Photoresponsive aptamer, photoactivated DNA polymerase and application thereof

Abstract

The application discloses a light-responsive nucleic acid aptamer, a light-activated DNA polymerase, a kit, a method for preparing the light-activated DNA polymerase, a method for activating the light-activated DNA polymerase, and application of the light-activated DNA polymerase. The light-responsive nucleic acid aptamer comprises an aptamer nucleotide sequence and a photo-cleavable group modified in the aptamer nucleotide sequence, the photo-cleavable group is used for being broken after ultraviolet light irradiation to break the aptamer nucleotide sequence into two nucleotide sequences; the aptamer nucleotide sequence comprises a G-quadruplex nucleotide sequence capable of forming a G-quadruplex and a side nucleotide sequence capable of forming a hairpin structure, the G-quadruplex nucleotide sequence is used for being combined with a G-quadruplex binding peptide fused on a DNA polymerase, and the side nucleotide sequence is used for being combined with an active site of the DNA polymerase. By using the light-activated DNA polymerase provided in the application, the difference in amplification time between samples is reduced, non-specific amplification is avoided, and the accuracy of nucleic acid detection is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a light-responsive nucleic acid aptamer, a light-initiated DNA polymerase, a method for preparing a light-initiated DNA polymerase, a method for activating the activity of a light-initiated DNA polymerase, and the application of the light-initiated DNA polymerase in the detection or synthesis of nucleic acids. Background Technology

[0002] A key problem in nucleic acid detection technology is nonspecific amplification products caused by primer mismatch and non-template amplification. Nonspecific amplification strongly inhibits or reduces the amplification of the target template, thus lowering detection sensitivity. However, currently, there are few methods to address the problem of nonspecific amplification in isothermal amplification. Precisely initiating and controlling the reaction activity of DNA polymerase is an effective means to ensure the accuracy of DNA amplification and reduce nonspecific amplification.

[0003] Light, as a physical signal, can be precisely switched on and off using physical devices. The combination of light-responsive nucleic acid aptamers and G-quadruplex-binding peptides allows for light-controlled DNA polymerase activity, effectively addressing the problem of non-specific amplification in DNA amplification reactions. Developing light-responsive DNA polymerase activity is of significant value for genetic engineering research and applications, and its application prospects are very broad. Summary of the Invention

[0004] One of the objectives of this invention is to provide a light-responsive nucleic acid aptamer.

[0005] One of the objectives of this invention is achieved by the following technical solution: a light-responsive nucleic acid aptamer for regulating DNA polymerases with G-quadruplex binding peptides, comprising an aptamer nucleotide sequence and a photolytic cleavage group, wherein the photolytic cleavage group is modified in the aptamer nucleotide sequence for cleavage upon ultraviolet light irradiation to split the aptamer nucleotide sequence into two nucleotide sequences.

[0006] The aptamer nucleotide sequence includes a G-quadruplex nucleotide sequence capable of forming a G-quadruplex and a flanking nucleotide sequence capable of forming a hairpin structure. The G-quadruplex nucleotide sequence is used to bind to the G-quadruplex binding peptide fused to the DNA polymerase, and the flanking nucleotide sequence is used to bind to the active site of the DNA polymerase.

[0007] A second objective of this invention is to provide a light-initiated DNA polymerase, comprising a DNA polymerase having a G-quadruplex-binding peptide and the aforementioned light-responsive nucleic acid aptamer;

[0008] The DNA polymerase having a G-quadruplex-binding peptide comprises a DNA polymerase fragment and the G-quadruplex-binding peptide, wherein the G-quadruplex-binding peptide is modified and linked to the N-terminus of the DNA polymerase fragment.

[0009] The G-quadruplex nucleotide sequence is used to bind the G-quadruplex binding peptide; the flanking nucleotide sequence is used to bind to the active site of the DNA polymerase fragment to inhibit the activity of the photo-initiated DNA polymerase; the photolyzable cleavage group is used to break the aptamer nucleotide sequence into two nucleotide sequences under ultraviolet light irradiation, so that the flanking nucleotide sequence is released from the active site of the DNA polymerase fragment, thereby activating the activity of the photo-initiated DNA polymerase.

[0010] The third objective of this invention is to provide a method for preparing the above-mentioned photo-initiated DNA polymerase, comprising the following steps:

[0011] The light-responsive nucleic acid aptamer was dissolved in the first buffer solution to a final concentration of 15 uM to 25 uM, heated at 92 °C to 95 °C for 4 min to 6 min, and then cooled to 23 °C to 27 °C at a rate of 0.05 °C / s to 2 °C / s to form the first mixture.

[0012] DNA polymerase containing G-quadruplex-binding peptide was dissolved in the second buffer to a final concentration of 7 μM to 15 μM to form the second mixture.

[0013] The first mixture and the second mixture are mixed at a volume ratio of 1:1 and placed at 4°C for 25 min to 35 min to form a DNA-protein complex.

[0014] An equal volume of glycerol was added to the DNA-protein bound complex to obtain the photo-initiated DNA polymerase.

[0015] The fourth objective of this invention is to provide a method for activating the above-mentioned photo-initiated DNA polymerase activity, comprising the following steps:

[0016] At a preset temperature, the photoactivated DNA polymerase is irradiated with ultraviolet light for a preset time to activate its activity; wherein the wavelength of the ultraviolet light is greater than or equal to 300 nm and less than or equal to 380 nm, and the preset time is greater than or equal to 0.5 min.

[0017] The fifth objective of this invention is to provide an application of the above-mentioned photo-initiated DNA polymerase for detecting or synthesizing nucleic acids.

[0018] The inventiveness of this invention lies in: (1) This invention is based on the natural DNA polymerase and is a creative technical modification that enables the DNA polymerase, which is not affected by external light signals, to have photoresponsiveness; (2) When the photo-activated DNA polymerase constructed by this invention is applied to nucleic acid amplification technology, it can enable the reaction of all samples to start simultaneously at a set temperature, reduce the difference in amplification time between samples, and avoid non-specific amplification. Therefore, it can improve the accuracy of nucleic acid detection. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the principle of light-responsive nucleic acid aptamers regulating DNA polymerase activity with G-quadruplex binding peptides in this invention.

