CN116410980A - Cavity hairpin ThT optical nucleic acid switch and application thereof - Google Patents

Cavity hairpin ThT optical nucleic acid switch and application thereof Download PDF

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CN116410980A
CN116410980A CN202310198757.5A CN202310198757A CN116410980A CN 116410980 A CN116410980 A CN 116410980A CN 202310198757 A CN202310198757 A CN 202310198757A CN 116410980 A CN116410980 A CN 116410980A
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许文涛
兰欣悦
朱龙佼
陈可仁
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China Agricultural University
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Abstract

The invention provides a cavity hairpin-based ThT optical nucleic acid switch. Compared with the G-quadruplex and the like, the fluorescent excitation of the ThT is enhanced by 17-20 times compared with single-stranded nucleic acid, the fluorescent excitation of the ThT is enhanced by 2.5-5 times compared with complementary double-stranded nucleic acid, and the fluorescent excitation of the ThT is enhanced by 500 times compared with the autofluorescence of the ThT, meanwhile, based on the high programmability of hairpin complementary sequences, the non-enzyme, label-free fluorescence/proportion type ThT optical nucleic acid switch sensor for tetracycline and berberine is successfully constructed through the integration of an aptamer, the specificity identification of two targets at pM level can be realized within 16min, the fluorescent excitation of the T has good selectivity for target detection in milk and plasma samples, and the developed ThT optical nucleic acid switch has the practicability and general potential in the sensing field.

Description

Cavity hairpin ThT optical nucleic acid switch and application thereof
Technical Field
The invention belongs to the field of molecular biology, and relates to a cavity hairpin THT optical nucleic acid switch and application thereof.
Background
As early as 1973, single stranded DNA sequences rich in purine bases were demonstrated to enhance the fluorescence of thioflavin T (ThT) while pyrimidine bases did not. After the 21 st century, G-quadruplex (G4) was found to be a typical nucleic acid conformation that strongly stimulated ThT fluorescence. In 2013, after co-incubation of ThT with human telomere G4 in the k+ environment, jyotidmayee et al observed that approximately 2100-fold enhancement in ThT fluorescence was achieved. Correspondingly, a G-four plane in the G-four chain body can provide a proper groove for ThT combination, so that random torsion of the benzothiazole ring and the dimethylbenzyl ring under natural conditions is limited, and a mechanism for triggering the start of an optical switch is proposed; thereafter, G4 led ThT to become the dominant fluorescent indicator tool for higher order nucleic acid conformations. However, stable formation of G4 requires additional provision of an ionic environment, and large amounts of G4 in the genome cannot be specifically imaged and distinguished by Th T, which has prompted researchers to begin exploring other specific nucleic acid conformations that can excite ThT fluorescence.
Recently, non-G4 structures such as G-triplexes, i-motifs and double-stranded structures containing sites such as base aberrations, ridges, mismatches and empty sites, etc., have been found to enhance ThT fluorescence in succession, especially parallel double strands containing GA can be accompanied by more than 100-fold enhancement of ThT fluorescence, even better than some G4 sequences. Meanwhile, duplex conformations are easier to form and assemble than higher-order conformations such as G4 to integrate and amplify ThT signals, showing a wider prospect in the fields of sensing and mutation diagnosis. However, the excitation mechanism of how the double-stranded structure excites ThT fluorescence needs to be further clarified in order to provide more detailed guidance and reference on how to avoid large sample sequence screening during accurate mining of potential conformations; furthermore, most duplex structures useful for ThT illumination are unconventional, requiring additional creation of specific nucleic acid sequences or structures, such as empty sites and parallel duplex, etc., to support conformational formation.
Thus, it is necessary to explore the common nucleic acid duplex conformation for ThT photoexcitation enhancement and stable loading with few conformational formation constraints to facilitate its development in multiple research fields.
Disclosure of Invention
The invention provides a base pair mismatch cavity structure with the potential of exciting THT fluorescence, which consists of CGG and AAA mismatch, wherein each base forms an A-C/A-G/A-G mismatch cavity structure. CGG and AAA are contiguous nucleotides.
Wherein, the mismatched cavity structure of A-C/A-G/A-G is realized by interaction of pi-pi bond and Van der Waals force provided by A base in conformation with benzothiazole ring of ThT molecule, and interaction of C and G base provided hydrogen bond and pi-pi bond form with aminomethyl on ThT dimethylamino ring, thereby helping ThT to combine with cavity better, limiting free rotation of two ring planes thereof, and realizing enhanced fluorescence.
The continuous nucleotide composed of CGG and the continuous nucleotide composed of AAA can be on the same nucleotide chain to form a hairpin structure.
In another aspect, the invention provides a cavity hairpin sequence comprising the sequence set forth in seq id no:
A-C/A-G/A-G：5’-TTTTCGGTTTTAAAAAAAAAAAAAAAAA-3’，SEQ ID NO.10；
6H3M1：5’-TTTTTTTTTTTCGGTTTTTTTTTTTAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA-3’，SEQ ID NO.17；
6H3M2：5’-TTTTTTCGGTTTTTTTCGGTTTTTTAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA-3’，SEQ ID NO.18；
6H3M3：5’-CGGTTTTTTTTCGGTTTTTTTTCGGAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA-3’，SEQ ID NO.19。
the invention also provides a cavity hairpin sequence containing a tetracyclines (TET) bivalent aptamer, wherein the sequence hairpin loop is formed by adding 0-10A bases, the sequence secondary structures comprise the CGG-AAA mismatched cavity structures, and the cavity structures are distributed in the middle part or the tail part of the hairpin arm.
