CN114854736B - Circular nucleic acid molecule, method for producing same, nucleic acid probe, and method for detecting same - Google Patents
Circular nucleic acid molecule, method for producing same, nucleic acid probe, and method for detecting same Download PDFInfo
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Abstract
The invention discloses a circular 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 has a linear nucleotide sequence (GGTTATTATT) n Wherein n is not less than 3. The circular nucleic acid molecule disclosed by the invention only contains three bases and has a repeated sequence with a least secondary structure, RCA is carried out by taking the sequence as a template, and the circular nucleic acid molecule has higher amplification efficiency and higher efficiency of combining with a single-stranded DNA signal probe, so that the detection is more efficient and the sensitivity is higher. Can be widely applied to the fields of signal amplification, nano structure, biological sensing, molecular diagnosis and treatment and the like.
Description
Technical Field
The invention relates to the technical field of molecular detection, in particular to a circular nucleic acid molecule, a preparation method thereof, a nucleic acid probe and a detection method.
Background
Rolling circle amplification Rolling Circle Amplification (RCA) is a nucleic acid-based biological signal amplification technique that can replicate a circular nucleic acid molecule as a template by thousands of times under the action of a polymerase, enabling signal amplification mediated by low concentration target molecules to be detected. Compared with the most widely used PCR technology at present, RCA has been widely used for detection of biomarkers at present due to the advantages of no need of thermal cycling steps and no need of special equipment. The construction Of ultra-fast and highly sensitive RCA techniques is particularly important for Point-Of-Care Testing (POCT) and biosensing applications. But the efficiency of existing rolling circle amplification is lower.
Disclosure of Invention
The invention aims at providing a circular 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, takes the sequence as a template to carry out RCA, has higher amplification efficiency, and can be widely applied to the fields of signal amplification, nano structure, biosensing, molecular diagnosis and treatment and the like.
The invention is realized in the following way:
in a first aspect, the present invention provides a circular nucleic acid molecule suitable for use in RCA reactions as a template to obtain a long single stranded nucleic acid molecule which does not contain base C and which has a linear nucleotide sequence of (GGTTATTATT) n Wherein n is greater than or equal to 3, and n is an integer.
The 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 annular nucleic acid molecule containing three bases and having a least secondary structure, and RCA (round robin) is carried out by taking the novel sequence as a template, so that the amplification efficiency is high, the invention can be widely applied to the fields of signal amplification, nano structure, biosensing, molecular diagnosis and treatment and the like, and provides a molecular basis 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 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 of linear structure, which can form a circular nucleic acid molecule as described above by a loop forming reaction.
Alternatively, in some embodiments, the nucleotide sequence of the precursor nucleic acid molecule is P- (GGTTATTATT) n Wherein n is more than or equal to 3, n is an integer, and P is a phosphoric acid group.
Optionally, in some embodiments, n=4-6, preferably n=5.
In a third aspect, the present invention provides a method for preparing a circular nucleic acid molecule as described above, comprising: a step of a ring-forming reaction,
the cyclization reaction step comprises the following steps: the precursor nucleic acid molecules as described above are placed in a loop-forming reaction system for reaction to obtain the circular nucleic acid molecules.
The synthesis of the above-mentioned cyclic nucleic acid molecule is carried out by ligating the above-mentioned precursor nucleic acid molecule having a phosphate group modified at the 5' -end by the catalytic action of a nucleic acid single-stranded ligase.
Optionally, in some embodiments, the above-described preparation method further comprises a purification step. For example, after the ligation product of the cyclization reaction step is subjected to PAGE electrophoresis, a nucleic acid band is stained with fluorescence by using a SYBR GREEN I postdyeing method, a circular nucleic acid template is cut out by using a gel cutting instrument, and a circular nucleic acid molecule is purified by using a PAGE purification kit, so that a pure circular nucleic acid molecule is obtained. Of course, it is within the scope of the invention for the person skilled in the art to carry out other purification methods to obtain the circular nucleic acid molecule.
Optionally, in some embodiments, the cyclization reaction system contains a cycloligase, ATP, manganese chloride, and a buffer.
