阅读说明：本技术 一种核酸接枝半导体聚合物、核酸探针及其制备方法和应用 (Nucleic acid grafted semiconductor polymer, nucleic acid probe, and preparation method and application thereof ) 是由 田雷蕾 肖凡 于 2020-12-11 设计创作，主要内容包括：本发明提供一种核酸接枝半导体聚合物、核酸探针及其制备方法和应用,所述核酸接枝半导体聚合物包括如式I所示结构的重复单元,具有两亲性,能够在水溶液中自组装,形成表面密集排布有单链DNA的核酸组装体,该核酸组装体能够作为一个高效的能量传递平台用于核酸的成像和检测中。包含上述核酸组装体的核酸探针中,染料修饰DNA单链与核酸组装体表面的DNA进行杂交,发生高效的从半导体聚合物到染料的能量传递,形成荧光能量共振转移。所述核酸探针用于靶标核酸的检测时,由于核酸分子的杂交替换,荧光能量共振转移的荧光比率发生变化,通过荧光比率和靶标核酸浓度的关系,实现了靶标核酸的高灵敏度和高特异性的定量检测。(The invention provides a nucleic acid grafted semiconductor polymer, a nucleic acid probe, a preparation method and application thereof, wherein the nucleic acid grafted semiconductor polymer comprises a repeating unit with a structure shown in a formula I, has amphipathy, can be self-assembled in an aqueous solution to form a nucleic acid assembly with densely distributed single-stranded DNA on the surface, and can be used as an efficient energy transfer platform for imaging and detecting nucleic acid. In the nucleic acid probe comprising the nucleic acid assembly, the dye-modified DNA single strand hybridizes with DNA on the surface of the nucleic acid assembly, and energy transfer from the semiconductor polymer to the dye occurs efficiently, thereby forming fluorescence energy resonance transfer. When the nucleic acid probe is used for detecting target nucleic acid, the fluorescence ratio of fluorescence energy resonance transfer is changed due to hybridization replacement of nucleic acid molecules, and the quantitative detection of the target nucleic acid with high sensitivity and high specificity is realized through the relation between the fluorescence ratio and the concentration of the target nucleic acid.)

1. A nucleic acid grafted semiconducting polymer comprising a repeat unit having a structure according to formula I:

wherein R is¹、R²Each independently selected from C2 to C10 straight or branched chain alkylene;

L¹、L²each independently selected from a single bond, CH ═ CH, or C ≡ C;

ar is selected from substituted or unsubstituted C6-C20 arylene, substituted or unsubstituted C2-C20 heteroarylene; the substituted substituent in Ar is selected from C1-C10 straight chain or branched chain alkyl, C1-C10 straight chain or branched chain alkoxy;

s1 is single-stranded DNA;

n is 0 or 1.

2. The nucleic acid-grafted semiconducting polymer according to claim 1, wherein R is¹、R²Each independently selected from C4 to C8 linear or branched alkylene, preferably C4 to C8 linear alkylene;

preferably, said R is¹、R²Are the same group;

preferably, said L¹、L²Each independently selected from a single bond or CH ═ CH;

preferably, said L¹、L²Are the same group;

preferably, Ar is selected from any one of the following groups:

wherein the dotted line represents the attachment site of the group;

R_A1、R_A2、R_A3、R_A4each independently selected from hydrogen, C1-C10 straight chain or branched chain alkyl, C1-C10 straight chain or branched chain alkoxy.

3. The nucleic acid grafted semiconducting polymer according to claim 1 or 2, wherein said nucleic acid grafted semiconducting polymer comprises any one or a combination of at least two of the following recurring units a 1-a 6:

preferably, the number of the basic groups of the single-stranded DNA is 5-15;

preferably, the nucleic acid sequence of S1 is: 5'-TAGCTTATCAGACTG-3', 5'-TCCCTGAGACCCTAA-3', or 5'-TGAGGTAGTAGGTTG-3'.