A cavity hairpin sequence of a tetracycline bivalent aptamer comprising the sequence shown below:
6H3Mm-TET：5’-CGGTGGTGCGGTGGTGAAAAAACACCAAAACACC ACCG-3’，SEQ ID NO.23；
6H3Mt-TET：5’-CGGTGGTGCGGTGGTGAAAAAACACCACCGCACCA AAA-3’，SEQ ID NO.24；
6H3Md-TET：5’-CGGTGGTGCGGTGGTGAAAAAACACCAAAACACC AAAA-3’，SEQ ID NO.25；
H3Md-TET：5’-CGGTGGTGCGGTGGTGCACCAAAACACCAAAA-3’，SEQ ID NO.32；
1H3Md-TET：5’-CGGTGGTGCGGTGGTGACACCAAAACACCAAAA-3’，SEQ ID NO.33；
2H3Md-TET：5’-CGGTGGTGCGGTGGTGAACACCAAAACACCAAAA-3’，SEQ ID NO.34；
3H3Md-TET：5’-CGGTGGTGCGGTGGTGAAACACCAAAACACCAAA A-3’，SEQ ID NO.35；
4H3Md-TET：5’-CGGTGGTGCGGTGGTGAAAACACCAAAACACCAA AA-3’，SEQ ID NO.36；
8H3Md-TET：5’-CGGTGGTGCGGTGGTGAAAAAAAACACCAAAACA CCAAAA-3’，SEQ ID NO.37；
10H3Md-TET：5’-CGGTGGTGCGGTGGTGAAAAAAAAAACACCAAA ACACCAAAA-3’，SEQ ID NO.38。
the invention also provides a cavity hairpin THT optical nucleic acid switch tetracycline fluorescence sensor, which comprises the cavity hairpin sequence of the tetracycline bivalent aptamer. Preferably, the sequence is as shown in SEQ ID NO. 35.
The invention also provides a cavity hairpin THT optical nucleic acid switch tetracycline fluorescence sensor which has good quantitative detection capability and linear relation for tetracyclines within the concentration range of 1nM-50 mu M, and the minimum detection limit is 0.64nM.
In another aspect, the present invention provides a fluorescent aptamer cavity hairpin sequence comprising Berberine (BB), comprising the sequence shown below:
H3Mm-BB：5’-ACATAATTTAATACGGATGTTAACATAAATATTAAA TTATGT-3’,SEQ ID NO.28；
H3Mt-BB：5’-ACATAACGGAATATTTATGTTAACATAAATATTAAAT TATGT-3’，SEQ ID NO.29；
H3Md-BB：5’-ACATAACGGAATACGGATGTTAACATAAATATTAAA TTATGT-3’，SEQ ID NO.30。
the sequence secondary structures comprise 1-2 mismatched cavity structures, and the cavity structures are distributed in the middle part or the tail part of the hairpin arm.
The invention also provides a cavity hairpin THT optical nucleic acid switch berberine proportional sensor, which comprises the following preparation steps:
(1) Dissolving a cavity hairpin sequence containing berberine fluorescent aptamer to 50-100 mu M by using ultrapure water, carrying out thermal melting at 90-98 ℃ for 3-10 min by using a PCR instrument, and naturally cooling to room temperature for standby;
preferably, dissolving the cavity hairpin sequence containing berberine fluorescent aptamer to 100 mu M with ultrapure water, carrying out thermal melting at 95 ℃ for 5min by a PCR instrument, and naturally cooling to room temperature for standby;
(2) Adding the aptamer into Tris-HCl buffer solution, adding berberine solution to be detected into the system, performing mediation mixing uniformly, incubating for 3-30 min under room temperature condition, adding ThT into the system before detection, performing incubation under room temperature condition, and then reading fluorescence;
preferably, the aptamer is added into Tris-HCl buffer solution, berberine solution to be detected is added into the system, the mixture is uniformly mixed, incubation is carried out for 3min at room temperature, thT is added into the system before detection, the mixture is uniformly mixed at room temperature, and fluorescence is read;
(3) The result judging method comprises the following steps: the ratio (I490/I530) of the fluorescence value of ThT and berberine corresponding to 490nm and 530nm is made under the 445nm excitation wavelength before and after berberine is added.
The berberine fluorescent aptamer cavity hairpin sequence is selected from the above sequences. Preferably, the sequence shown in SEQ ID No.30 is selected.
The cavity hairpin ThT optical nucleic acid switch berberine proportional sensor has good quantitative detection capability and linear relation to berberine within the concentration range of 100 nM-50 mu M, and the minimum detection limit reaches 24.99nM.
The invention also provides a cavity hairpin THT optical nucleic acid switch berberine fluorescence sensor, which comprises the berberine fluorescence aptamer cavity hairpin sequence. Preferably, the sequence shown in SEQ ID No.30 is selected.
The invention provides the application of the cavity hairpin sequence in exciting ThT.
The invention also provides the use of a cavity hairpin sequence comprising a tetracycline bivalent aptamer in tetracycline detection.
The invention also provides application of the cavity hairpin sequence containing the tetracycline bivalent aptamer in preparing a tetracycline detection kit.
The invention also provides application of the cavity hairpin ThT optical nucleic acid switch tetracycline fluorescence sensor in tetracycline detection.
The invention also provides application of the cavity hairpin THT optical nucleic acid switch tetracycline fluorescence sensor in preparation of a tetracycline detection kit.
The invention also provides application of the berberine fluorescent aptamer cavity hairpin sequence in berberine proportional type and fluorescent type detection.
The invention also provides application of the berberine fluorescent aptamer cavity hairpin sequence in preparation of berberine proportional type fluorescent detection kit.
The invention also provides application of the cavity hairpin THT optical nucleic acid switch berberine proportional sensor in berberine detection.
The invention also provides an application of the cavity hairpin THT optical nucleic acid switch berberine proportional sensor in preparation of berberine detection kit.
The invention also provides application of the cavity hairpin THT optical nucleic acid switch berberine fluorescence sensor in berberine detection.
The invention also provides application of the cavity hairpin THT optical nucleic acid switch berberine fluorescence sensor in preparation of berberine detection kit.
Based on the problems of a plurality of restriction conditions, insufficient application universality and the like of nucleic acid conformation formation factors for ThT fluorescence excitation, the invention aims to solve the technical problems that: the novel double-stranded nucleic acid conformation has the advantages of strong fluorescence excitation and loading stability of the ThT, less restriction factors caused by self conformation formation and the like, can be used as a core element, enables the ThT to serve as a fluorescence reporter molecule, and is developed into a universal platform for non-enzyme label-free small molecule sensing.