Alternatively, in some embodiments, the cyclization reaction is performed under the following conditions: incubation at 58-62℃for 0.5-1 hour and at 80-99℃for 8-12 minutes to inactivate the enzyme.
In a fourth aspect, the invention provides a method of performing an RCA reaction to obtain a long single stranded nucleic acid molecule comprising: RCA reactions were performed using the circular nucleic acid molecules as described above as templates.
For example, the addition of circular nucleic acid molecules to the RCA reaction mixture results in long single stranded nucleic acid molecules with minimal secondary structure. The RCA reaction mixture includes polymerase buffer, dntps, primers, nucleic acid polymerase. The reaction conditions were 37℃incubation (the length of time depends on the requirements of the detection conditions), 95℃incubation for 10 minutes to inactivate.
Alternatively, in some embodiments, the primer sequences used to carry out the RCA reaction are set forth in SEQ ID NO. 4. The primer can obtain a long single-stranded nucleic acid molecule through RCA reaction, and the length of the long single-stranded nucleic acid molecule can be controlled by a person skilled in the art through regulating the reaction time and the like according to actual requirements.
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 need in the art, and the length thereof can be controlled by adjusting it as will be clear to those skilled in the art.
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 main nucleic acid strand is the long single-stranded nucleic acid molecule of claim 10.
The signal nucleic acid strand is directly or indirectly bound to the main nucleic acid strand, and the signal nucleic acid strand is modified with a detectable signal label;
the ends of the main nucleic acid strand also have a binding moiety for binding to a detection molecule that specifically binds to a target molecule.
Alternatively, in some embodiments, the signal nucleic acid strand is directly complementary to the main nucleic acid strand.
Alternatively, in some embodiments, the signal nucleic acid strand is from a long single stranded nucleic acid molecule resulting from the RCA reaction described above.
Alternatively, in some embodiments, the signal nucleic acid strand is indirectly bound to the main nucleic acid strand through at least one branch nucleic acid strand;
each of the strands comprising: a head region and a tail region, said head region being complementarily bound to said main nucleic acid strand, said tail region being complementarily bound to at least one of said signal nucleic acid strands.
Alternatively, in some embodiments, the signal nucleic acid strand is indirectly bound 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 header region that is complementarily bound to said main nucleic acid strand, and a first tail region that is complementarily bound to at least one of said second nucleic acid strands;
each of the second nucleic acid strands comprises: a second head region complementarily binding to the first tail region of the first nucleic acid strand, and a second tail region complementarily binding to at least one of the signal nucleic acid strands.
Alternatively, in some embodiments, the target binding region is located 5' to the main nucleic acid strand.
Optionally, in some embodiments, the signal tag is a quantum dot. The signal label may be other labels such as fluorescent protein, radioisotope, etc.
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 the polypeptide.
Alternatively, in some embodiments, the target molecule is a nucleic acid fragment and the detection molecule is a nucleic acid fragment that is capable of complementary binding to the nucleic acid fragment.
In a seventh aspect, the present invention provides a nucleic acid probe conjugate comprising a detection molecule, and the above nucleic acid probe attached to the detection molecule, wherein the detection molecule is capable of specifically binding to a target molecule.
The target molecule may be any polypeptide, protein or nucleic acid fragment of interest in the art, etc. Accordingly, the detection molecule may be an antibody that specifically binds to the protein or the polypeptide, or a nucleic acid fragment that complementarily binds to the nucleic acid fragment.
In an eighth aspect, the invention provides an antibody-nucleic acid probe conjugate comprising an antibody, and a nucleic acid probe as described above attached to the antibody, the nucleic acid probe having a terminus attached to the antibody.
In a ninth aspect, the present invention provides a method for single molecule ultrasensitive POCT detection of a protein, comprising: detection is performed 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, i.e. the target molecule, in the detection system is detectable by the specific binding of the detection molecule, e.g. an antibody, to the target protein, which is detected by the person skilled in the art by detection of the signal marker.