[0020] Figure 2 The pCold-I-G4P-Bst plasmid pattern provided in Embodiment 1 of the present invention;

[0021] Figure 3A This is a schematic diagram of the structure of the Bst DNA polymerase provided in Embodiment 1 of the present invention, wherein G4P-Bst is a large fragment of Bst DNA polymerase with G-quadruplex binding protein (G4P) fused to its N-terminus.

[0022] Figure 3B This is a schematic diagram of the secondary structure of the photoresponsive nucleic acid aptamer provided in Embodiment 1 of the present invention;

[0023] Figure 3C The figure shows the primer extension experiment results provided in Embodiment 2 of the present invention before and after binding with the light-responsive nucleic acid aptamer, and under conditions of UV irradiation or no irradiation.

[0024] Figure 3D for Figure 3C Graph showing the results of DNA polymerase activity quantification under different conditions;

[0025] Figure 3E This is a graph showing the photolysis efficiency of photoresponsive nucleic acid aptamers at different times of ultraviolet light irradiation in the presence of photopromoted DNA polymerase G4P-Bst, as provided in Embodiment 2 of the present invention.

[0026] Figure 3F This is a graph showing the results of G4P-Bst activity at different times of ultraviolet irradiation in the presence of photo-initiated DNA polymerase G4P-Bst, as provided in Example 2 of the present invention.

[0027] Figure 4A This is a schematic diagram of the PC-linker provided in Embodiment 3 of the present invention at different sites on a light-responsive nucleic acid aptamer;

[0028] Figure 4BThe results of primer extension experiments were performed on G4P-Bst blocked with photoresponsive nucleic acid aptamers of different PC linkers provided in Example 3 of the present invention after exposure to no light (UV-) and UV light for 2 minutes (UV+).

[0029] Figure 4C This is a schematic diagram of light-responsive nucleic acid aptamers with different hairpin lengths provided in Embodiment 3 of the present invention;

[0030] Figure 4D The results of primer extension experiments were performed on G4P-Bst blocked with light-responsive nucleic acid aptamers of different hairpin lengths provided in Embodiment 3 of the present invention after exposure to no light (UV-) and UV light for 2 minutes (UV+).

[0031] Figure 5 The pCold-I-G4P-Taq plasmid map provided in Embodiment 4 of the present invention;

[0032] Figure 6 The pCold-I-G4P-Bsu plasmid pattern provided in Embodiment 4 of the present invention;

[0033] Figure 7A The diagram shows the G4P-fused Taq DNA polymerase (G4P-Taq) and Bsu DNA polymerase (G4P-Bsu) provided in Embodiment 4 of the present invention, and the principle of the interaction between these enzymes and light-responsive nucleic acid aptamers.

[0034] Figure 7B The primer extension experiment provided in Example 4 of this invention was used to detect the activity of G4P-Taq. Among them, the control sample using an enzyme without a light-responsive nucleic acid aptamer was marked as CTRL, the sample using an enzyme bound to a light-responsive nucleic acid aptamer but not irradiated with ultraviolet light was marked as UV(-), and the sample using an enzyme bound to a light-responsive nucleic acid aptamer and irradiated with ultraviolet light for 2 minutes was marked as UV(+).

[0035] Figure 7C The primer extension experiment provided in Example 4 of this invention is used to detect the activity of G4P-Bsu; wherein, the control sample using an enzyme without a light-responsive nucleic acid aptamer is labeled CTRL, the sample using an enzyme bound to a light-responsive nucleic acid aptamer but not irradiated with ultraviolet light is labeled UV(-), and the sample using an enzyme bound to a light-responsive nucleic acid aptamer and irradiated with ultraviolet light is labeled UV(+).

[0036] Figure 8AThis is a schematic diagram of the operation of a loop-mediated isothermal amplification (LAMP) reaction using a light-initiated device provided in Embodiment 5 of the present invention; wherein, the LAMP reaction is carried out on a heating block, and a 360-370nm LED light source is irradiated from a height of 0.5cm from the reaction tube cap;

[0037] Figure 8B The image shows the results of detecting human papillomavirus (HPV) type 45 E6-E7 gene DNA using light-activated visual color-changing LAMP, as provided in Embodiment 5 of the present invention.

[0038] Figure 8C This is an electrophoresis result of a sample used in Embodiment 5 of the present invention to detect human papillomavirus (HPV) type 45 E6-E7 gene DNA using light-activated LAMP.

[0039] Figures 9A-9C This is a result diagram of light-started PCR using light-controlled Taq DNA polymerase provided in Embodiment Six of the present invention; wherein, Figure 9A The control group using enzymes that do not contain light-responsive nucleic acid aptamers is labeled CTRL. Figure 9B Samples containing enzymes with light-responsive nucleic acid aptamers but without UV irradiation were labeled as UV(-). Figure 9C Samples that used enzymes incorporating light-responsive nucleic acid aptamers and were irradiated with ultraviolet light were labeled UV(+). Detailed Implementation

[0040] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0041] This invention provides a light-responsive nucleic acid aptamer for regulating DNA polymerases with G-quadruplex-binding peptides. The aptamer includes an aptamer nucleotide sequence and a photo-cleaving group. The photo-cleaving group is modified into the aptamer nucleotide sequence and is used to break the aptamer nucleotide sequence into two nucleotide sequences after ultraviolet light irradiation.

[0042] The aptamer nucleotide sequence includes a G-quadruplex nucleotide sequence that can form a G-quadruplex and a flanking nucleotide sequence that can form a hairpin structure. The G-quadruplex nucleotide sequence is used to bind the G-quadruplex binding peptide fused to DNA polymerase, and the flanking nucleotide sequence is used to bind the active site of DNA polymerase.

