template C1 and the primer P1 initiates MSS-RCA reaction, the combination of the circular template C2 and the primer P2 initiates SS-RCA reaction, the combination of the circular template C3 and the primer P1 initiates Hybrid-RCA-1 reaction, and the combination of C3 and the primer P2 initiates Hybrid-RCA-2 reaction were carried out in the same system and the amplification efficiencies were compared respectively. The reaction system was 80. mu.L, 5. mu.L of sample was taken every 5 minutes at 37 ℃ and incubated for 10 minutes at 95 ℃ to inactivate the Phi29 amplification enzyme and stop the amplification reaction. The reaction contained 20nM circular template and a final concentration of 200nM primer, and the concentration of the remaining components of the RCA reaction was identical to that of example 2.
The results show (FIG. 7) that the amplification efficiency of the SS-RCA reaction is the slowest, the amplification efficiency of Hybrid-RCA-2 initiated by the circular template 3 bound to primer P2 is the second, and the amplification efficiency of the MSS-RCA reaction is comparable to that of Hybrid-RCA-1 initiated by the circular template C3 bound to primer P1. This result further verifies that the SS-RCA reaction in the above example has the lowest fluorescence signal after 1 hour of amplification. Furthermore, the efficiency of amplification of Hybrid-RCA-2 initiated by the circular template 3 bound to the primer P2 was lower than that of the Hybrid-RCA-1 initiated by the circular template C3 bound to the primer P1, which suggests that the secondary structure of the primer hinders the amplification reaction, probably because the binding efficiency of the primer having a secondary structure was not as high at room temperature as that of the primer having no secondary structure. The result of MSS-RCA reaction and primer P1 combined with circular template C3 to initiate Hybrid-RCA-1 reaction is probably that the primer is saturated due to high primer concentration. Therefore, the ratio of the primer to the circular template was changed in the subsequent example 4, and the specific study compared the reaction efficiency of MSS-RCA and Hybrid-RCA-1.
Example 4
In order to specifically study and compare the reaction efficiency of MSS-RCA and Hybrid-RCA-1, the combination of the circular template C1 and the primer P1 initiates the MSS-RCA reaction, and the combination of the circular template C3 and the primer P1 initiates the Hybrid-RCA-1 reaction, which are implemented in the same system and respectively compare the amplification efficiency. The reaction system was 80. mu.L, 5. mu.L of sample was taken every 5 minutes at 37 ℃ and incubated for 10 minutes at 95 ℃ to inactivate the Phi29 amplification enzyme and stop the amplification reaction. The reaction contained 10nM circular template, primers at different final concentrations (2.5nM,5nM,10nM), and the concentrations of the remaining components of the RCA reaction were identical to those of example 2.
The results (FIG. 8) show that the amplification efficiency of Hybrid-RCA-1 is also improved when the concentration of primer is increased under the reaction conditions of this example, but the whole is lower than that of MSS-RCA reaction. In the MSS-RCA reaction, when the concentration of the primer is increased, the amplification efficiency is still almost the same, which shows that the MSS-RCA amplification efficiency is high, and the amplification efficiency cannot be influenced by changing the concentration of the primer in the concentration range. The result well proves that the MSS-RCA amplification efficiency is highest, and the MSS-RCA can reach higher sensitivity than other RCA reactions containing secondary structures under the same conditions and the same time. The experimental results prove that the MSS-RCA method is successfully established, and the amplification efficiency of the method is far higher than that of a control group of a circular template with a secondary structure. MSS-RCA enables the synthesis of linear DNA molecules up to at least 6 microns in length (see AFM picture, FIG. 9). Examples 5-9 provide the application of the resulting linear DNA molecules, for example as the main nucleic acid strand in examples 5-9, for the purpose of MSS-RCA amplification and branch signal amplification on the surface later, enabling single molecule detection, as will be seen in more detail below.