4. A method for preparing the nucleic acid-grafted semiconducting polymer according to any of claims 1 to 3, comprising the steps of:

(1) reacting a semiconductor polymer containing a repeating unit B, a cuprous catalyst and pore glass beads loaded with alkynyl modified nucleic acid in a solvent to obtain a product, wherein the product is loaded on the pore glass beads; the reaction formula is as follows:

wherein R is¹、R²、L¹、L²Ar, S1, n each independently have the same limitations as in formula I;

(2) and (2) separating the product obtained in the step (1) from the porous glass beads under the action of alkali liquor to obtain the nucleic acid grafted semiconductor polymer.

5. The method according to claim 4, wherein the molar ratio of the repeating unit B to the alkynyl-modified nucleic acid in the step (1) is (1-10): 1;

preferably, the cuprous catalyst of step (1) comprises CuI and/or CuBr;

preferably, the molar ratio of the alkynyl modified nucleic acid to the cuprous catalyst in the step (1) is 1 (0.1-1);

preferably, the raw materials for the reaction of step (1) further comprise a stabilizer;

preferably, the molar ratio of the stabilizing agent to the alkynyl modified nucleic acid is (0.1-1): 1;

preferably, the catalyst is CuBr and the stabilizer is tris ((1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl) amine;

preferably, the solvent of step (1) comprises a mixture of dichloromethane, dimethylformamide and dimethylsulfoxide;

preferably, the dosage of the solvent is 0.5-2 mL based on 1 mu mol of the alkynyl modified nucleic acid;

preferably, the reaction of step (1) is carried out under stirring conditions;

preferably, the temperature of the reaction in the step (1) is 30-50 ℃;

preferably, the reaction time in the step (1) is 5-24 h;

preferably, the reaction in step (1) further comprises a washing step after the reaction is completed;

preferably, the washing reagent comprises any one of dichloromethane, dimethylformamide or dimethyl sulfoxide or a combination of at least two of the same;

preferably, the alkali liquor in step (2) comprises sodium hydroxide solution, potassium hydroxide solution or ammonia water;

preferably, the temperature of the action in the step (2) is 50-60 ℃;

preferably, the action time of the step (2) is 5-24 h;

preferably, the separation of the step (2) further comprises the steps of purification and drying;

preferably, the purification is performed by a centrifugal filter.

6. The method according to claim 4 or 5, comprising the following steps:

(1) stirring and reacting a semiconductor polymer containing a repeating unit B, a cuprous catalyst, a stabilizer and pore glass beads loaded with alkynyl modified nucleic acid in a solvent at 30-50 ℃ for 5-24 h to obtain a product, wherein the product is loaded on the pore glass beads; the molar ratio of the repeating unit B to the alkynyl modified nucleic acid is (1-10): 1; the dosage of the alkynyl modified nucleic acid is 1 mu mol, the dosage of the cuprous catalyst is 0.1-1 mu mol, and the dosage of the stabilizer is 0.1-1 mu mol;

(2) and (2) mixing the product obtained in the step (1) with alkali liquor, treating for 5-24 hours at 50-60 ℃, separating the product from the porous glass beads, and purifying and drying to obtain the nucleic acid grafted semiconductor polymer.

7. A nucleic acid probe, characterized in that the nucleic acid probe comprises a nucleic acid assembly body and a dye modified DNA single strand S2 hybridized on the nucleic acid assembly body; the nucleic acid assembly is self-assembled by the nucleic acid-grafted semiconductor polymer according to any one of claims 1 to 3.

8. The nucleic acid probe of claim 7, wherein the nucleic acid assembly is a spherical nanoparticle;

preferably, the hydration radius of the nucleic acid assembly is 20-100 nm;

preferably, the molar ratio of the nucleic acid on the nucleic acid assembly to the dye modified DNA single strand S2 is 1 (0.1-0.2);

preferably, the number of the bases of the DNA single strand S2 is 20-25;

preferably, the nucleic acid sequence of the DNA single strand S2 is: 5'-TCAACATCAGTCTGATAAGCTA-3', respectively;

preferably, the dye comprises cyanine dye, further preferably Cy3, Cy5 or Cy 7;

preferably, the absorption spectrum of the dye overlaps with the emission spectrum of the nucleic acid-grafted semiconducting polymer.