[0043] As one implementation, the G-quadruplex nucleotide sequence is SEQ ID NO: 1; and / or,

[0044] The chemical formula of the photolytically cleavable group is:

[0045]

[0046] The connection relationship between the photolytically cleavable group and the upstream and downstream bases is as follows:

[0047]

[0048] In one implementation, the length of the double-stranded pairing region of the hairpin structure formed by the flanking nucleotide sequence is greater than or equal to 5 bp and less than or equal to 20 bp.

[0049] As one implementation, when the length of the double-stranded pairing region of the hairpin structure formed by the flanking nucleotide sequence is greater than or equal to 5 bp and less than or equal to 8 bp, the light-responsive nucleic acid aptamer can inhibit the activity of DNA polymerase with G-quadruplex-binding peptide within a temperature range of greater than or equal to 15°C and less than or equal to 40°C.

[0050] As one implementation, when the length of the double-stranded pairing region of the hairpin structure formed by the flanking nucleotide sequence is greater than or equal to 9 bp and less than or equal to 20 bp, the light-responsive nucleic acid aptamer can inhibit the activity of DNA polymerase within a temperature range of greater than or equal to 15°C and less than or equal to 65°C.

[0051] In one implementation, the G-quadruple chain has a first connecting ring, a second connecting ring, and a third connecting ring, and the hairpin structure has a fourth connecting ring;

[0052] Photolytically cleavable groups are modified in the middle of the first linker ring, the second linker ring, the third linker ring, the fourth linker ring, or the G-quadruplex and the hairpin structure.

[0053] In one implementation, when a photolytic cleavage group is modified between the G-quadruplex and the hairpin structure, the photoresponsive nucleic acid aptamer, under ultraviolet light irradiation, restores the activity of the DNA polymerase containing the G-quadruplex-binding peptide within a temperature range greater than or equal to 30°C and less than or equal to 65°C.

[0054] In one implementation, when a photolytic cleavage group is modified onto the first, second, third, or fourth linker ring, the photoresponsive nucleic acid aptamer, under ultraviolet light irradiation, restores the activity of DNA polymerase with G-quadruplex-binding peptide within a temperature range greater than or equal to 50°C and less than or equal to 65°C.

[0055] The present invention also provides a light-initiated DNA polymerase, comprising a DNA polymerase having a G-quadruplex-binding peptide and the above-mentioned light-responsive nucleic acid aptamer;

[0056] DNA polymerases with G-quadruplex-binding peptides include a DNA polymerase fragment and a G-quadruplex-binding peptide, with the G-quadruplex-binding peptide modified and linked to the N-terminus of the DNA polymerase fragment.

[0057] The G-quadruplex nucleotide sequence is used to bind the G-quadruplex binding peptide; the flanking nucleotide sequence is used to bind the active site of the DNA polymerase fragment to inhibit the activity of photoinitiated DNA polymerase; the photocleavable group is used to break the aptamer nucleotide sequence into two nucleotide sequences under ultraviolet light irradiation, so that the flanking nucleotide sequence is released from the active site of the DNA polymerase fragment, thereby initiating the activity of photoinitiated DNA polymerase.

[0058] As one implementation method, the amino acid sequence of the G-quadruplex-binding peptide is shown in SEQ ID NO: 2.

[0059] As one implementation method, the DNA polymerase fragment is derived from any one of Bst DNA polymerase, Bsu DNA polymerase, or Taq DNA polymerase.

[0060] In one implementation, the DNA polymerase fragment is derived from Bst DNA polymerase, and the amino acid sequence of the DNA polymerase fragment is shown in SEQ ID NO: 3; or,

[0061] The DNA polymerase fragment is derived from Bsu DNA polymerase, and the amino acid sequence of the DNA polymerase fragment is shown in SEQ ID NO: 4; or,

[0062] The DNA polymerase fragment is derived from Taq DNA polymerase, and the amino acid sequence of the DNA polymerase fragment is shown in SEQ ID NO: 5.

[0063] The present invention also provides a method for preparing the above-mentioned photo-initiated DNA polymerase, comprising the steps of:

[0064] The light-responsive nucleic acid aptamer was dissolved in the first buffer solution to a final concentration of 15 uM to 25 uM, heated at 92 °C to 95 °C for 4 min to 6 min, and then cooled to 23 °C to 27 °C at a rate of 0.05 °C / s to 2 °C / s to form the first mixture.

[0065] DNA polymerase containing G-quadruplex-binding peptide was dissolved in the second buffer to a final concentration of 7 μM to 15 μM to form the second mixture.

[0066] The first and second mixtures were mixed at a volume ratio of 1:1 and placed at 4°C for 25 to 35 minutes to form a DNA-protein complex.

[0067] An equal volume of glycerol was added to the DNA-protein complex to obtain photo-initiated DNA polymerase.

[0068] In one embodiment, the first buffer comprises 10 mM Tris-HCl (pH 7.4), 75 mM KCl, 0.5 mM EDTA, and 0.2 mg / ml bovine serum albumin; and / or,

[0069] The second buffer consists of 10 mM Tris-HCl (pH 7.4), 75 mM KCl, 0.5 mM EDTA, 0.2 mg / ml bovine serum albumin, 1 mM dithiothreitol, 1 mM benzyl sulfonyl fluoride, and 1% Triton X-100.

[0070] The present invention also provides a method for initiating the activity of the above-mentioned photo-initiated DNA polymerase, comprising the steps of:

[0071] At a preset temperature, ultraviolet light is used to irradiate the photoactivated DNA polymerase for a preset time to activate its activity; wherein,

[0072] The wavelength of the ultraviolet light is greater than or equal to 300nm and less than or equal to 380nm, and the preset time is greater than or equal to 0.5min.

[0073] As one implementation method, a photo-cleavage group can be modified between the G-quadruplex and the hairpin structure, with a preset temperature greater than or equal to 30°C and less than or equal to 65°C; or,

[0074] The G-quadruplex has a first linker ring, a second linker ring, and a third linker ring. The hairpin structure has a fourth linker ring. Photolytically cleavable groups can be modified onto the first linker ring, the second linker ring, the third linker ring, or the fourth linker ring. The preset temperature is greater than or equal to 50°C and less than or equal to 65°C.