Alternatively, in some embodiments, the class of antibody is selected based on the nature of the protein of interest, so long as it is capable of binding or specifically binding to the protein of interest. For example, the antibody can be used as a primary antibody for directly detecting target protein, and can also be used as a secondary antibody, wherein the antibody is an anti-primary antibody and indirectly binds to the target protein to realize detection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the calculation of the structure and thermodynamic parameters of the binding of different circular templates C1 to the primers using Mfold;
FIG. 2 is a flow chart showing the reaction of the MSS-RCA signal amplification method with other control RCA methods in example 2;
FIG. 3 is a schematic representation of the secondary structure of MSS-RCA and other control RCA method nucleic acid loop templates;
FIG. 4 is a chart showing the results of cyclic template PAGE of the DNAs of different RCAs generated by the ligase reaction of example 2;
FIG. 5 is a fluorescence standard curve of the highly sensitive and accurate quantitative purified nucleic acid of example 2.
FIG. 6 is an agarose gel plot of the amplification reaction for different RCAs in example 2.
FIG. 7 is a graph showing the comparison of amplification efficiencies of amplification reactions for different RCAs in example 3.
FIG. 8 is a graph showing comparison of amplification efficiencies of MSS-RCA (C1 circular template) and hybrid RCA (C3 circular template) using different concentrations of P1 as primers in example 4.
FIG. 9 is an AFM image of a linear DNA molecule of example 4.
FIG. 10 is a schematic diagram showing the structure of a nucleic acid probe in examples 5 to 8.
FIG. 11 is a schematic diagram showing the binding of the antibody-nucleic acid probe conjugate of example 9 to a target protein molecule.
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 branch nucleic acid strand, 231-first head region, 232-first tail region, 24-second branch 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 more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
Design of nucleic acid sequence this example designed a single-stranded DNA with a minimum of two-stage structure containing only three bases labeled with phosphate groups at one end, which was ligated into a loop to give a circular nucleic acid molecule which was subjected to rapid and efficient rolling circle amplification.
To design a nucleic acid circular template with a minimum secondary structure. In this example, the nucleic acid circular template with a minimum secondary structure is limited by the characteristics of the ligase, and the ligation activity of the single-stranded nucleic acid ligase is related to the length of the nucleic acid, and the ligation activity is related to the circular shape of the nucleic acidThe length of the circular template (MSS-RCA template) with the minimum secondary structure is generally designed to be tens of bases in consideration of the cost of the circular template for nucleic acids, the base at the 5 'end is G and modifies the phosphate group, the base at the 3' end is T, and the sequence is an integer multiple of the repeating unit with the minimum secondary structure. The primer is designed to pair with a circular template formed after the ligation reaction. The Tm value of the primer sequence design is considered to be suitable for the temperature of the amplification reaction. Considering the characteristics and cost of single-stranded nucleases that cannot be ligated below 15 bases, the circular template sequence (C1) in this example is designed as a single-stranded DNA sequence with a total length of 50 bases, which contains 5 repeats with a minimal secondary structure: (GGTTATTATT) 5 The linear nucleic acid sequences were:
GGTTATTATTGGTTATTATTGGTTATTATTGGTTATTATTGGTTATTATT(SEQ ID NO.1)；
precursor structure:
in this example, single-stranded DNA (MSS-RCA-precursor) with a minimum secondary structure having a 5' phosphate group, and Primer 1 complementarily paired therewith were designed;
as a control, the SS-RCA-precursor contains four bases, comprising 5 repeats with a secondary structure, and a Primer 2 complementary thereto.
As a control group for transition states, hybrid-RCA-precursors contained 2 repeated sequences of MSS-RCA-precursors, and 3 repeated sequences of SS-RCA-precursors. The length of the portion of the primer to be paired with the circular template was designed to be 20 bases, and the Tm value was 52 ℃.
The other end of the primer can be designed into any sequence matched with the target to be detected so as to be designed into a biosensor capable of detecting 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 MSS-RCA circular template C1 and primers P1, SS-RCA circular template C2 and primers P2, hybrid-RCA circular template C3 and primers P1, C3 combined with primer P2 calculated by using Mfold, and shows that Tm values are higher than 60 ℃ and can be completely complementary paired at room temperature.