The invention has the technical effects that:
(1) The invention obtains a CGG-AAA mismatched cavity structure with ThT binding and fluorescence excitation capability.
(2) The invention utilizes the CGG-AAA mismatched cavity structure to construct the hairpin, which can realize the effect of enhancing the fluorescence excitation of the ThT by 17-20 times compared with single-stranded nucleic acid, 2.5-5 times compared with complementary double-stranded nucleic acid and 500 times compared with the autofluorescence enhancement of the ThT; has a more convenient formation pattern compared to the G-quadruplex and other nucleic acid conformations found to excite Th T.
(3) The invention provides a cavity hairpin THT optical nucleic acid switch fluorescence difference/ratio sensing platform based on CGG-AAA mismatch, which has good hairpin target segment sequence programmability and detection universality for different small molecule substances.
(4) The invention does not need to additionally introduce fluorescent color-developing substances such as fluorescent markers or enzymes, avoids complex operation and additional cost, and realizes non-enzyme label-free sensing of tetracycline and berberine.
(5) The invention can realize the tetracycline detection within the concentration range of 1nM-50 mu M, and the detection limit is as low as 640pM; meanwhile, the kit has no obvious nonspecific signal change to several common antibiotics such as kanamycin, ampicillin, doxycycline, terramycin, gentamicin, chloramphenicol, lincomycin and the like, and has sensitive sensing and strong specificity.
(6) The invention can realize berberine detection within the concentration range of 1 nM-10 mu M, and the detection limit is as low as 550pM; meanwhile, the sensor has no obvious nonspecific signal change to several common natural products and bioactive substances such as baicalin, capsaicin, betaine, cannabidiol, glycine, glutamine, tyrosine, ascorbic acid and the like, and has sensitive sensing and strong specificity.
(7) The invention can realize the rapid detection of the tetracycline residue in the milk sample within 16min, has good practical value and is convenient for popularization.
(8) The invention can realize the rapid detection of berberine residues in plasma samples within 10min, has good practical value and is convenient for popularization.
Drawings
FIG. 1 shows the results of molecular docking simulation between ThT and four cavities of single base pair (A), double base pair (B), triple base pair (C), CGG-AAA (D). The darkened box represents the intended docking area, while the darkened area represents the actual docking pocket.
FIG. 2 is a 2D graph of receptor-ligand interactions resulting from molecular docking between a ThT and a cavity hairpin.
Fig. 3 is a diagram showing the evaluation of the ThT effect by the CGG-AAA cavity structure without the actual sequence. The corresponding secondary prediction structure of double chains (A) and hairpins (B) containing different numbers of cavities. The actual fluorescence excitation capacity (C) and electrophoresis result (D) of the structure to ThT/SGI.
FIG. 4 is a graph showing the excitation of the ThT effect and the self-complementarity evaluation by the duplex structure. (A) Predicted secondary structures of B-TET, ds-TE T and 6H-TET. The intensity of ThT fluorescence excited by the sequence (B). SGI fluorescence intensity of the sequence of (C) and electrophoresis results.
FIG. 5 is an actual sequence carrying a functional fragment to evaluate the effect of CGG-AAA cavity structure on ThT. Predicted secondary structures carrying (A) TET and (D) BB aptamer corresponding to the cavity hairpin nucleic acid sequence. ThT excitation ability of the corresponding cavity hairpin nucleic acid sequences carrying (B) TET and (E) BB aptamer. SGI fluorescence intensity and electrophoresis results carrying (C) TET and (F) BB aptamer corresponding to the cavity hairpin nucleic acid sequence.
Fig. 6 is a view of optimizing the number of hair clamping rings of the cavity hair clamp ThT optical switch. (A) A secondary prediction structure containing different base numbers of hairpin loops. The bases added to form a loop are marked with protrusions. (B) ThT excitation capacity of hairpin (dark color) and fluorescence difference before and after response to TET (light color). (C) SGI fluorescence intensity change of the spring and electrophoresis result.
FIG. 7 is a TET-cavity hairpin ThT photoswitch sensor system optimization. (A) optimization of incubation time of ThT with 3H3Md-TE T. (B) optimization of the reaction time of TET binding to 3H3 Md-TET. (C) the reaction ratio of 3H3Md-TET to ThT is optimized. (D) optimization of buffer and ion concentration.
FIG. 8 is an evaluation of TET-cavity hairpin ThT optical switch sensor detection capability. (A) TET-mediated fluorescence spectra of ThT at different concentrations. The solid line represents the ThT fluorescence curve without TET. (B) The fluorescence difference (I0-IF) is linearly related to the logarithm of the TET concentration. (C) evaluation of the response of the sensor to the interfering substance.
Fig. 9 is an emission spectrum of BB and ThT at different excitation wavelengths.
FIG. 10 is a BB-cavity hairpin ThT photoswitch sensing system optimization. (A) optimization of incubation time of ThT with H3 Md-BB. (B) optimization of reaction time of BB binding to H3 Md-BB. And (C) optimizing the reaction ratio of H3Md-BB and ThT. (D) optimization of buffer and ion concentration.
FIG. 11 is an evaluation of detection capability of BB-cavity hairpin THT optical switch ratio sensor. (A) Different concentrations of BB mediated variation spectra of ThT fluorescence and BB fluorescence. The solid line represents the T fluorescence curve without BB. (B) The fluorescence ratio (I490/I530) is linear with the logarithm of BB concentration. (C) evaluation of the response of the sensor to the interfering substance.
FIG. 12 is an evaluation of detection capability of BB-cavity hairpin THT optical switch sensor. (A) different concentrations of BB mediated ThT fluorescence spectra. The solid line represents the ThT fluorescence curve without BB. (B) The fluorescence difference (I0-IF) is linearly related to the logarithm of the TET concentration. (C) evaluation of the response of the sensor to the interfering substance.