[0075] The present invention also provides the application of the above-mentioned photo-initiated DNA polymerase for detecting or synthesizing nucleic acids.

[0076] The light-initiated DNA polymerase provided by this invention is formed by combining a DNA polymerase containing a G-quadruplex-binding peptide with a light-responsive nucleic acid aptamer containing a G-quadruplex. The light-responsive nucleic acid aptamer includes a G-quadruplex nucleotide sequence (as shown in SEQ ID NO: 1), flanking nucleotide sequences, and a photolyzable cleavage group (also represented by PClinker). The G-quadruplex formed by the light-responsive nucleic acid aptamer through the G-quadruplex nucleotide sequence binds to the G-quadruplex-binding peptide of the DNA polymerase containing the G-quadruplex-binding peptide. A hairpin structure (i.e., double-stranded "hairpin" DNA) formed by the flanking nucleotide sequence binds to the active site of the DNA polymerase containing the G-quadruplex-binding peptide, thereby inhibiting the activity of the DNA polymerase containing the G-quadruplex-binding peptide. When a DNA polymerase with a G-quadruplex-binding peptide that binds to a light-responsive nucleic acid aptamer is exposed to ultraviolet light, the photolyzable cleavage group on the light-responsive nucleic acid aptamer breaks, causing the competitive substrate to detach from the active site of the DNA polymerase with the G-quadruplex-binding peptide. In other words, the hairpin structure formed by the flanking nucleotide sequence detaches from the active site of the DNA polymerase with the G-quadruplex-binding peptide, thereby restoring the activity of the DNA polymerase with the G-quadruplex-binding peptide. Figure 1 This diagram illustrates the principle of how a light-responsive nucleic acid aptamer regulates the activity of a G-quadruplex-binding peptide DNA polymerase. The light-initiated DNA polymerase provided by this invention exhibits activity strictly inhibited by the bound light-responsive nucleic acid aptamer, becoming active only upon irradiation with ultraviolet light of a specific wavelength. When applied to nucleic acid amplification technology, it allows the reaction of all samples to start simultaneously without producing non-specific amplification; therefore, it has significant application value in nucleic acid amplification technology. Using the light-initiated Bst DNA polymerase provided by this invention, the problem of non-specific amplification in the detection of HPV DNA using LAMP reactions has been successfully solved. Furthermore, the light-initiated Taq DNA polymerase constructed using this invention, along with light-initiated PCR technology, can improve the success rate of PCR.

[0077] Example 1:

[0078] Construction of Bst DNA polymerase with G-quadruplex-binding peptide and preparation of light-responsive G4P-Bst:

[0079] The pCold-I-G4P-Bst plasmid map can be found in [link to image]. Figure 2 The plasmid pCold-I-G4P-Bst was transformed into Escherichia coli strain BL21, induced, expressed, and purified. Then, G4P-Bst was stored in a buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, and 50% glycerol. Figure 3AThe diagram shows the structures of Bst DNA polymerase, Bst-LF, and G4P-Bst.

[0080] Bst-LF is derived from residues 291-878 of DNA polymerase I of *Bacillus stearothermophilus* (ARA98840.1), specifically the portion of the *Bacillus stearothermophilus* DNA polymerase lacking the 5′-3′ exonuclease domain. The amino acid sequence of Bst-LF is shown in SEQ ID NO: 3. G-quadruplex-binding peptide (G4P) is a small protein composed of 64 amino acids. G4P has a strong G-quadruplex binding affinity, and its amino acid sequence is shown in SEQ ID NO: 2.

[0081] Figure 3B A schematic diagram of the secondary structure of the light-responsive nucleic acid aptamer is shown. The light-responsive nucleic acid aptamer DNA (PC-1) listed in Table 1 was dissolved in buffer to a final concentration of 20 μM, heated at 95 °C for 5 min, and then cooled to 25 °C at a rate of 0.1 °C / s. The buffer contained 10 mM Tris-HCl (pH 7.4), 75 mM KCl, 0.5 mM EDTA, and 0.2 mg / ml bovine serum albumin. G4P-Bst was dissolved in a buffer containing 10 mM Tris-HCl (pH 7.4), 75 mM KCl, 0.5 mM EDTA, 0.2 mg / ml bovine serum albumin, 1 mM dithiothreitol, 1 mM benzyl sulfonyl fluoride, and 1% Triton X-100, to a final concentration of 10 μM. The above light-responsive nucleic acid aptamer and G4P-Bst were mixed at a 1:1 volume ratio and incubated at 4 °C for 30 min to form a DNA-protein binding complex. Then, an equal volume of glycerol was added to obtain G4P-Bst, a pre-bound light-responsive aptamer. This DNA polymerase can be stored at -20 to -30°C.

[0082] Table 1

[0083]

[0084] Example 2:

[0085] Primer extension assays were used to detect the regulatory effect of light-responsive nucleic acid aptamers on the activity of DNA polymerases with G-quadruplex-binding peptides.

[0086] The activity of the light-responsive G4P-Bst was detected by primer extension assay. The reaction system was 25 μL, including 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween-20, 2.5 mM dNTP, 100 nM primer, 100 nM template DNA, and 100 nM of the above DNA polymerase. The sequences of the primers and template DNA are shown in Table 2. Primer extension was performed at 15–65 °C for 5 minutes, and immediately after completion, four volumes of stop buffer (99% formamide, 0.1% SDS, and 20 mM EDTA) were added to terminate the reaction. The sample was denatured at 95 °C for 5 minutes and then electrophoresed on a 1×TBE 12% urea denaturing polyacrylamide gel. Primers and full-length products were photographed using the ChemiDoc MP imaging system (Bio-Rad), and digital quantification was performed using Image Quant 5.2 software.

[0087] Table 2

[0088]

[0089] Figure 3C and Figure 3D The image shows the results of primer extension experiments for G4P-Bst. G4P-Bst exhibits polymerase activity in the 15-65℃ range, but its activity is significantly inhibited upon binding to the photoresponsive nucleic acid aptamer (PC-1), showing only weak activity above 55℃. The inhibition of G4P-Bst activity by the photoresponsive nucleic acid aptamer can be relieved by UV irradiation. The reaction results of samples containing the photoresponsive nucleic acid aptamer and G4P-Bst under 365nm UV irradiation show that the activity of G4P-Bst bound to the nucleic acid aptamer is significantly restored in the 15-65℃ range, approaching the activity of G4P-Bst without the photoresponsive nucleic acid aptamer.