FIG. 2 shows the secondary structure of each single-stranded circular template predicted by NUPACK software. The results show that the C1 template for MSS-RCA has no secondary structure production, the free energy is 0kcal/mol, the C2 template for SS-RCA has secondary structure production, the free energy is-9.96 kcal/mol, the C3 template for Hybrid-RCA has less secondary structure production than C2, and the free energy is-4.98 kcal/mol.
Example 2
As shown in FIG. 2, the process of the rolling circle amplification technology of the circular template based on the nucleic acid with the minimum secondary structure is mainly realized by the steps of synthesis of the circular template, purification of the circular template and RCA reaction.
FIG. 3 shows the secondary structure prediction results of MSS-RCA and other control RCA method nucleic acid circular templates.
The specific process of each step is as follows:
(1) Synthesis of circular template the ligation reaction 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 circular precursor single-stranded template, 2.5. Mu.L of 100U/. Mu.L nucleic acid single-stranded ligase (ssDNA/RNA circular). Incubation at 60℃for 1 hour and at 95℃for 10 minutes inactivated the nucleic acid single-stranded ligase. The ligation product was stored at-20 ℃.
The results of the ligation reaction were verified by non-denaturing polyacrylamide gel electrophoresis (15% PAGE gel). As a result, the lanes from left to right are, respectively, 20bp DNA ladder, single-stranded C1 template of MSS-RCA without nucleotidyl ligase addition, C1 template of MSS-RCA with nucleotidyl ligase addition, single-stranded C2 template of SS-RCA without nucleotidyl ligase addition, C2 template of SS-RCA with nucleotidyl ligase addition, single-stranded C3 template of Hybrid-RCA without nucleotidyl ligase addition, and C3 template of Hybrid-RCA with nucleotidyl ligase addition, as shown in FIG. 4. As a result of denaturing PAGE electrophoresis, when no nucleic acid single-stranded ligase was added in the ligation reaction, the templates were all in a linear state with only one band. After the ligation reaction, a nucleic acid single-stranded ligase is added to form a loop, a new band appears on the single-stranded template, and the band can be determined to be the nucleic acid circular template which is connected to form a loop because the circular template runs slower than a linear shape in electrophoresis. The above results demonstrate that the circular nucleic acid template product is produced in the system after the single-stranded nucleic acid ligase reaction.
(2) The purification of the circular template, namely preparing 15% PAGE denaturing gel, adding an equal volume of formamide into the sample, incubating for 20 minutes at 95 ℃ for denaturation, and adding loading dye of 1/5 of the volume of the sample. Running electrophoresis at 100V until the dark blue band runs out of the gel plate. mu.L 10000 Xsybr green I is added into 20ml of water to prepare a dye solution with 1 time of the final concentration of the sybr green I, and after the glue is removed from the glue plate, the glue is added into the dye solution for incubation for 20-30 minutes. After removal of the glue, it was placed on a blue light emitting glue cutter, the annular template strip was carefully cut off, collected in an EP tube and weighed. 1-2 volumes of diffusion buffer (100 mg of gum added 100-150ml diffusion buffer) were added, and the mixture was centrifuged at 600rpm at 50℃for 2 hours and at 12,000rpm for 20 minutes. Taking the supernatant, adding 3 times of absolute ethyl alcohol, and standing at-80 ℃ for 1 hour. Centrifuge at 14,000rpm at 4℃for 20 min. The supernatant was discarded. 70% ethanol was added and the mixture was centrifuged at 12,000rpm at 4℃for 20 minutes. The supernatant was discarded. And (5) drying by using an ultra clean bench. Adding a proper amount of water for dissolution. The DNA was again purified using a nucleic acid purification column. Obtaining the pure nucleic acid circular template.