9. A method for preparing the nucleic acid probe according to claim 7 or 8, comprising: the nucleic acid grafted semiconducting polymer of any of claims 1 to 3 self-assembles in an aqueous solution to obtain a nucleic acid assembly; hybridizing the nucleic acid assembly with a dye modified DNA single strand S2 to obtain the nucleic acid probe;

preferably, the concentration of the nucleic acid grafted semiconductor polymer in the aqueous solution is 0.1-10 mu g/mL.

10. Use of a nucleic acid probe according to claim 7 or 8 in biosensing, bioimaging or nucleic acid detection;

preferably, the method for detecting nucleic acid comprises the steps of:

(A) adding target nucleic acid into the solution of the nucleic acid probe for hybridization replacement to obtain a solution to be detected;

(B) performing fluorescence test on the solution to be tested obtained in the step (A), and realizing quantitative detection of the target nucleic acid according to the fluorescence ratio of fluorescence energy resonance transfer;

preferably, the target nucleic acid comprises DNA, messenger RNA or MicroRNA.

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a nucleic acid grafted semiconductor polymer, a nucleic acid probe, and preparation methods and applications thereof.

Background

Nucleic acid is a marker biomolecule. By detecting nucleic acids, specific diseases can be diagnosed with high precision. Therefore, a method for detecting nucleic acid with high sensitivity and high specificity is required. The nucleic acid detection method commonly used at present relies on biological amplification, such as Polymerase Chain Reaction (PCR), to amplify the amount of target nucleic acid, and requires trained personnel and specialized laboratories, and is time-consuming and costly. In response to this problem, fluorescence-based biosensing exhibits unique advantages, including high sensitivity, high temporal resolution. The amplification of the fluorescence signal is an important strategy for improving the sensitivity of the detection system, and the Semiconductor Polymer (SP) is a functional material meeting the above requirements.

SP is a macromolecule containing a backbone of pi-conjugated building blocks. SP has at least two advantages due to electron delocalization: (1) compared with small molecule analogues, SP has strong light collection capacity; (2) the exciton energy efficiently migrates along the polymer backbone, facilitating efficient energy transfer to low energy acceptors or fluorescence quenchers, a process also referred to as the "molecular wire" effect. Matching with suitable energy receptors, efficient energy transfer can be achieved, which can improve the sensitivity of detection. The application of SP to a biosensing system requires solving the problem of water solubility, and methods for solving the problem are divided into two categories: one is to introduce charged side chains to the SP, such as cationic quaternary ammonium salts or anionic sulfonates; and secondly, preparing the SP into polymer dots by a nano precipitation method.

Many researchers have been working on fluorescence-sensing nucleic acid detection, and one of the ideas is to use SP with cationic side chains for nucleic acid detection, which is based on the principle that SP with cationic side chains can form a complex with negatively charged nucleic acid through electrostatic interaction, and fluorescein-labeled neutral peptide nucleic acid (PNA, a synthetic DNA mimic with a neutral backbone) as a probe, which can form a complex duplex with complementary target single-stranded DNA (ssdna). Initially, there was no electrostatic interaction between the probe and the cation SP in solution, and therefore Fluorescence Resonance Energy Transfer (FRET) could not occur; upon addition of complementary ssDNA, a negatively charged ssDNA/probe complex duplex is created as a result of molecular hybridization, which further interacts electrostatically with cationic SP to form a SP/ssDNA/probe ternary complex. The recombination draws the distance between the SP and the probe, and in addition, the spectra of the fluorescein on the SP and the probe can also be effectively overlapped, so that efficient FRET occurs between the SP and the probe, and the emission of the fluorescein is enhanced. When non-complementary ssDNA is added, electrostatic interactions only occur between the SP and ssDNA, but the distance between the SP and the probe is still too large for FRET to occur, so only weak fluorescein signals can be detected ("DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes", Brent S. Gaylord et al, Proceedings of the National Academy of Sciences of the United States of America, 2002, 99, 17, 10954). The method can detect the target ssDNA, but the cation SP in the ssDNA is combined with the negatively charged DNA through nonspecific electrostatic interaction, and if other charged macromolecules exist in the detection system to participate in the electrostatic interaction, the specificity and the sensitivity of the system can be influenced.