[0090] Under ultraviolet light irradiation, the efficiency of aptamer DNA fragmentation affects the recovery ability of DNA polymerase activity. For example... Figure 3E As shown, the photoresponsive nucleic acid aptamer bound to G4P-Bst was largely cleaved after 0.5 minutes of UV irradiation, and the DNA cleavage efficiency approached 100% after irradiation for more than 1 minute. Furthermore, the DNA cleavage efficiency of the photoresponsive nucleic acid aptamer remained consistent across different temperatures. Accordingly, the inventors tested the activity of G4P-Bst pre-bound with the photoresponsive nucleic acid aptamer under different UV irradiation times. Figure 3FAs shown, G4P-Bst pre-bound with the light-responsive nucleic acid aptamer was added to the sample for primer extension experiment after UV irradiation for a specified time. The results showed that G4P-Bst pre-bound with the light-responsive nucleic acid aptamer began to recover its activity after 0.5 minutes of UV irradiation; its activity was completely restored after irradiation time exceeding 1 minute. This result is consistent with... Figure 3E The DNA fragmentation rate is consistent in light-responsive aptamers.

[0091] The results above demonstrate that the nucleic acid aptamer containing photolytic activity can inhibit the polymerase activity of G4P-Bst within the temperature range of 15-65℃. Its rapid photolytic cleavage allows the DNA polymerase bound to the aptamer to quickly regain its activity under light irradiation. Therefore, G4P-Bst bound to the nucleic acid aptamer exhibits the characteristics of a photoinitiated reaction.

[0092] Example 3: Effects of photolytic cleavage sites and hairpin length of photoresponsive nucleic acid aptamers on the activity of DNA polymerases with G-quadruplex-binding peptides.

[0093] G4P-Bst blocks containing photoresponsive nucleic acid aptamers with different photolytic cleavage sites (PC-linker) were prepared according to the method in Example 2, and their activity was detected by primer extension assay. The sequences of photoresponsive nucleic acid aptamers with different PC-linker sites are shown in Table 3.

[0094] Table 3

[0095]

[0096] Figure 4A and 4B The results showed that light-responsive nucleic acid aptamers modified with PC linkers at different sites could inhibit the DNA polymerase activity of G4P-Bst. After UV irradiation, the activity of G4P-Bst blocked by the light-responsive nucleic acid aptamer (PC-1) with the PC linker located between the G-quadruplex and the hairpin structure was completely restored between 30-65℃. G4P-Bst blocked by light-responsive nucleic acid aptamers with PC linkers at other sites (PC-2, PC-3, PC-4, PC-5) required UV irradiation at temperatures above 50℃ to restore activity. The G-quadruplex has a first, second, and third linking loop, and the hairpin structure has a fourth linking loop. In this embodiment, light-responsive nucleic acid aptamers with PC linkers at other sites refer to those with PC linkers located in the first, second, third, or fourth linking loop.

[0097] The results of this embodiment demonstrate that the PC linker located in the aptamer (PC-1) connecting the G-quadruplex and the hairpin structure allows G4P-Bst activity to be controlled solely by ultraviolet light. In contrast, the PC linker located in the linker loop of the hairpin structure and the linker loop of the G-quadruplex allows G4P-Bst activity to be controlled simultaneously by ultraviolet light and ambient temperature. In other words, the PC linker located in the first, second, third, or fourth linker loop allows G4P-Bst activity to be controlled simultaneously by ultraviolet light and ambient temperature.

[0098] Photoresponsive nucleic acid aptamers with different hairpin structures were prepared into G4P-Bst aptamer-blocked aptamers according to the method in Example 2, and their activity was detected by primer extension assay. The sequences of photoresponsive nucleic acid aptamers with different hairpin lengths are shown in Table 4.

[0099] Table 4

[0100]

[0101]

[0102] Figure 4C and 4D The results showed that when the aptamer hairpin structure length was 7 bp, the aptamer could inhibit the activity of G4P-Bst within the temperature range of 15-40℃. When the aptamer hairpin structure length was 10-16 bp, the aptamer could inhibit the activity of G4P-Bst over a wider temperature range of 15-65℃. After UV irradiation, the G4P-Bst blocked by aptamers of different hairpin lengths recovered its activity within the temperature range of 30-65℃. This result indicates that aptamers with a hairpin length of 10-16 bp allow the activity of G4P-Bst to be controlled by UV light over a wider temperature range.

[0103] Example 4:

[0104] Expanding applications of photo-initiated DNA polymerase.

[0105] DNA polymerases G4P-Taq and G4P-Bsu, containing G-quadruplex-binding peptides, were constructed according to the construction method in Example 1. Specifically, the sequence encoding the large fragment of Bst DNA polymerase in plasmid pCold-I-G4P-Bst was replaced with the gene sequence encoding Taq DNA polymerase or Bsu DNA polymerase to construct plasmids pCold-I-G4P-Taq and pCold-I-G4P-Bsu. The plasmid maps are shown below. Figure 5 and Figure 6 As shown. Among them, Figure 7AThe diagram illustrates G4P-Taq and G4P-Bsu, and the principle of how these enzymes interact with light-responsive nucleic acid aptamers.

[0106] The activity of G4P-Taq was detected according to the method in Example 2, using a sample without light-responsive nucleic acid aptamers as a control. Reactions were performed at different temperatures for 15 minutes. The reaction buffer consisted of 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, and 0.08% Nonidet P40. The primer extension system was 10 μL, comprising 2.5 mM dNTPs, 100 nM primers, 100 nM template DNA, and 100 nM of the aforementioned DNA polymerase.