Since the detection sensitivity of the nanodrop is generally at ng/. Mu.L, the concentration of the nucleic acid circular template obtained by the purification method of this example is generally at ng/. Mu.L or even lower than ng/. Mu.L, and thus the purified nucleic acid circular template cannot be precisely quantified by the nanodrop. If the nucleic acid loop template cannot be precisely quantified, the subsequent comparison and measurement of RCA efficiency will be affected. Therefore, in this example, the purified nucleic acid circular template was highly sensitively and precisely quantified using the more sensitive sybr gold nucleic acid dye. The quantitative standard curve system was 50. Mu.L, and the system contained 10. Mu.L of single-stranded nucleic acid templates at different concentrations, 1. Mu.L of 100 Xsybr gold. The standard curve results are shown in FIG. 5, and the results show that the fluorescence intensity of the single-stranded C1 template of MSS-RCA is highest as a whole after the single-stranded C1 template is combined with sybr gold, and the fluorescence intensity is high when the single-stranded C1 template is combined with sybr gold, namely, the fluorescence intensity is high 3. It was also verified that the single-stranded C1 template of MSS-RCA has the least secondary structure. The single-stranded C3 template of Hybrid-RCA combined with the C2 template of sybr gold with higher fluorescence intensity than that of SS-RCA demonstrated that the single-stranded C3 template of Hybrid-RCA had more secondary structure than that of 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 low-concentration-level nucleic acid circular template can be accurately quantified with high sensitivity.
(3) RCA reaction the reaction system contained 2. Mu.L of 10 XPhi 29 DNA polymerase buffer, 1. Mu.L of 10mM dNTP, 5U Phi29 DNA polymerase, 2. Mu.L of 100nM purified nucleic acid circular template, primers of different concentrations. Reaction (time according to need) at 37℃and incubation at 95℃for 10 min.
Binding of circular template C1 to primer P1 elicited MSS-RCA reaction, binding of circular template C2 to primer P2 elicited SS-RCA reaction, binding of circular template C3 to primer P1 elicited Hybrid-RCA-1 reaction, and binding of C3 to primer P2 elicited Hybrid-RCA-2 reaction was performed to verify the feasibility of RCA reaction. The reaction system was 20. Mu.L, reacted at 37℃for 1 hour, and incubated at 95℃for 10 minutes. The remainder of the RCA reaction was identical to that of example 2 in a circular template containing a final concentration of 20nM MSS-RCA, primer at a final concentration of 200 nM. The results showed that after adding 2x sybr gold to the DNA product of 10. Mu. LRCA, fluorescent signals were generated under blue light irradiation, and that the binding of circular template C2 to primer P2 gave the lowest fluorescent signal to trigger the SS-RCA reaction (as shown in FIG. 6).
The above results can be explained: 1. the SS-RCA reaction produces a DNA product with many secondary structures and thus results in low fluorescence intensity after sybr gold binding; 2. and the C2 template contains more secondary structure, so that the DNA product generated by RCA amplification is insufficient.
The amplification results from the above procedure were verified by electrophoresis on a 0.6% agarose gel and the agarose gel results were irradiated with blue light using the sybr gold spot method. The results are shown in FIG. 6. Lanes from left to right are DNA ladder, circular template C1 bound to primer P1 to trigger MSS-RCA reaction, circular template C2 bound to primer P2 to trigger SS-RCA reaction, circular template C3 bound to primer P1 to trigger Hybrid-RCA-1 reaction, and C3 bound to primer P2 to trigger Hybrid-RCA-2 reaction, respectively. As shown by agarose electrophoresis results, the circular templates designed and synthesized in this example and the corresponding primers were used, and long-chain DNA products were produced after the RCA reaction for 1 hour.