Another nucleic acid detection method based on fluorescence sensing is performed by semiconductor polymerization sites (SP dots), and a representative method is as follows: modifying DNA on the surface of SP dots, taking PicoGreen (PG) dye as a receptor, wherein the emission spectrum of SP and the absorption spectrum of PG are well overlapped, and PG can be embedded in a DNA double strand but not in a single strand. Therefore, in the initial stage, because complementary double-stranded DNA is not formed, the distance between the PG dye and the SP dots in the solution is far, and FRET cannot occur; when the target DNA is present in the solution and becomes double-stranded with the SP dots surface-modified DNA, the PG dye in the solution intercalates into the DNA double strand, and at this time, the distance between PG and SP dots is shortened to cause FRET, so that the PG signal is amplified (Conjugated Polymer Nanoparticles for Label-Free and bioconjugated-Recojected DNA Sensing in Serum ", Bao Biqing et al, Advanced Science 2015, 2,3, 1400009). Although the method can realize the detection of the target ssDNA, the efficiency of grafting DNA on the SP dots surface is not high, the quantity of the surface DNA is not enough, and the hybridization efficiency of the target DNA and the SP dots surface DNA is low; furthermore, insufficient density of DNA on the surface of the semiconducting polymer dots not only reduces the stability of SP dots, but also results in a small amount of PG dye that can be captured by the surface, thus resulting in poor FRET efficiency and limited sensitivity.

Therefore, the development of a nucleic acid probe material and a detection method with high sensitivity, good specificity and reliable detection result is the focus of research in the field.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a nucleic acid grafted semiconductor polymer, a nucleic acid probe, a preparation method and application thereof, wherein the nucleic acid grafted semiconductor polymer has amphipathy, single-stranded DNA is mutually connected with a side chain of the semiconductor polymer through a stable chemical bond, self-assembly can be carried out in a water phase, a nucleic acid assembly body with densely arranged single-stranded DNA on the surface is formed, and the nucleic acid grafted semiconductor polymer can be used as an efficient energy transfer platform for detecting and imaging nucleic acid.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a nucleic acid grafted semiconducting polymer comprising a repeat unit having a structure according to formula I:

in the formula I, R¹、R²Each independently selected from C2 to C10 linear or branched alkylene.

The C2-C10 linear chain or branched chain alkylene can be C2, C3, C4, C5, C6, C7, C8, C9 or C10 linear chain or branched chain alkylene; exemplary include, but are not limited to:therein, deficiency of bloodThe line represents the attachment site of the group and m is selected from an integer of 2 to 10, such as 2,3, 4, 5, 6, 7, 8, 9 or 10.

In the formula I, L¹、L²Each independently selected from a single bond, CH ═ CH, or C ≡ C; said L¹Is a single bond, meaning that the fluorene ring is directly connected to Ar by a single bond; said L²Is a single bond, meaning that Ar is directly single-bonded to another repeat unit.

In the formula I, Ar is selected from substituted or unsubstituted C6-C20 arylene, substituted or unsubstituted C2-C20 heteroarylene; the substituted substituent in Ar is selected from C1-C10 straight chain or branched chain alkyl and C1-C10 straight chain or branched chain alkoxy.