Detailed Description
The invention discloses a construction of a cavity hairpin THT optical nucleic acid switch and a development method for a small-molecule non-enzyme label-free sensor and an intracellular imaging platform, and the construction method can be implemented by appropriately improving process parameters by a person skilled in the art by referring to the content of the specification. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Example 1 rational design and molecular docking simulation screening of Cavity Structure with ThT binding and fluorescence excitation Capacity
In view of the optical switching mechanism that can excite ThT conformation, and the mutation and transcriptional bubbling structure that DNA accompanies during transcription processing, this experiment explores in advance whether there is a potentially double-stranded mismatched cavity structure that can achieve ThT molecule insertion and excitation, and structure screening based on computer molecular modeling docking. Firstly, a DNA hairpin formed by complementary pairing of A-T base groups is taken as a skeleton, a single base cavity hairpin formed by A-A, A-G and A-C mismatched base pairs is designed (the sequence is shown in table 1), a hairpin sequence is predicted to be in a 2D structure through an MC fold server (https:// major.ica/MC-Pipeline /), secondary structure information is converted into a tertiary structure through an RNA compound server (https:// rnacompe. Poznan. Plnan /), the obtained tertiary structure U base is replaced with a T base through Discovery studio 2021 software, a DNA nucleic acid acceptor molecule in a dbp.
As a result, see FIG. 1A, it is expected that binding of ThT to the A-A mismatch cavity (boxed region) does not occur, i.e., the mismatch pattern is not the preferred binding site for ThT to the sequence; while even though ThT binds to a-G and a-C mismatched cavities, it does not perfectly match the insertion of ThT, and complementary base pairs to the cavity clinic also interact with ThT (fig. 2A). Indicating that single base mismatched cavities do not completely limit molecular torsion of ThT. Thus, considering the molecular size of ThT and the stability of cavity conformation formation, the present study considered that the cavity size, or consisting of 2-3 mismatched base pairs, could be used for ThT full insertion and efficient fluorescence excitation.
A-C, A-G single base mismatch cavity with better butt joint effect is selected as a basis, construction and molecular simulation butt joint of double base mismatch cavity hairpins are carried out (sequences are shown in table 1, the analysis mode is the same as above), CScore score and interaction of receptor-ligand are analyzed, the A-C/A-G double base mismatch cavity shows higher possibility of combining with ThT than the single base mismatch cavity, and simultaneously, two loop planes of the ThT molecule are better in parallelism with a base plane (figures 1B and 2), so that the cavity pocket is more suitable for ThT insertion.
Continuing to construct a three-base mismatch cavity based on A-C/A-G, and FIG. 1C and FIG. 2 show that the interaction forces of the receptor and the ligand in the three-base mismatch cavity group tend to be similar, on one hand, the consistency of the docking mode suggests that the docking result of the three-base mismatch cavity group is more reliable on the basis that the randomness of the molecular simulation analysis cannot be eliminated; on the other hand, it was suggested that the three base mismatched cavity was sized or optimally seated by ThT, while the a-C/a-G (CGG-AAA) set did show the best docking results (fig. 1D, fig. 2) and was used for subsequent practical sequence validation and analysis. Meanwhile, based on the force analysis results of FIG. 2, it was determined that fluorescence enhancement of ThT benefits from the interaction of pi-pi bonds and Van der Waals forces provided by the A base of the CGG-AAA cavity with the benzothiazole ring of the ThT molecule, and the interaction of the C and G bases with the aminomethyl group on the ThT dimethylamino ring in a form that provides hydrogen bonds and pi-pi bonds; ext> furtherext>,ext> basedext> onext> theext> sizeext> effectext> ofext> theext> Aext> -ext> Cext> (ext> purineext> -ext> pyrimidineext>)ext> baseext> mismatchext>,ext> aext> properext> pocketext> isext> providedext> forext> theext> insertionext> ofext> theext> ThText>,ext> whenext> theext> ThText> interactsext> withext> theext> Aext> -ext> Cext> cavityext>,ext> theext> moleculesext> canext> enhanceext> theext> actingext> forceext> collisionext> betweenext> theext> Gext> baseext> andext> theext> paraext> Aext> baseext> soext> asext> toext> promoteext> theext> insertionext> positioningext> ofext> theext> moleculesext>,ext> andext> theext> ThText> isext> betterext> combinedext> withext> theext> cavityext> byext> relyingext> onext> theext> supportingext> functionext> similarext> toext> aext> baseext> ofext> theext> Gext> -ext> Aext> mismatchext>,ext> soext> thatext> theext> freeext> rotationext> ofext> twoext> ringext> planesext> ofext> theext> ThText> isext> limitedext>,ext> andext> fluorescenceext> enhancementext> isext> realizedext>.ext>
Table 1 design of simulation screening sequences
Figure BDA0004108211980000091
Example 2CGG-AAA mismatched Cavity hairpin actual sequence ThT Capacity validation
First, double-stranded and hairpin sequences containing 0-3 CGG-AAA mismatched cavities based on A-T complementation pairing were synthesized (see Table 2 for sequences), and the nucleic acid secondary structure was mapped using NUPACK server (http:// www.nupack.org/part/new /) (FIGS. 3A, 3B). Dissolving the synthesized sequence to 100 mu M with ultrapure water, carrying out thermal melting at 95 ℃ for 5min by a PCR instrument, and naturally cooling to room temperature for standby. The sequence was added to 10mM Tris-HCI buffer, pH=7.4, to a final concentration of 1. Mu.M, and to the system was added the final concentration of 2. Mu.M to mix well before detection. And immediately placing 100 mu L of the detection system in a fluorescence spectrophotometer, selecting a fluorescence measurement mode, setting the width of an emission and excitation slit to be 10nm, the excitation wavelength of fluorescent molecules to be 445nm, the monitoring range of the fluorescence emission spectrum to be 470-600nm, and selecting the position of 490nm to be the maximum emission light intensity so as to verify the actual excitation effect of the fluorescence detection system on the THT.