Example 3
To compare the amplification efficiencies of different RCA reaction systems, the loop template C1 and primer P1 combined to trigger the MSS-RCA reaction, the loop template C2 and primer P2 combined to trigger the SS-RCA reaction, the loop template C3 and primer P1 combined to trigger the Hybrid-RCA-1 reaction, and C3 and primer P2 combined to trigger the Hybrid-RCA-2 reaction were performed in the same system and the amplification efficiencies were compared, respectively. The reaction system was 80. Mu.L, and 5. Mu.L of the sample was taken out every 5 minutes at 37℃and incubated at 95℃for 10 minutes to inactivate the Phi29 amplification enzyme to stop the amplification reaction. The reaction contained 20nM circular template, primer with a final concentration of 200nM, 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 was the slowest, and the amplification efficiency of Hybrid-RCA-2 initiated by the circular template 3 bound to the primer P2 was the second, whereas the MSS-RCA reaction was comparable to the reaction efficiency of Hybrid-RCA-1 initiated by the circular template C3 bound to the primer P1. This result further verifies that the SS-RCA reaction in the above examples has the lowest fluorescence signal after 1 hour of amplification. Furthermore, the efficiency of amplification of Hybrid-RCA-2 by circular template 3 bound to primer P2 was lower than that of Hybrid-RCA-1 by circular template C3 bound to primer P1, which suggests that the secondary structure of the primer impedes the amplification reaction, probably because the binding efficiency of the primer with the secondary structure was not as high at room temperature as that of the primer without the secondary structure. The MSS-RCA reaction was comparable to the reaction efficiency of primer P1 in combination with circular template C3 to initiate Hybrid-RCA-1 reaction, probably due to the high primer concentration, which saturated the primer. Thus, the ratio of primer to circular template was changed in the subsequent example 4, and specific studies compared MSS-RCA with Hybrid-RCA-1 in reaction efficiency.
Example 4
To compare the efficiency of MSS-RCA reaction with Hybrid-RCA-1 for specific studies, the binding of circular template C1 to primer P1 triggered MSS-RCA reaction, and the binding of circular template C3 to primer P1 triggered Hybrid-RCA-1 reaction were performed in the same system and the amplification efficiencies were compared, respectively. The reaction system was 80. Mu.L, and 5. Mu.L of the sample was taken out every 5 minutes at 37℃and incubated at 95℃for 10 minutes to inactivate the Phi29 amplification enzyme to stop the amplification reaction. The reaction contained 10nM of circular template, primers with different final concentrations (2.5 nM,5nM,10 nM), and the concentrations of the remaining components of the RCA reaction were identical to those of example 2.
The results show (FIG. 8) that when the concentration of the primer is increased under the reaction conditions of this example, the amplification efficiency of Hybrid-RCA-1 is also improved correspondingly, 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 about, which indicates that the MSS-RCA amplification efficiency is high, and the concentration of the primer in the concentration range is changed so as not to influence the amplification efficiency. This result is a good demonstration of the highest amplification efficiency of MSS-RCA, and can indicate that under the same conditions and at the same time, MSS-RCA can achieve a higher sensitivity than other RCA reactions containing secondary structures. 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 with a circular template with a secondary structure. MSS-RCA is capable of synthesizing linear DNA molecules up to at least 6 microns (see AFM, FIG. 9). The basis for the subsequent amplification of MSS-RCA and the amplification of branched signals on the surface, for example as the main nucleic acid strand in examples 5-9, examples 5-9 provide the use of the resulting linear DNA molecules, see in particular below.
Example 5
The present embodiment 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 combined with the main nucleic acid strand 20, and the signal nucleic acid strand 21 is modified with fluorescent quantum dots 211.
The end (5' end) of the main nucleic acid strand 20 has a target binding region 201, and the nucleic acid probe can specifically bind to a target molecule (e.g., an antibody) modified with a binding nucleic acid strand complementarily bound to the target binding region 201 via the target binding region 201.
Example 6
This example provides a nucleic acid probe carrying a fluorescent signal (e.g., A1, L1 in FIG. 10 represents the nucleotide length, d1 represents the complementary length), which has substantially the same structure as that of 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 the main nucleic acid strand 20. The fluorescence signal was significantly stronger and easier to detect than example 5, which carried more fluorescence signal.
Example 7
The present embodiment 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 fluorescent quantum dots 211, and the signal nucleic acid strand 21 is indirectly bound 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 nucleic acid strand 22 includes: a head region 221 and a tail region 222, each of the nucleic acid strands 22 is complementarily bound to the main nucleic acid strand 20 via its head region 221, and the tail region 222 of each of the nucleic acid strands 22 is complementarily bound to the plurality of signal nucleic acid strands 21.