The arylene group of C6-C20 may be an arylene group of C6, C9, C10, C12, C14, C16, C18, C20, etc., and exemplarily includes but is not limited to: phenylene, biphenylene, naphthylene, anthracenylene, phenanthrenylene, fluorenylene, or indenylene, and the like.

The C2-C20 heteroarylene group can be a C2, C3, C4, C5, C6, C7, C8, C10, C1, C14, C16, C18 or C20 heteroarylene group, and the like, and exemplarily includes but is not limited to: furanylene, thiophenylene, benzodithiophenylene, benzodifuranylene, benzothiadiazolylene, or benzoxadiazolylene, and the like.

The C1-C10 linear chain or branched chain alkyl can be C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10 linear chain or branched chain alkyl, and exemplarily comprises but is not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl or n-decyl, and the like.

The C1-C10 linear or branched alkoxy group can be a C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10 linear or branched alkoxy group, and exemplarily comprises but is not limited to: methoxy, ethoxy, propoxy, butoxy, or the like.

In formula I, S1 is single-stranded DNA.

The nucleic acid grafted semiconductor polymer provided by the invention comprises a repeating unit with a structure shown in a formula I, and a nucleic acid S1 is connected with a side chain of the semiconductor polymer through a stable chemical bond (triazole); the nucleic acid grafted semiconductor polymer has amphipathy, can be subjected to self-assembly in an aqueous solution to form a spherical nucleic acid assembly, single-stranded DNA is densely distributed on the surface of the nucleic acid assembly, and can be used as an efficient energy transfer platform for imaging and detecting nucleic acid based on the molecular wire property of the semiconductor polymer.

Preferably, said R is₁、R₂Each independently selected from C4-C8 linear or branched alkylene, more preferably C4-C8 linear alkylene; namely the R₁、R₂Each independently selected fromWherein m is 4, 5, 6, 7 or 8.

Preferably, said R is₁、R₂Are the same group.

Preferably, said L¹、L²Each independently selected from a single bond or CH ═ CH.

Preferably, said L¹、L²Are the same group.

Preferably, Ar is selected from any one of the following groups:

wherein the dotted line represents the attachment site of the group.

R_A1、R_A2、R_A3、R_A4Each independently selected from hydrogen, C1-C10 (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10) linear or branched alkyl, C1-C10 (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10) linear or branched alkoxy.

Preferably, the nucleic acid-grafted semiconducting polymer comprises any one or a combination of at least two of the following repeating units a 1-a 6:

preferably, the number of bases of the single-stranded DNA is 5 to 15, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

Preferably, the nucleic acid sequence of S1 is: 5'-TAGCTTATCAGACTG-3' (SEQ ID NO.1), 5'-TCCCTGAGACCCTAA-3' (SEQ ID NO.2) or 5'-TGAGGTAGTAGGTTG-3' (SEQ ID NO. 3).

In another aspect, the present invention provides a method for preparing the nucleic acid-grafted semiconducting polymer according to the first aspect, comprising the steps of:

(1) reacting a semiconductor polymer containing a repeating unit B, a cuprous catalyst and pore glass beads loaded with alkynyl modified nucleic acid in a solvent to obtain a product, wherein the product is loaded on the pore glass beads; the reaction formula is as follows:

wherein R is¹、R²、L¹、L²Ar, S1, n each independently have the same limitations as in formula I;

(2) and (2) separating the product obtained in the step (1) from the porous glass beads under the action of alkali liquor to obtain the nucleic acid grafted semiconductor polymer.

According to the preparation method provided by the invention, the semiconductor polymer containing the repeating unit B and alkynyl modified nucleic acid are subjected to click chemical reaction (click reaction) under the action of a cuprous catalyst, so that the nucleic acid (S1) is efficiently grafted on the side chain of the semiconductor polymer, and the amphiphilic nucleic acid grafted semiconductor polymer is obtained.