[0107] Figure 7B The results showed that G4P-Taq exhibited DNA polymerase activity in the range of 25℃ to 65℃. When bound by a light-responsive nucleic acid aptamer, its activity was significantly inhibited by the light-responsive nucleic acid aptamer at temperatures below 55℃ without light exposure. At temperatures above 55℃, its activity was partially restored. When G4P-Taq pre-bound with a light-responsive nucleic acid aptamer was used for primer extension experiments under ultraviolet light, its polymerase activity at 25℃ to 65℃ was almost completely restored, comparable to that of G4P-Taq without a light-responsive nucleic acid aptamer.

[0108] The activity of G4P-Bsu was also detected using the method described in Example 2, with a sample without light-responsive nucleic acid aptamers as a control. Reactions were performed at different temperatures for 15 minutes each. The reaction buffer consisted of 10 mM Tris-HCl (pH 8.8), 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT. The total reaction volume was 10 μL, including 2.5 mM dNTPs, 100 nM primers, 100 nM template DNA, and 100 nM of the aforementioned DNA polymerase G4P-Bsu.

[0109] like Figure 7C As shown, G4P-Bsu exhibits DNA polymerase activity in the range of 20℃ to 50℃. When it is bound by a light-responsive nucleic acid aptamer, its activity is significantly inhibited by the light-responsive nucleic acid aptamer in the absence of light and at a temperature below 40℃. When G4P-Taq pre-bound with the light-responsive nucleic acid aptamer is used for primer extension experiments under ultraviolet light, its polymerase activity at 20℃ to 50℃ is almost completely restored, comparable to that of G4P-Bsu without the light-responsive nucleic acid aptamer.

[0110] Therefore, the binding strategy of G-quadruplex-binding peptides and light-responsive nucleic acid aptamers containing G-quadruplexes is also applicable to Taq DNA polymerase and Bsu DNA polymerase, and they can be modified into light-initiated DNA polymerases.

[0111] Example 5:

[0112] Photoactivated Bst DNA polymerase was used to detect human papillomavirus (HPV) type 45 E6-E7 gene DNA.

[0113] The pH-mediated colorimetric LAMP reaction system includes 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween-20, 1.6 mM dNTPs, 100 nM G4P-Bst with or without photoresponsive aptamer binding, 0.013% (w / v) cresol red, 1.6 μM FIP / BIP primers, 0.4 μM FLP / BLP primers, 0.2 μM F3 / B3 primers, and plasmids containing different copy numbers of human papillomavirus (HPV) 45 E6-E7 gene DNA. Figure 8A As shown, the LAMP reaction was carried out on a metal bath at a temperature of 65°C, with a 360-370nm LED light source irradiating the tube vertically from a height of 0.5cm above the tube cap for 0.5-8 minutes.

[0114] Plasmids containing human papillomavirus (HPV) type 45 E6-E7 gene DNA were added to different samples after being serially diluted 10-fold. Figure 8B and Figure 8C In the middle, from left to right, the copy numbers of E6-E7 gene DNA in the samples are 1,000,000, 100,000, 10,000, 100, 10, 1, 0, respectively. In the absence of light-responsive aptamers, the LAMP reaction caused all samples to change from red to yellow, indicating that significant DNA amplification occurred in the samples regardless of the presence of the HPV45 DNA template. Figure 8B In the Control group, and in the G4P-Bst group of pre-bound photoresponsive nucleic acid aptamers, no color change was observed in any sample without UV irradiation, indicating that no DNA amplification reaction occurred. Figure 8B Group (1); while the G4P-Bst group samples pre-bound with photoresponsive nucleic acid aptamers were irradiated with ultraviolet light for 0.5, 1, 2, 4, and 8 minutes at the initial stage of the reaction (corresponding to respectively) Figure 8B In groups (2-6) of the study, a significant color change was observed in samples containing more than 10 copies of HPV45 DNA, with the most pronounced effect observed after 2-4 minutes of illumination. Figure 8B Group (4-5)

[0115] Furthermore, the amplification products of the Control group, group (1), and group (4) were analyzed by electrophoresis. Figure 8C The results showed that all samples in the Control group exhibited electrophoretic bands. The bands in the template-free samples showed a significant positional difference from those in the high-concentration template samples, suggesting they might be non-specific amplification products. Samples in group (1) showed no DNA bands, indicating that DNA could not be amplified without light. In group (4), samples exposed to light for two minutes showed DNA amplification bands with similar characteristics in samples containing more than 10 copies of HPV45 DNA. Since the activity of the pre-bound light-responsive nucleic acid aptamer G4P-Bst was inhibited before light exposure, it did not extend mismatched primers at low temperatures, resulting in no amplification bands in the template-free samples, which was completely consistent with the results of color-changing LAMP.

[0116] This result demonstrates that photo-initiated LAMP reactions can avoid non-specific amplification products and improve the accuracy of LAMP reactions.

[0117] Example 6:

[0118] Light-started PCR was performed using light-started Taq DNA polymerase.

[0119] Using HeLa cell genomic DNA as a template, PCR reactions were performed targeting eight different sites of the NPM1 gene. The PCR products were detected by agarose gel electrophoresis. The primer sequences for the eight different sites of the NPM1 gene are shown in Table 5.

[0120] Table 5

[0121]

[0122]

[0123] The PCR reaction system consisted of: 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 250 μM dNTP, 4% DMSO, 40 ng HeLa cell genomic DNA, 0.8 μM upstream primer, 0.8 μM downstream primer, and 5 nM G4P-Taq. The reaction program was as follows: incubation at 65°C for 3 minutes, followed by denaturation at 95°C for 5 minutes; then 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and a final extension at 72°C for 5 minutes. UV irradiation was performed at 65°C in the first step.

[0124] Figure 9AThe results showed that using G4P-Taq for PCR, the NPM1 gene produced target bands at 6 different sites; however, when using G4P-Taq with pre-bound light-responsive nucleic acid aptamers and without UV irradiation, no target bands were produced in any of the samples. Figure 9B ); Figure 9C The results showed that, using the pre-bound light-responsive nucleic acid aptamer G4P-Taq and after two minutes of UV irradiation at 65°C at the initial reaction temperature, all eight different sites of the NPM1 gene were detected with the target PCR bands.