The results show that ThT highest fluorescence emission was stimulated by hairpin conformation in the same cavity number group (fig. 3C). To this end, the extent of formation of the double-stranded structure of the sequence was verified by analyzing 2% agarose gel electrophoresis strips and adding SYBR Gre en I (SG I) fluorochromes to a 1. Mu.M final concentration in a 1. Mu.M final concentration buffer (pH=7.4, 10mM Tris-HCI) to a total volume of 100. Mu.L, then placing the strips in a fluorescence spectrophotometer, selecting a fluorescence measurement mode, setting the width of the emission and excitation slit to 10nm, the excitation wavelength of the fluorescent molecule to 497nm, the monitoring range of the fluorescence emission spectrum to 510-620nm, and selecting the maximum intensity of the emitted light at 525 nm.
The results show that in the same cavity number group, the hairpin has stronger SGI fluorescence and clearer and brighter electrophoresis bands (figures 3C and 3D) compared with the double-chain group, namely, the introduction of the hairpin loop or the pairing of two complementary arms with higher probability than the double-chain is facilitated, so that the double-chain and the cavity can be formed better.
Further, when only one CGG-AAA mismatch cavity is introduced in the duplex conformation, the complementarity of the hairpin is not significantly affected, and nearly 2.5-fold ThT fluorescence enhancement is obtained over the full complement hairpin, suggesting a positive effect of CGG-AAA mismatch on ThT fluorescence excitation. As the number of CGG-AAA mismatch units increases, the complementarity of the duplex decreases. In particular, for the double-stranded set, thT fluorescence intensities with 2 or 3 cavity units are nearly as close to single-stranded ployT (CGG) 2/3, suggesting that double-stranded and cavity formation is limited and cannot provide more ThT excitation. However, the 6H3M2 and 6H3M3 groups still showed significantly better fluorescence enhancement than the double and single stranded conformations of the same number of mismatched units and the complementary hairpin, although their complementarity was also not affected much and the corresponding ThT fluorescence excitation was compromised compared to 6H3M1, but still sufficient to demonstrate the value of the existence of CGG-AAA mismatched cavities and hairpin loops.
Therefore, the experimental result shows that the CGG-AAA mismatched cavity hairpin can realize more excellent ThT fluorescence excitation than the full-complementary duplex structure as long as the complementary degree of the duplex structure is ensured, namely the introduced quantity of the cavity units is reasonably controlled.
TABLE 2 mismatch cavity actual verification sequence design
Figure BDA0004108211980000101
Figure BDA0004108211980000111
After non-functional actual sequence verification, 8nt TET single-chain aptamer (comprising CGG cavity unit) cut by Kwon et al is further selected, and the construction of the mismatched cavity hairpin containing CGG-AAA is tried. Particularly, in the construction process, the original 8nt sequence is a G-rich sequence for the fluorescence excitation of the ThT, so that the 8nt short sequence is extended to be a 16nt bivalent aptamer in the construction process, thereby further promoting the formation of G4 and better comparing the fluorescence excitation effects of the G4 and the cavity on the ThT; meanwhile, complementary double-strand hairpins and complementary double-strand hairpins are designed, and the excitation effect of the cavities in the actual functional sequences is better verified by increasing the number of the cavities (the sequences are shown in Table 2) and changing the positions of the cavities.
From the results of FIGS. 4B and 4C, it can be seen that the best ThT fluorescence excitation and duplex formation are both from the hairpin structure (6H-TET), again confirming the conclusion that the hairpin loop can better pair double strands with complementary potential into a duplex structure to enhance ThT fluorescence.
Since the 6H-TET sequence contains two repeated CGG units, the effect of the location and number of CGG-AAA cavities on the thT excitation was first studied (FIG. 5A). As shown in fig. 5B and 5C, hairpins with a single cavity in the middle (6H 3 Mm-TET) or tail (6H 3 Md-TET) have similar ThT fluorescence intensities and hairpin complementarity indicating that the cavity position does not unduly interfere with ThT fluorescence excitation as long as hairpin complementarity does not change significantly. While for a double-cavity hairpin (6H 3 Md-TET), although the degree of complementarity is slightly affected, a THT lighting effect of 17 times more than B-TET, 5 times more than 6H-TET, and 1.7 times more than that of a single-cavity hairpin can still be achieved, and it is proved that the existence of a CGG-AAA mismatched cavity at the hairpin arm can effectively light the THT and even far exceeds a single-stranded sequence with potential for forming G4.
Furthermore, to better verify the versatility of the cavity, experimental analogy 6H3Md-TET, 6H3Mt-TET, 6H3Mm-TET was constructed in a manner designed to include the hairpin sequence of berberine (BB) fluorescent aptamer sequence BBR4S3 (see Table 2 for sequence containing the cavity constituting basic unit AAA) (FIG. 5D). The corresponding fluorescence excitation effect is consistent with the above results (FIGS. 5E, F), and H3Md-BB shows higher efficacy on ThT fluorescence excitation.
Example 3CGG-AAA mismatched luminal hairpin for tetracycline sensor construction and optimization
The results of the analysis based on example 2 show that the introduction of hairpin loops promotes hairpin pairing via the ortho effect. Therefore, hairpin sequences containing different numbers of loop bases including 2CGG-AAA mismatched cavities (see Table 3) were synthesized and validated, and the nucleic acid secondary structure was mapped with a NUPACK server (http:// www.nupack.org/part/new /) (FIG. 6A) in an attempt to search for the optimal complementary hairpin by optimizing the number of bases of the hairpin loop, thereby improving ThT fluorescence excitation in the absence of target, reducing background signals during analysis, and achieving as sensitive detection as possible. In particular, since the verification should be established under the condition that the difficulty of damaging the hairpin by TET is consistent, MCfold is firstly used to predict hairpin secondary structures corresponding to hairpin loops with different base numbers before synthesizing sequences so as to ensure the consistency of complementary hairpin arms and CGG-AAA cavity conformations. Hairpin pairing initiated by hairpin loops consisting of 0-2 bases was found to be quite different from its structure (FIG. 6A), so that no subsequent verification was performed.