Example 5
This example provides a nucleic acid probe (e.g., a0 in fig. 10) carrying a fluorescent signal, which includes a main nucleic acid strand 20 and a signal nucleic acid strand 21, wherein the signal nucleic acid strand 21 is directly bonded to the main nucleic acid strand 20, and the signal nucleic acid strand 21 is modified with fluorescent quantum dots 211.
The main nucleic acid strand 20 has a target binding region 201 at its end (5' end), and the nucleic acid probe can specifically bind to a target molecule (e.g., an antibody) via the target binding region 201, and the target molecule is modified with a binding nucleic acid strand that binds to the target binding region 201 complementarily.
Example 6
This example provides a nucleic acid probe carrying a fluorescent signal (e.g., A1, L1 for nucleotide length, d1 for complementary length in FIG. 10), which has substantially the same structure as in example 5, but has a plurality of signal nucleic acid strands 21, and the plurality of signal nucleic acid strands 21 are independently complementarily bound to a main nucleic acid strand 20. Compared with the embodiment 5 which carries more fluorescence signals, the fluorescence signals are obviously stronger and are easier to detect.
Example 7
This example provides a nucleic acid probe carrying a fluorescent signal (refer to A2 in FIG. 10) comprising one main nucleic acid strand 20, a plurality of signal nucleic acid strands 21, and a plurality of branch nucleic acid strands 22.
The signal nucleic acid strand 21 is modified with a fluorescent quantum dot 211, and the signal nucleic acid strand 21 is indirectly joined to the main nucleic acid strand 20 through the branch nucleic acid strand 22. The end (5' -end) of the main nucleic acid strand 20 has a target binding region 201.
Each of the branch nucleic acid strands 22 includes: a head region 221 and a tail region 222, each of the branch nucleic acid strands 22 being complementarily bound to the main nucleic acid strand 20 by the head region 221 thereof, the tail region 222 of each of the branch nucleic acid strands 22 being complementarily bound with the plurality of signal nucleic acid strands 21.
The fluorescence signal of the nucleic acid probe with the structure is obviously amplified and can be detected more easily.
Example 8
This example provides a nucleic acid probe carrying a fluorescent signal (refer to A3 in FIG. 10) comprising a main nucleic acid strand 20, a plurality of signal nucleic acid strands 21, a plurality of first branch nucleic acid strands 23, and a plurality of second branch nucleic acid strands 24;
the signal nucleic acid strand 21 is modified with a fluorescent quantum dot, and the signal nucleic acid strand 21 is indirectly joined to the main nucleic acid strand 20 through the first nucleic acid strand 23 and the second nucleic acid strand 24. The end (5' -end) of the main nucleic acid strand 20 has a target binding region 201.
Each first nucleic acid strand 23 includes: a first head region 231 to which the main nucleic acid strand 20 is complementarily bound, and a first tail region 232 to which a plurality of second nucleic acid strands 24 are complementarily bound;
each second nucleic acid strand 24 comprises: a second head region 241 complementarily binding to the first tail region 232 of the first nucleic acid strand 23, and a second tail region 242 complementarily binding to the plurality of signal nucleic acid strands 21.
The fluorescent signal of the nucleic acid probe with the structure is amplified more obviously and is detected more easily.
The formula for calculating the amplification factor of the nucleic acid probes of examples 5 to 8 is shown in FIG. 10, and An represents the amplification factor of a fluorescence signal; ln represents the nucleotide length of the tail region of the main nucleic acid strand or the branch nucleic acid strand; dn represents the complementary paired nucleotide length between the two nucleic acid strands; kn is a constant for each stage of signal branching. As can be seen from the figure, the more branches, the stronger the fluorescence signal, and the easier it is to detect.
The signal amplification factor can be adjusted by the nucleotide length Ln of the main nucleic acid strand and the branch nucleic acid strand, and the length dn of the complementary pairing part between the two nucleic acid strands.