[0125] Therefore, it is evident that after G4P-Taq binds to a light-responsive aptamer, the aptamer inhibits G4P-Taq activity, preventing it from performing subsequent PCR reactions. However, upon UV irradiation, the photolyzable cleavage group on the nucleic acid of the light-responsive aptamer breaks, allowing the competitive substrate to detach from the active site of G4P-Taq, thus restoring its polymerase activity. Because the activity of G4P-Taq pre-bound with the light-responsive aptamer is inhibited before light irradiation, it will not extend mismatched primers during the low-temperature stage, thereby avoiding the generation of erroneous amplification products and the ineffective consumption of primers, improving the accuracy and success rate of PCR reactions.

[0126] In summary, the binding strategy of G-quadruplex-binding peptides and light-responsive nucleic acid aptamers containing G-quadruplexes is applicable to a variety of DNA polymerases. DNA polymerases can be modified to be regulated by light-responsive nucleic acid aptamers, i.e., photo-initiated DNA polymerases, giving them the properties of photoinitiation. Furthermore, different photo-initiated DNA amplification technologies can be developed through photo-initiated DNA polymerases.

[0127] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A light-responsive nucleic acid aptamer for regulating DNA polymerases with G-quadruplex-binding peptides, characterized in that, It includes an aptamer nucleotide sequence and a photolyzable cleavage group, wherein the photolyzable cleavage group is modified in the aptamer nucleotide sequence for cleavage after ultraviolet light irradiation to break the aptamer nucleotide sequence into two nucleotide sequences; The aptamer nucleotide sequence includes a G-quadruplex nucleotide sequence capable of forming a G-quadruplex and a flanking nucleotide sequence capable of forming a hairpin structure. The G-quadruplex nucleotide sequence is used to bind to the G-quadruplex binding peptide fused to the DNA polymerase, and the flanking nucleotide sequence is used to bind to the active site of the DNA polymerase. When the photolytic cleavage group breaks the aptamer nucleotide sequence into two nucleotide sequences under ultraviolet light irradiation, the flanking nucleotide sequence detaches from the active site of the DNA polymerase. The G-quadruplex nucleotide sequence is SEQ ID NO: 1; The chemical formula of the photolytically cleavable group is: ； The length of the double-stranded pairing region of the hairpin structure formed by the flanking nucleotide sequence is greater than or equal to 7 bp and less than or equal to 16 bp.

2. The light-responsive nucleic acid aptamer according to claim 1, characterized in that, When the length of the double-stranded pairing region of the hairpin structure formed by the flanking nucleotide sequence is greater than or equal to 7 bp and less than or equal to 8 bp, the light-responsive nucleic acid aptamer can inhibit the activity of the DNA polymerase with the G-quadruplex-binding peptide within a temperature range of greater than or equal to 15°C and less than or equal to 40°C.

3. The light-responsive nucleic acid aptamer according to claim 1, characterized in that, When the length of the double-stranded pairing region of the hairpin structure formed by the flanking nucleotide sequence is greater than or equal to 9 bp and less than or equal to 16 bp, the light-responsive nucleic acid aptamer can inhibit the activity of the DNA polymerase with the G-quadruplex-binding peptide within a temperature range of greater than or equal to 15°C and less than or equal to 65°C.

4. The light-responsive nucleic acid aptamer according to any one of claims 1 to 3, characterized in that, The sequence of the light-responsive nucleic acid aptamer is as follows: 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTT(SEQ ID NO: 6) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 7); Or, 5'-CGCAGACCAGTT(SEQ ID NO: 10) / PC-linker / TTCTGGTCTGCGTTTTTTTGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 11); Or, 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTGGG(SEQ ID NO: 12) / PC-linker / TGGGTGGGTGGGT-3'(SEQ ID NO: 13); Or, 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTGGGTGGG(SEQ ID NO: 14) / PC-linker / TGGGTGGGT-3'; Or, 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTGGGTGGGTGGG(SEQ ID NO: 15) / PC-linker / TGGGT-3'; Or, 5'-AGACCAGTTTTCTGGTCTTTTTTT (SEQ ID NO: 16) / PC-linker / TGGGTGGGTGGGTGGGT-3' (SEQ ID NO: 17); Or, 5'-CGACGCAGACCAGTTTTCTGGTCTGCGTCGTTTTTT(SEQ ID NO: 20) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 21); Or, 5'-CCACGACGCAGACCAGTTTTCTGGTCTGCGTCGTGGTTTTTT(SEQ ID NO: 22) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 23); PC-linker is the photolytically cleavable group.

5. The light-responsive nucleic acid aptamer according to claim 4, characterized in that, When the sequence of the light-responsive nucleic acid aptamer is 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTT(SEQ ID NO: 6) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 7), or 5'-AGACCAGTTTTCTGGTCTTTTTTT(SEQ ID NO: 16) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 17), or 5'-CGACGCAGACCAGTTTTCTGGTCTGCGTCGTTTTTT(SEQ ID NO: 20) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 21), or 5'-CCACGACGCAGACCAGTTTTCTGGTCTGCGTCGTGGTTTTTT(SEQ ID NO: 22) / PC-linker / TGGGTGGGTGGGTGGGT-3' ...PC-linker / TGGGTGGGTGGGTG 23) When the photoresponsive nucleic acid aptamer is irradiated with ultraviolet light, the DNA polymerase with G-quadruplex-binding peptide recovers its activity within a temperature range of 30°C or higher and 65°C or lower.

6. The light-responsive nucleic acid aptamer according to claim 4, characterized in that, When the sequence of the light-responsive nucleic acid aptamer is 5'-CGCAGACCAGTT (SEQ ID NO: 10) / PC-linker / TTCTGGTCTGCGTTTTTTTGGGTGGGTGGGTGGGT-3' (SEQ ID NO: 11), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTGGG (SEQ ID NO: 12) / PC-linker / TGGGTGGGTGGGT-3' (SEQ ID NO: 13), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGG (SEQ ID NO: 14) / PC-linker / TGGGTGGGT-3', or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGG (SEQ ID NO: 14) / PC-linker / TGGGTGGGT-3', or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGGG (SEQ ID NO: 10 ...GGTGGGG (SEQ ID NO: 10) / PC-linker / TGGGTGGGT-3', or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGGG ( When the photoresponsive nucleic acid aptamer is irradiated with ultraviolet light, the DNA polymerase containing the G-quadruplex binding peptide recovers its activity within a temperature range of 50°C or higher and 65°C or lower.