TABLE 3 design of TET label-free sensor optical nucleic acid switch sequences
Figure BDA0004108211980000121
Figure BDA0004108211980000131
The results of the study showed that in the absence of TET, the SGI fluorescence decreased significantly as the number of constituent loop bases increased to 6 (fig. 6C), indicating optimal stability of hairpin complementation with 3 or 4 base hairpin loops. Accordingly, 3H3Md-TET showed the optimal ThT enhancement and the most pronounced fluorescence change upon target addition, while 10H3Md-TET showed better ThT enhancement and detection potential than 8H3Md-TET (fig. 6B), it may benefit from promotion of the a group in the hairpin loop, resulting in rebound of ThT fluorescence. Thus, 3H3Md-TET was selected as the cavity hairpin ThT photonucleic acid switch (Cavity hairpin ThT-light nucleic acid switches, CHTLNAS) of the TET label-free sensor, and the reaction conditions of the sensor were further optimized.
(1) Optimal time for ThT insertion into 3H3Md-TET cavity the thermally annealed 3H3Md-TET sequence was added to 10mm tris-HCl buffer at ph=7.4 to a final concentration of 1 μm, thT to a final concentration of 2 μm was added to the system before detection and mixed well. And placing 100 mu L of the detection system into a fluorescence spectrophotometer, selecting a measurement program into a fluorescence dynamics scanning mode, setting the width of an emission and excitation slit to be 10nm, setting the excitation wavelength to be 445nm, and collecting the maximum emission light intensity at 490nm for 15min and collecting the emission light intensity at 0.02s intervals. As shown in FIG. 7A, the fluorescence intensity of ThT was increased continuously after the addition of the system until no significant change was observed after 8min, indicating that ThT could be rapidly bound to the hairpin and kept the system fluorescence stable.
(2) The tetracycline incubation time is optimized, the 3H3Md-TET sequence after thermal annealing treatment is added into 10mM Tris-HCl buffer solution with pH=7.4, the final concentration is 1 mu M, the ThT with the final concentration of 2 mu M is added into the system, after uniform mixing for 10min, the TET solution with the final concentration of 1 mu M is added into the system, 100 mu L of the detection system is immediately placed into a fluorescence spectrophotometer, the measurement program is selected as a fluorescence dynamics scanning mode, the width of an emission and excitation slit is set to be 10nm, the excitation wavelength is 445nm, the maximum emission light intensity is collected at 490nm, the collection time is 15min, and the collection interval is 0.02s. As a result, as shown in FIG. 7B, fluorescence decreased rapidly after TET was added to the reaction system, and two consecutive small peaks were observed during the first 30 seconds (FIG. 7B inset), and fluctuation of fluorescence suggested that TET destroyed the competition between hairpin activity and ThT binding cavity. Whereas the curve remains unchanged after 8min of fluctuation, 8min was chosen as the optimal reaction time between TET and sequence.
(3) 3H3Md-TET sequence and concentration ratio of ThT reaction System optimization the thermally annealed 3H3Md-TET sequence was added to 10mM Tris-HCl buffer, pH=7.4, to a final concentration of 1. Mu.M, and ThT, 0.1. Mu.M, 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M was added to the system before detection, followed by mixing. Immediately placing 100 mu L of detection system into a fluorescence spectrophotometer, selecting a fluorescence measurement mode, setting the width of an emission and excitation slit to be 10nm, setting the excitation wavelength of fluorescent molecules to be 445nm, and collecting the maximum emitted light intensity at 490 nm. The results show, as shown in fig. 7C, that as ThT concentration increases from 0.1 μm to 2 μm, the fluorescence intensity increases continuously, while further increases in ThT concentration, the fluorescence value tends to decrease with increasing ThT concentration, indicating that ThT molecules tend to form dimers or polymers at high concentrations, while being limited by molecular structure and size, do not match well with the cavity in the hairpin, and fluoresce. The fluorescence response of ThT to the sequence was suggested to be saturated, so 1:2 was chosen as the optimal ratio for the subsequent experiments.
(4) Reaction system and ion concentration optimization the 3H3Md-TET sequence aptamer sequence after thermal annealing treatment is added into ultrapure water, 5, 10, 20, 50, 100mM Tris-HCl, tris-KCl, tris-NaCl and Trisl-MgCl2 buffer solution with pH=7.4 respectively, the final concentration is 1 mu M, and ThT with the final concentration of 2 mu M is added into the detection forward system and is uniformly mixed. Immediately placing 100 mu L of detection system into a fluorescence spectrophotometer, selecting a fluorescence measurement mode, setting the width of an emission and excitation slit to be 10nm, setting the excitation wavelength of fluorescent molecules to be 445nm, and collecting the maximum emitted light intensity at 490 nm. As shown in FIG. 7D, the fluorescence measurement results showed the largest change in fluorescence intensity of ThT under the condition of 10mM Tris-HCl buffer, suggesting that the condition is the optimal buffer environment for the sensor to realize conformational free conversion.
As shown in FIG. 8A, the detection performance of 3H3Md-TET was explored under the optimal experimental conditions. In the absence of TET, the reaction system showed the strongest ThT fluorescence. After addition of TET, the fluorescence intensity decreased with increasing TET concentration and showed a good linear relationship between the logarithm of the concentration and the ThT fluorescence difference (I0-IF) before and after TET addition (y= 65.26516log x-54.5016, r2= 0.99526) in the range of 1nM-50 μm (fig. 8B), and LOD of the sensor was calculated to be 0.64nM according to formula 3 sigma/k.
1.25. Mu.M TET and 12.5. Mu.M antibiotics each, which may remain in milk, were selected, namely kanamycin, ampicillin, doxycycline, terramycin, gentamicin, chloramphenicol, lincomycin, to investigate the interference resistance of the TET assay. As shown in fig. 8C, the common antibiotic residues did not significantly interfere with the detection, indicating good sensor selectivity.