The main nucleic acid strand may be a single-stranded DNA obtained by the RCA reaction described above, for example, a single-stranded DNA obtained by the RCA reaction of the circular template C1 and the primer P1; the sequences of the branch nucleic acid strand and the signal nucleic acid strand are also readily available.
Example 9
This example provides an antibody-nucleic acid probe conjugate, the structure of which is shown in FIG. 11, comprising: the antibody and the fluorescent probe which is combined on the antibody and is described in any one of embodiments 5-8 are incubated together with a target protein molecule, the antibody-nucleic acid probe combination is combined with the target protein molecule, and the detection of the target protein molecule can be realized through the detection of the fluorescent quantum dot. The relevant fluorescence detection means are conventional technical means well known in the art and will not be described in detail herein.
In summary, in the prior art, the in vitro screening method is used for finding that the DNA sequence with high A and C contents has the characteristic of increasing the reaction efficiency when being used as the circular template of the RCA reaction, and the theory that the A and C contents of the DNA circular template are related to the amplification efficiency is proposed. It has also been shown that the efficiency of RCA amplification is not only related to the sequence of the circular template, but also to size, secondary structure and topology. In the above examples, the inventors considered that the amplification efficiency of RCA was mainly related to the secondary structure, and designed a DNA circular template with a minimum secondary structure from which one base was deleted to perform RCA reaction. The results show that the amplification efficiency of the RCA using the DNA sequence with the minimum secondary structure as the circular template (C1) provided by the above embodiment is indeed higher than that of other RCA amplification methods containing secondary structures, and a new, more efficient and more sensitive signal amplification nucleic acid molecular material, a new method and a research tool are provided for the bioanalysis method depending on the RCA signal amplification in the fields of biosensing, molecular diagnosis and treatment and the like.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> hong Kong Chinese university (Shenzhen)
<120> circular nucleic acid molecule, method for producing the same, nucleic acid probe and detection method
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
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<213> Artificial sequence (Artificial sequence)
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<213> Artificial sequence (Artificial sequence)
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gagacgcggt gagacgcggt gagacgcggt gagacgcggt gagacgcggt 50
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<211> 50
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
ggttattatt ggttattatt gagacgcggt gagacgcggt gagacgcggt 50
<210> 4
<211> 55
<212> DNA
<213> Artificial sequence (Artificial sequence)
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atcctcctcc tctgagctgt catttaccaa taataaccaa taataaccaa taata 55
<210> 5
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<212> DNA
<213> Artificial sequence (Artificial sequence)
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atcctcctcc tctgagctgt catttaccgc gtctcaccgc gtctcaccgc gtctc 55
Claims (20)
1. A circular nucleic acid molecule suitable for use in an RCA reaction as a template for obtaining a long single-stranded nucleic acid molecule, characterized in that it does not contain a base C and has a linear nucleotide sequence (GGTTATTATT) n Wherein n is more than or equal to 3; n is an integer.
2. The circular nucleic acid molecule according to claim 1, wherein n-4-6, preferably n-5.
3. The circular nucleic acid molecule according to claim 1 or 2, wherein the linear nucleotide sequence of the circular nucleic acid molecule is as shown in SEQ ID No. 1: GGTTATTATTGGTTATTATTGGTTATTATTGGTTATTATTGGTTATTATT are provided.
4. A precursor nucleic acid molecule for preparing the circular nucleic acid molecule of any one of claims 1 to 3, which is a linear structure that forms the circular nucleic acid molecule of any one of claims 1 to 3 by a cyclization reaction;
preferably, the nucleotide sequence of the precursor nucleic acid molecule is P- (GGTTATTATT) n Wherein n is not less than 3, n is an integer, and P is a phosphate group.
5. The precursor nucleic acid molecule according to claim 4, wherein n-4-6, preferably n-5.
6. A method for preparing a circular nucleic acid molecule according to any of claims 1 to 3, comprising: a step of ring-forming reaction,
the cyclization reaction step comprises: placing the precursor nucleic acid molecule of claim 4 or 5 in a loop reaction system to perform a reaction to obtain the circular nucleic acid molecule.