7. A photo-initiated DNA polymerase, characterized in that, Includes DNA polymerases having G-quadruplex-binding peptides and light-responsive nucleic acid aptamers as described in any one of claims 1 to 6; The DNA polymerase having a G-quadruplex-binding peptide comprises a DNA polymerase fragment and the G-quadruplex-binding peptide, wherein the G-quadruplex-binding peptide is modified and linked to the N-terminus of the DNA polymerase fragment. The G-quadruplex nucleotide sequence is used to bind the G-quadruplex binding peptide; the flanking nucleotide sequence is used to bind to the active site of the DNA polymerase fragment to inhibit the activity of the photo-initiated DNA polymerase; the photolyzable cleavage group is used to break the aptamer nucleotide sequence into two nucleotide sequences under ultraviolet light irradiation, so that the flanking nucleotide sequence is released from the active site of the DNA polymerase fragment, thereby activating the activity of the photo-initiated DNA polymerase. The DNA polymerase fragment is derived from Bst DNA polymerase, and the amino acid sequence of the DNA polymerase fragment is shown in SEQ ID NO: 3; or, The DNA polymerase fragment is derived from Bsu DNA polymerase, and the amino acid sequence of the DNA polymerase fragment is shown in SEQ ID NO: 4; or, The DNA polymerase fragment is derived from Taq DNA polymerase, and the amino acid sequence of the DNA polymerase fragment is shown in SEQ ID NO:

5.

8. The photo-initiated DNA polymerase according to claim 7, characterized in that, The amino acid sequence of the G-quadruplex-binding peptide is shown in SEQ ID NO:

2.

9. A method for preparing the photo-initiated DNA polymerase as described in claim 7 or 8, comprising the steps of: The light-responsive nucleic acid aptamer was dissolved in the first buffer to a final concentration of 15 uM to 25 uM, heated at 92°C to 95°C for 4 min to 6 min, and then cooled to 23°C to 27°C at a rate of 0.05°C / s to 2°C / s to form the first mixture. DNA polymerase containing G-quadruplex-binding peptide was dissolved in the second buffer to a final concentration of 7 μM to 15 μM to form the second mixture. The first mixture and the second mixture are mixed at a volume ratio of 1:1 and placed at 4°C for 25 min to 35 min to form a DNA-protein binding complex. An equal volume of glycerol was added to the DNA-protein bound complex to obtain the photo-initiated DNA polymerase.

10. The method according to claim 9, characterized in that, The first buffer comprises 10 mM Tris− HCl pH 7.4, 75 mM KCl, 0.5 mM EDTA, and 0.2 mg / ml bovine serum albumin; and / or, The second buffer solution comprises 10 mM Tris− HCl pH 7.4, 75 mM KCl, 0.5 mM EDTA, 0.2 mg / ml bovine serum albumin, 1 mM dithiothreitol, 1 mM benzyl sulfonyl fluoride, and 1% Triton X-100.

11. A method for initiating the activity of a photo-initiated DNA polymerase as described in claim 7 or 8, characterized in that, Including the following steps: At a preset temperature, the photoactivated DNA polymerase is irradiated with ultraviolet light for a preset time to activate its activity. Wherein, the wavelength of the ultraviolet light is greater than or equal to 300nm and less than or equal to 380nm, the preset time is greater than or equal to 0.5 min, and the preset temperature is greater than or equal to 30°C and less than or equal to 65°C.

12. The method according to claim 11, characterized in that, When the sequence of the light-responsive nucleic acid aptamer is 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTT(SEQ ID NO: 6) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 7), or 5'-AGACCAGTTTTCTGGTCTTTTTTT(SEQ ID NO: 16) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 17), or 5'-CGACGCAGACCAGTTTTCTGGTCTGCGTCGTTTTTT(SEQ ID NO: 20) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 21), or 5'-CCACGACGCAGACCAGTTTTCTGGTCTGCGTCGTGGTTTTTT(SEQ ID NO: 22) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 21), or 5'-CCACGACGCAGACCAGTTTTCTGGTCTGCGTCGTGGTTTTTT(SEQ ID NO: 22) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 22), the sequence of the light-responsive nucleic acid aptamer is 5'-CGCAGACCAGTTTTCTGGTCTGCGTCGTGGTTTTTT(SEQ ID NO: 22) / PC-linker / TGGGTGGGTGGGTGGGT-3'(SEQ ID NO: 22). When NO: 23), the preset temperature is greater than or equal to 30°C and less than or equal to 65°C; or, When the sequence of the light-responsive nucleic acid aptamer is 5'-CGCAGACCAGTT (SEQ ID NO: 10) / PC-linker / TTCTGGTCTGCGTTTTTTTGGGTGGGTGGGTGGGT-3' (SEQ ID NO: 11), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTGGG (SEQ ID NO: 12) / PC-linker / TGGGTGGGTGGGT-3' (SEQ ID NO: 13), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGG (SEQ ID NO: 14) / PC-linker / TGGGTGGGT-3', or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGT ...G (SEQ ID NO: 11), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGG (SEQ ID NO: 12), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGG (SEQ ID NO: 13), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGG (SEQ ID NO: 14), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGG (SEQ ID NO: 15), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGG (SEQ ID NO: 15), or 5'-CGCAGACCAGTTTTCTGGTCTGCGTTTTTTTTTGGGTGGG (SEQ ID NO: 15), or 5'-CGCAGACCAGTTTTCT When NO:15) / PC-linker / TGGGT-3' is present, the preset temperature is greater than or equal to 50°C and less than or equal to 65°C. PC-linker is the photolytically cleavable group.

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