2mL of the pure milk preparation was diluted to 5mL with ultrapure water, then 2mL of 10% trichloroacetic acid and 2mL of chloroform were added, and mixed in a vortexing state for 1min to precipitate proteins and dissolve fats and other organic substances in the sample matrix. To remove proteins, fats and other organics, the mixture was then sonicated at 20 ℃ for 10min and centrifuged at 12,000rpm for 10min to separate the sediment. Next, the supernatant was transferred to another centrifuge tube, centrifuged at 12,000rpm for 10min, and the sediment was removed again to obtain the final supernatant, which was diluted 5-fold, and TET was dissolved to prepare a stock solution for labeling. Adding the standard adding stock solutions with different concentrations into 10mM Tris-HCl buffer solution with final concentration of 1 mu M3H 3Md-TET and pH=7.4, carrying out vortex mixing, incubating for 8min at room temperature, adding ThT with final concentration of 2 mu M into the system before detection, mixing for 8min, placing the detection sample with total volume of 100 mu L into a fluorescence spectrophotometer, selecting a fluorescence measurement mode, selecting fluorescence values corresponding to the ThT in milk samples with different TET concentrations at 490nm, carrying out parallel repetition for 3 times, analyzing the brought standard curve, calculating the recovery concentration, and obtaining the relation between the recovery concentration and the actual standard adding concentration. Taking the difference value between the obtained fluorescence intensity value and the THT fluorescence intensity under the condition of no target, taking the difference value into a standard curve, and calculating to obtain the concentration of the residual tetracycline in the milk sample. The relationship between the recovered concentration and the actual labeled concentration is obtained as shown in Table 4. It can be seen that the recovery rate of the method is between 98.6% and 105.8%, namely the repeatability and the accuracy of the sensor are good. It is shown that the sensor has the capability of detecting tetracycline in an actual milk sample.
Table 4 milk sample addition recovery
Figure BDA0004108211980000151
Example 4CGG-AAA mismatched luminal hairpin for berberine sensor set-up
Since the number of bridging bases constituting hairpin loops influences the degree of complementarity of hairpin arms and thus the sensitivity of the sensor, and at the same time, in example 3, the obtained optimal hairpin loop base number is verified to be 3, while in the CHTLNAS design for berberine, it was found that the secondary structure of H3Md-BB had no normal complementarity of 2 base pairs at the hairpin loop before any bridging base was added additionally, therefore, taking into account the complementary stability of hairpin and the flexibility of mediating conformational changes after BB addition, the conformation of H3Md-BB was studied to have maximally approached the optimal conformational conditions of the sensor without further introduction of bridging bases, so as not to influence the sensitivity of fluorescence reaction.
Since H3Md-BB contains a BB fluorescent aptamer sequence, and ThT and BB have the potential to simultaneously obtain excellent fluorescence emission at the same excitation wavelength, research is devoted to constructing BB-ratio fluorescent sensors. As shown in fig. 9, BB and ThT show good fluorescence emission at the optimum excitation wavelength (365 nm/445 nm), respectively, whereas when the optimum excitation wavelength is exchanged, both fluorescence emissions are very weak. Therefore, neglecting the fluorescence effect of BB fluorescence on ThT at a maximum excitation wavelength of 445nM, the reaction condition optimization of the sensor was performed as in the 3H3Md-TET optimized format: FIG. 10 shows that in 10mM Tris-HCl (pH=7.4), the concentration ratio of H3Md-BB to ThT is 1:2, incubation times of ThT and BB are 3min and 6min, respectively, and the sensor shows the best detection performance.
Further, on the basis of ensuring the consistency of the reaction conditions, the fluorescence effect of ThT and BB at the respective optimal excitation wavelengths is comprehensively considered, the ThT concentration is reduced to 100nM, a clear expected bimodal fluorescence change curve is obtained at 445nM excitation wavelength (fig. 11A), it can be seen that as the BB concentration increases, the fluorescence emission peak of ThT at 490nM decreases, the fluorescence emission peak of BB at 530nM increases, the value of I490/I530 gradually decreases, a significant linear relationship exists between the corresponding ratio and the logarithm of BB concentration between 100nM and 50 μm, the linear regression equation is y= -0.44197logx+2.28556, r2= 0.99265 (fig. 11B), LOD is 24.99nM, and the feasibility of constructing a label-free ratio fluorescence sensor based on H3Md-BB is shown.
However, since the maximum emission peaks of ThT and BB are very close to [ (EM (max) tht=490 nm, EM (max) bb=530 nm) ], and the emission peak of BB appears to be significantly blue shifted with decreasing concentration, indicating interference between the two fluorescent signals. Meanwhile, in order to ensure that the ratio sensor is bimodal and simultaneously appears, the ThT doping concentration which has to be reduced also weakens the response sensitivity of the sensor to a certain extent, and the detection range can be limited. Therefore, considering sensitivity of fluorescence change and practicality of detection, thT was selected as a single signal output of the sensor, and as shown in fig. 12A, the higher the BB concentration, the weaker the fluorescence emission peak of ThT at 490 nm. When BB concentration reached 500nM, the fluorescence curve flattened, suggesting that the system detection capacity reached saturation. The fluorescence intensity at 490nM was plotted against BB concentration (FIG. 12B), and it was found that in the range of 1nM to 10. Mu.M, there was a linear relationship between the fluorescence difference (I0-IF) and the BB concentration logarithm (Y=30.556logX+1.467, R2= 0.99756), and LOD was 0.55nM.
The anti-interference performance of BB detection was studied by selecting 1.25. Mu.M BB and 12.5. Mu.M each of various plant extracts including baicalein, capsaicin, betaine, cannabidiol and various common natural biochemical molecules including glycine, threonine, tyrosine and ascorbic acid. As shown in FIG. 12C, 12.5. Mu.M of natural products and bioactive substances induced fluorescence response was weak compared to 1.25. Mu.M BB, showing good sensor selectivity.