7. The method according to claim 6, wherein the cyclization reaction system comprises a cyclization ligase, ATP, manganese chloride, and a buffer.
8. The production method according to claim 6 or 7, wherein the cyclization reaction is carried out under conditions of: incubating at 58-62 deg.C for 0.5-1 hr, and incubating at 80-99 deg.C for 8-12 min.
9. A method of performing an RCA reaction to obtain a long single-stranded nucleic acid molecule, comprising: performing an RCA reaction using the circular nucleic acid molecule of any one of claims 1 to 3 as a template;
preferably, the primer sequence used for carrying out the RCA reaction is shown in SEQ ID NO. 4.
10. A long-chain nucleic acid molecule obtained by the method of claim 9.
11. A nucleic acid probe carrying a detection signal, comprising:
at least one main nucleic acid strand, and at least one signal nucleic acid strand; wherein the primary nucleic acid strand is the long single-stranded nucleic acid molecule of claim 10;
the signal nucleic acid chain is directly or indirectly combined with the main nucleic acid chain, and is modified with a detectable signal marker;
the ends of the primary nucleic acid strand are used to bind to detection molecules that can specifically bind to target molecules.
12. The nucleic acid probe of claim 11, wherein the signal nucleic acid strand is in direct complementary binding with the primary nucleic acid strand;
preferably, the signal nucleic acid strand is from the long single-stranded nucleic acid molecule of claim 10.
13. The nucleic acid probe of claim 11, wherein the signal nucleic acid strand is indirectly joined to the main nucleic acid strand through at least one branch nucleic acid strand;
each of the nucleic acid strands includes: a head region complementarily binding to said primary nucleic acid strand and a tail region complementarily binding to at least one of said signal nucleic acid strands.
14. The nucleic acid probe of claim 11, wherein the signal nucleic acid strand is indirectly joined to the main nucleic acid strand through at least one first nucleic acid strand and at least one second nucleic acid strand;
each of the first nucleic acid strands comprises: a first head region that complementarily binds said primary nucleic acid strand, and a first tail region that complementarily binds at least one of said secondary nucleic acid strands;
each of the second nucleic acid strands comprises: a second head region that complementarily binds to the first tail region of said first nucleic acid strand, and a second tail region that complementarily binds to at least one of said signal nucleic acid strands.
15. The nucleic acid probe of any one of claims 11-14, wherein the target-binding region is located at the 5' end of the primary nucleic acid strand.
16. The nucleic acid probe of any one of claims 11-14, wherein the signaling tag is a quantum dot.
17. The nucleic acid probe of claim 16, wherein the quantum dots are selected from the group consisting of Q525, Q565, Q585, Q605, Q625, 655, Q705, and Q800;
preferably, the target molecule is a protein or a polypeptide, and the detection molecule is an antibody capable of specifically binding to the protein or the polypeptide;
preferably, the target molecule is a nucleic acid fragment and the detection molecule is a nucleic acid fragment that can complementarily bind to the nucleic acid fragment.
18. A nucleic acid probe conjugate comprising a detection molecule that specifically binds to a target molecule and a nucleic acid probe according to any one of claims 11 to 17 attached to the detection molecule.
19. An antibody-nucleic acid probe conjugate comprising an antibody, and the nucleic acid probe of any one of claims 11-17 attached to the antibody, wherein the end of the nucleic acid probe is attached to the antibody, and wherein the detection molecule is the antibody.
20. A method for detecting protein by using single-molecule super-sensitivity POCT (point of care testing), which is characterized by comprising the following steps: detection is carried out using a nucleic acid probe according to any of claims 11 to 17, or a nucleic acid probe conjugate according to claim 18, or an antibody-nucleic acid probe conjugate according to claim 19.
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