The fluorescent signal of the nucleic acid probe with the structure is obviously amplified and is better and easier to detect.
Example 8
The present embodiment provides a nucleic acid probe carrying a fluorescent signal (refer to A3 in FIG. 10) comprising one main nucleic acid strand 20, a plurality of signal nucleic acid strands 21, a plurality of first nucleic acid strands 23, and a plurality of second nucleic acid strands 24;
the signal nucleic acid strand 21 is modified with fluorescent quantum dots, and the signal nucleic acid strand 21 is indirectly bound to the main nucleic acid strand 20 via 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 complementarily binding to the main nucleic acid strand 20, and a first tail region 232 complementarily binding to the plurality of second nucleic acid strands 24;
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 obviously amplified and is easier to detect.
The calculation formula of the amplification factor of the nucleic acid probe of examples 5 to 8 is shown in the formula in FIG. 10, an represents the amplification factor of the fluorescent signal; ln represents the nucleotide length of the tail region of the main nucleic acid strand or the branched nucleic acid strand; dn denotes the nucleotide length of the complementary pair between two nucleic acid strands; kn is a constant for each stage of signal branches. It can be seen from the figure that the more branches, the stronger the fluorescent signal, and also the easier it is to detect.
By the nucleotide length Ln of the main nucleic acid strand and the branch nucleic acid strand, and the length dn of the complementary pair portion between the two nucleic acid strands, the signal amplification factor can be adjusted.
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 with the primer P1; the sequences of the branched nucleic acid strand and the signal nucleic acid strand are also readily available.
Example 9
This example provides an antibody-nucleic acid probe conjugate having a structure as shown in FIG. 11, comprising: the antibody and the fluorescent probe combined on the antibody are incubated together with the target protein molecules, the antibody-nucleic acid probe conjugate is combined to the target protein molecules, and the detection of the target protein molecules can be realized through the detection of fluorescent quantum dots. The related fluorescence detection means are conventional technical means known in the art, and are not described in detail herein.
In summary, the existing research discovers that the DNA sequence with high content of A and C is used as the circular template of RCA reaction by using an in vitro screening method, has the characteristic of increasing the reaction efficiency, and proposes the theory that the content of A and C of the DNA circular template is related to the amplification efficiency. Studies have also shown that the efficiency of RCA amplification is related not only 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 is mainly related to the secondary structure, and designed a circular template of DNA with a minimum secondary structure in which one base was deleted for RCA reaction. The result shows that the RCA amplification efficiency of the circular template (C1) with the DNA sequence with the least secondary structure provided by the embodiment is higher than that of other RCA amplification methods with the secondary structure, and the novel, more efficient and sensitive signal amplifier nucleic acid molecule materials, novel methods and research tools are provided for biological analysis methods which rely on RCA signal amplification in the fields of biosensing, molecular diagnosis and treatment and the like.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> university of hong Kong Chinese (Shenzhen)
<120> circular nucleic acid molecule, method for producing the same, nucleic acid probe and method for detecting the same
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
ggttattatt ggttattatt ggttattatt ggttattatt ggttattatt 50
<210> 2
<211> 50
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gagacgcggt gagacgcggt gagacgcggt gagacgcggt gagacgcggt 50
<210> 3
<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)
<400> 4
atcctcctcc tctgagctgt catttaccaa taataaccaa taataaccaa taata 55
<210> 5
<211> 55
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
atcctcctcc tctgagctgt catttaccgc gtctcaccgc gtctcaccgc gtctc 55
Claims (24)
1. A circular nucleic acid molecule suitable for use in RCA reactions as a template to obtain a long single stranded nucleic acid molecule, characterized in that it does not contain base C and has a linear nucleotide sequence (GGTTATTATT) n Wherein n is greater than or equal to 3; n is an integer.
2. The circular nucleic acid molecule of claim 1, wherein n = 4-6.
3. The circular nucleic acid molecule of claim 2, wherein n = 5.
4. A precursor nucleic acid molecule for the preparation of a circular nucleic acid molecule according to any one of claims 1 to 3, characterized in that it is of a linear structure which forms a circular nucleic acid molecule according to any one of claims 1 to 3 by a loop forming reaction;
the nucleotide sequence of the precursor nucleic acid molecule is P- (GGTTATTATT) n Wherein n is greater than or equal to 3, n isInteger, P is a phosphate group.