3mL of chloroform was added to 500. Mu.L of BB-labeled human plasma, mixed for 2min under vortex, and centrifuged at 4,000rpm for 10min. The organic phase was then transferred to a centrifuge tube and evaporated to dryness at 40 ℃ under a gentle stream of nitrogen. The residue was redissolved in a mobile phase consisting of acetonitrile and water (1:2, v/v), vortexed for 30s, sonicated for 10min, and centrifuged at 4000rpm for 5min. The supernatant was used as the final sample solution. Adding the sample into 10mM Tris-HCl buffer solution with final concentration of 1 mu M H3Md-TET and pH=7.4, uniformly stirring, incubating for 6min at room temperature, adding ThT with final concentration of 2 mu M into the system before detection, uniformly mixing for 3min, placing the detection sample with total volume of 100 mu L into a fluorescence spectrophotometer, selecting a fluorescence measurement mode, selecting fluorescence values corresponding to the ThT in plasma samples with different BB concentrations at 490nm, and after 3 times of parallel repetition, analyzing the carried standard curve to calculate the recovery concentration, thereby obtaining the relation between the recovery concentration and the actual standard concentration. Taking the difference value between the obtained fluorescence intensity value and the THT fluorescence intensity under the condition of no target, taking the difference value into a standard curve, and calculating to obtain the concentration of residual berberine in the plasma sample. The relationship between the recovered concentration and the actual labeled concentration is obtained as shown in Table 5. The relation between the actual addition concentration and the recovery concentration is determined to be between 104.7 and 106.4 percent, and the reliability and the practicability of the method are proved.
TABLE 5 recovery of plasma samples by labelling
Figure BDA0004108211980000171
Figure BDA0004108211980000181
Reagents and apparatus for use in the methods of the present invention are commercially available. The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A base pair mismatched cavity structure that excites ThT fluorescence potential, wherein the base pair mismatched cavity structure consists of CGG and AAA mismatches, wherein each base forms a-C/a-G mismatched cavity structure.
2. The base pair mismatched cavity structure according to claim 1, wherein the mismatched cavity structure of a-C/a-G is achieved by interaction of pi-pi bonds and van der waals forces provided by the a base in the conformation with the benzothiazole ring of ThT molecule, and interaction of the C and G bases with the aminomethyl groups on the ThT dimethylamino ring in the form of hydrogen bonds and pi-pi bonds, thereby helping ThT to better bind to the cavity and limiting free rotation of its two ring planes.
3. The base pair mismatched cavity structure according to claim 1 or 2, wherein the continuous nucleotides of CGG and AAA are on the same nucleotide chain to form a hairpin structure.
4. A cavity hairpin sequence, characterized in that the sequence is as follows:
A-C/A-G/A-G：5’-TTTTCGGTTTTAAAAAAAAAAAAAAAAA-3’，SEQ ID NO.10；
6H3M1：5’-TTTTTTTTTTTCGGTTTTTTTTTTTAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA-3’，SEQ ID NO.17；
6H3M2：5’-TTTTTTCGGTTTTTTTCGGTTTTTTAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA-3’，SEQ ID NO.18；
6H3M3：5’-CGGTTTTTTTTCGGTTTTTTTTCGGAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA-3’，SEQ ID NO.19。
5. a cavity hairpin sequence comprising a tetracycline bivalent aptamer, wherein the sequence hairpin loop is formed by adding 0-10 a bases, the sequence secondary structures comprise 1-2 CGG-AAA mismatched cavity structures according to claim 1, and the cavity structures are distributed in the middle or tail of the hairpin arm.
6. A cavity hairpin sequence of claim 5, wherein the sequence is as follows:
6H3Mm-TET：5’-CGGTGGTGCGGTGGTGAAAAAACACCAAAACACC ACCG-3’，SEQ ID NO.23；
6H3Mt-TET：5’-CGGTGGTGCGGTGGTGAAAAAACACCACCGCACCA AAA-3’，SEQ ID NO.24；
6H3Md-TET：5’-CGGTGGTGCGGTGGTGAAAAAACACCAAAACACC AAAA-3’，SEQ ID NO.25；
H3Md-TET：5’-CGGTGGTGCGGTGGTGCACCAAAACACCAAAA-3’，S EQ ID NO.32；
1H3Md-TET：5’-CGGTGGTGCGGTGGTGACACCAAAACACCAAAA-3’，SEQ ID NO.33；
2H3Md-TET：5’-CGGTGGTGCGGTGGTGAACACCAAAACACCAAAA-3’，SEQ ID NO.34；
3H3Md-TET：5’-CGGTGGTGCGGTGGTGAAACACCAAAACACCAAA A-3’，SEQ ID NO.35；
4H3Md-TET：5’-CGGTGGTGCGGTGGTGAAAACACCAAAACACCAA AA-3’，SEQ ID NO.36；
8H3Md-TET：5’-CGGTGGTGCGGTGGTGAAAAAAAACACCAAAACA CCAAAA-3’，SEQ ID NO.37；
10H3Md-TET：5’-CGGTGGTGCGGTGGTGAAAAAAAAAACACCAAA ACACCAAAA-3’，SEQ ID NO.38。
7. a cavity hairpin sequence comprising berberine fluorescent aptamer, wherein the sequence secondary structures comprise 1-2 CGG-AAA mismatched cavity structures according to claim 1, and the cavity structures are distributed in the middle or tail of the hairpin arm.
8. A cavity hairpin sequence of claim 7, wherein the sequence is as follows:
H3Mm-BB：5’-ACATAATTTAATACGGATGTTAACATAAATATTAAA TTATGT-3’,SEQ ID NO.28；
H3Mt-BB：5’-ACATAACGGAATATTTATGTTAACATAAATATTAAAT TATGT-3’，SEQ ID NO.29；
H3Md-BB：5’-ACATAACGGAATACGGATGTTAACATAAATATTAAA TTATGT-3’，SEQ ID NO.30。
9. use of a base pair mismatched cavity structure according to any one of claims 1 to 3, a cavity hairpin sequence according to claim 4 and/or a cavity hairpin sequence according to any one of claims 5 to 8 for exciting ThT.
10. Use of a cavity hairpin sequence according to claim 5 or 6 in tetracycline detection.
CN202310198757.5A 2023-03-03 2023-03-03 Cavity hairpin ThT optical nucleic acid switch and application thereof Pending CN116410980A (en)

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