Preferably, the molar ratio of the repeating unit B to the alkynyl modified nucleic acid in step (1) is (1-10): 1, for example, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1 or 10: 1.

Preferably, the cuprous catalyst of step (1) comprises CuI and/or CuBr.

Preferably, the molar ratio of the alkynyl modified nucleic acid to the cuprous catalyst in step (1) is 1 (0.1-1), and may be, for example, 1:0.15, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, or 1: 0.95.

Preferably, the raw materials for the reaction of step (1) further comprise a stabilizer.

Preferably, the molar ratio of the stabilizer to the alkynyl modified nucleic acid is (0.1-1): 1, e.g., 0.15:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 0.95: 1.

Preferably, the catalyst is CuBr and the stabilizer is tris ((1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl) amine (TBTA).

Preferably, the solvent of step (1) comprises a mixture of Dichloromethane (DCM), Dimethylformamide (DMF) and Dimethylsulfoxide (DMSO).

Preferably, the amount of the solvent is 0.5-2 mL, such as 0.6mL, 0.8mL, 1mL, 1.1mL, 1.3mL, 1.5mL, 1.7mL, or 1.9mL, based on 1. mu. mol of the alkynyl modified nucleic acid, and specific values therebetween, which are not intended to be exhaustive for the invention and are included in the range for brevity.

Preferably, the reaction of step (1) is carried out under stirring conditions.

Preferably, the reaction temperature in step (1) is 30-50 ℃, for example 31 ℃, 33 ℃, 35 ℃, 37 ℃, 39 ℃, 40 ℃, 41 ℃, 43 ℃, 45 ℃, 47 ℃ or 49 ℃, and the specific values therebetween are limited by space and for the sake of brevity, and the invention is not exhaustive.

Preferably, the reaction time in step (1) is 5-24 h, such as 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h, and the specific values therebetween are limited by space and for the sake of brevity, and the invention is not exhaustive.

Preferably, the reaction in step (1) further comprises a washing step after completion.

Preferably, the washing reagent comprises any one of dichloromethane, dimethylformamide or dimethylsulfoxide, or a combination of at least two thereof.

Preferably, the alkali solution in step (2) comprises sodium hydroxide solution, potassium hydroxide solution or ammonia water.

Preferably, the temperature of the action in the step (2) is 50-60 ℃, for example, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃ or 59 ℃, and the specific values therebetween are limited by space and for the sake of brevity, and the invention is not exhaustive.

Preferably, the action time of step (2) is 5-24 h, for example, 6h, 8h, 10h, 12h, 13h, 14h, 16h, 18h, 20h, 21h or 23h, and the specific values therebetween are limited by space and for the sake of brevity, and the invention is not exhaustive.

Preferably, the separation in step (2) further comprises purification and drying steps.

Preferably, the purification is performed by a centrifugal filter.

Preferably, the preparation method specifically comprises the following steps:

(1) stirring and reacting a semiconductor polymer containing a repeating unit B, a cuprous catalyst, a stabilizer and pore glass beads loaded with alkynyl modified nucleic acid in a solvent at 30-50 ℃ for 5-24 h to obtain a product, wherein the product is loaded on the pore glass beads; the molar ratio of the repeating unit B to the alkynyl modified nucleic acid is (1-10): 1; the dosage of the alkynyl modified nucleic acid is 1 mu mol, the dosage of the cuprous catalyst is 0.1-1 mu mol, and the dosage of the stabilizer is 0.1-1 mu mol;

(2) and (2) mixing the product obtained in the step (1) with alkali liquor, treating for 5-24 hours at 50-60 ℃, separating the product from the porous glass beads, and purifying and drying to obtain the nucleic acid grafted semiconductor polymer.

In another aspect, the present invention provides a nucleic acid assembly self-assembled by the nucleic acid-grafted semiconducting polymer according to the first aspect.