5. The precursor nucleic acid molecule of claim 4, wherein n = 4-6.
6. The precursor nucleic acid molecule of claim 5, wherein n = 5.
7. A method of preparing a circular nucleic acid molecule according to any one of claims 1 to 3, comprising: a step of a ring-forming reaction,
the cyclization reaction step comprises the following steps: placing the precursor nucleic acid molecule of any one of claims 4-6 in a loop forming reaction system for reaction to obtain the circular nucleic acid molecule.
8. The method according to claim 7, wherein the cyclization reaction system comprises a cyclase, ATP, manganese chloride and a buffer.
9. The process according to claim 7 or 8, wherein the cyclization reaction is carried out under the following conditions: incubation is carried out at 58-62 ℃ for 0.5-1 hour, and at 80-99 ℃ for 8-12 minutes.
10. A method of performing an RCA reaction to obtain a long single stranded nucleic acid molecule, comprising: an RCA reaction using the circular nucleic acid molecule of any one of claims 1 to 3 as a template.
11. The method of claim 10, wherein the RCA reaction is performed using a primer sequence set forth in SEQ ID No. 4.
12. A long single stranded nucleic acid molecule obtainable by the method of claim 10 or 11.
13. 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 main nucleic acid strand is the long single-stranded nucleic acid molecule of claim 12;
the signal nucleic acid strand is directly or indirectly bound to the main nucleic acid strand, and the signal nucleic acid strand is modified with a detected signal marker;
the ends of the main nucleic acid strand are used to bind to a detection molecule that specifically binds to a target molecule.
14. The nucleic acid probe of claim 13, wherein the signal nucleic acid strand is directly complementary to the main nucleic acid strand.
15. The nucleic acid probe of claim 13, wherein the signal nucleic acid strand is indirectly bound to the main nucleic acid strand through at least one branch nucleic acid strand;
each of the strands comprising: a head region and a tail region, said head region being complementarily bound to said main nucleic acid strand, said tail region being complementarily bound to at least one of said signal nucleic acid strands.
16. The nucleic acid probe of claim 13, wherein the signal nucleic acid strand is indirectly bound 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 header region that is complementarily bound to said main nucleic acid strand, and a first tail region that is complementarily bound to at least one of said second nucleic acid strands;
each of the second nucleic acid strands comprises: a second head region complementarily binding to the first tail region of the first nucleic acid strand, and a second tail region complementarily binding to at least one of the signal nucleic acid strands.
17. The nucleic acid probe of any one of claims 13-16, wherein the target binding region is located 5' to the main nucleic acid strand.
18. The nucleic acid probe of any one of claims 13-16, wherein the signal label is a quantum dot.
19. The nucleic acid probe of claim 18, wherein the quantum dot is selected from the group consisting of Q525, Q565, Q585, Q605, Q625, 655, Q705, and Q800.
20. The nucleic acid probe of claim 18, wherein the target molecule is a protein or polypeptide and the detection molecule is an antibody that specifically binds to the protein or polypeptide.
21. The nucleic acid probe of claim 18, wherein the target molecule is a nucleic acid fragment and the detection molecule is a nucleic acid fragment capable of complementary binding to the nucleic acid fragment.
22. A nucleic acid probe conjugate comprising a detection molecule that specifically binds to a target molecule, and the nucleic acid probe of any one of claims 13-21 attached to the detection molecule.
23. An antibody-nucleic acid probe conjugate comprising an antibody, and the nucleic acid probe of any one of claims 13-21 attached to the antibody, wherein the nucleic acid probe is attached at the end to the antibody and the detection molecule is the antibody.
24. A method for single molecule ultrasensitive POCT detection of a protein, comprising: detection using a nucleic acid probe according to any one of claims 13 to 21, or a nucleic acid probe conjugate according to claim 22, or an antibody-nucleic acid probe conjugate according to claim 23.
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