The nucleic acid assembly is formed by assembling the nucleic acid grafted semiconductor polymer in an aqueous solution based on hydrophobic acting force and pi-pi stacking force, the space form of the nucleic acid assembly is spherical nano particles, DNA is densely distributed on the surfaces of the nano particles, and the nucleic acid assembly has the characteristic of spherical nucleic acid. The nucleic acid assembly serves as an efficient energy transfer platform and can be used for detecting target nucleic acid with high sensitivity and specificity.

In another aspect, the present invention provides a nucleic acid probe comprising a nucleic acid assembly, and a dye-modified single-stranded DNA S2 hybridized to the nucleic acid assembly; the nucleic acid assembly is self-assembled by the nucleic acid-grafted semiconducting polymer according to the first aspect.

In the nucleic acid probe, the nucleic acid assembly is formed by self-assembling the nucleic acid grafted semiconductor polymer, the single-stranded DNA (S1) is densely distributed on the surface of the nucleic acid assembly, and can be subjected to molecular hybridization with the dye modified DNA single strand (S2), so that the distance between the semiconductor polymer and the dye is shortened, high-efficiency energy transfer from the semiconductor polymer to the dye can be generated, fluorescence energy resonance transfer (FRET) is formed, and the high-specificity and high-sensitivity quantitative detection of target nucleic acid can be realized.

Preferably, the nucleic acid assembly is a spherical nanoparticle.

Preferably, the nucleic acid assembly has a hydration radius of 20-100 nm, such as 22nm, 25nm, 28nm, 30nm, 32nm, 35nm, 38nm, 40nm, 42nm, 45nm, 48nm, 50nm, 52nm, 55nm, 58nm, 60nm, 62nm, 65nm, 68nm, 70nm, 72nm, 75nm, 78nm, 80nm, 82nm, 85nm, 88nm, 90nm, 92nm, 95nm or 98nm, and the specific points between the above points are limited to space and for brevity, the invention is not exhaustive of the specific points included in the range.

The molar ratio of the nucleic acid to the dye-modified DNA single strand S2 in the nucleic acid assembly is preferably 1 (0.1 to 0.2), and may be, for example, 1:0.11, 1:0.12, 1:0.13, 1:0.14, 1:0.15, 1:0.16, 1:0.17, 1:0.18, or 1: 0.19.

Preferably, the number of bases of the DNA single strand S2 is 20 to 25, and for example, the number may be 20, 21, 22, 23, 24 or 25.

Preferably, the nucleic acid sequence of the DNA single strand S2 is: 5'-TCAACATCAGTCTGATAAGCTA-3' (SEQ ID NO. 4).

Preferably, the dye comprises cyanine dye, further preferably Cy3, Cy5 or Cy 7.

Preferably, the absorption spectrum of the dye overlaps with the emission spectrum of the nucleic acid-grafted semiconducting polymer.

In another aspect, the present invention provides a method for preparing the nucleic acid probe as described above, comprising: self-assembling the nucleic acid-grafted semiconducting polymer according to the first aspect in an aqueous solution to obtain a nucleic acid assembly; and hybridizing the nucleic acid assembly with a dye modified DNA single strand S2 to obtain the nucleic acid probe.

Preferably, the concentration of the nucleic acid grafted semiconductor polymer in the aqueous solution is 0.1-10 mu g/mL, for example, it may be 0.2. mu.g/mL, 0.5. mu.g/mL, 0.8. mu.g/mL, 1.0. mu.g/mL, 1.5. mu.g/mL, 2.0. mu.g/mL, 2.5. mu.g/mL, 3.0. mu.g/mL, 3.5. mu.g/mL, 4.0. mu.g/mL, 4.5. mu.g/mL, 5.0. mu.g/mL, 5.5. mu.g/mL, 6.0. mu.g/mL, 6.5. mu.g/mL, 7.0. mu.g/mL, 7.5. mu.g/mL, 8.0. mu.g/mL, 8.5. mu.g/mL, 9.0. mu.g/mL or 9.5. mu.g/mL, and the specific values between the foregoing, are not intended to be exhaustive or to limit the invention to the precise values encompassed within the scope, for reasons of brevity and clarity.

Preferably, the molar ratio of the surface nucleic acid of the nucleic acid assembly to the dye-modified DNA single strand S2 is 1 (0.1-0.2), and may be, for example, 1:0.11, 1:0.12, 1:0.13, 1:0.14, 1:0.15, 1:0.16, 1:0.17, 1:0.18, or 1: 0.19.

In another aspect, the present invention provides the use of a nucleic acid probe as described above in biosensing, bioimaging or nucleic acid detection.

Preferably, the method for detecting nucleic acid comprises the steps of:

(A) adding target nucleic acid into the solution of the nucleic acid probe for hybridization replacement to obtain a solution to be detected;

(B) and (C) carrying out fluorescence test on the solution to be tested obtained in the step (A), and realizing quantitative detection of the target nucleic acid according to the fluorescence ratio of fluorescence energy resonance transfer.

In the method for detecting nucleic acid, after target nucleic acid is added into a solution of a nucleic acid probe, because of competitive hybridization replacement of nucleic acid molecules, the target nucleic acid and a dye modified DNA single strand S2 on the nucleic acid probe form a double-stranded complex and are dispersed in the solution, at the moment, the distance between an energy acceptor dye and a nucleic acid assembly is increased, and FRET disappears; as more target nucleic acids are added, more dye-modified DNA single strand S2 is detached from the nucleic acid assembly, and thus the overall FRET efficiency continues to become lower; by calculating the relationship between the FERT efficiency and the concentration of the target nucleic acid, the target nucleic acid can be quantitatively detected with high sensitivity.

Preferably, the solvent of the solution is a buffer, and more preferably a PBS buffer.

Preferably, the target nucleic acid comprises DNA, messenger RNA or MicroRNA.

Compared with the prior art, the invention has the following beneficial effects:

(1) the nucleic acid grafted semiconductor polymer provided by the invention comprises a repeating unit with a structure shown in a formula I, and a nucleic acid S1 is connected with a side chain of the semiconductor polymer through a stable chemical bond; the nucleic acid grafted semiconductor polymer has amphipathy, self-assembly occurs in aqueous solution, and a nucleic acid assembly with densely arranged single-stranded DNA on the surface is formed.

(2) In the nucleic acid probe containing the nucleic acid assembly, the dye modified DNA single strand is subjected to molecular hybridization with DNA on the surface of the nucleic acid assembly, and the distance between the semiconductor polymer and the dye is shortened, so that efficient energy transfer from the semiconductor polymer to the dye is generated, and fluorescence energy resonance transfer is formed. When the nucleic acid probe is used for detecting target nucleic acid, the fluorescence ratio of fluorescence energy resonance transfer is changed due to hybridization replacement of nucleic acid molecules, and the quantitative detection of the target nucleic acid with high sensitivity and high specificity is realized through the relation between the fluorescence ratio and the concentration of the target nucleic acid.

Drawings

FIG. 1 is a diagram showing the result of agarose gel electrophoresis analysis of the nucleic acid-grafted semiconducting polymer provided in example 1;

FIG. 2 is a schematic diagram of the self-assembly of the nucleic acid-grafted semiconducting polymer provided in example 1;

FIG. 3 is a transmission electron micrograph of the nucleic acid assembly of example 2;

FIG. 4 is a graph showing a particle size distribution of the nucleic acid assembly in example 2;

FIG. 5 is a fluorescence spectrum of the nucleic acid probe in example 3;

FIG. 6 is a schematic diagram showing the principle of using a nucleic acid probe for nucleic acid detection in example 4;

FIG. 7 is a fluorescence spectrum of detection of a nucleic acid in example 4;

FIG. 8 is a graph of fluorescence ratio for nucleic acid detection versus concentration of target nucleic acid in example 4;

FIG. 9 is a graph showing the specificity of detection of nucleic acid in example 4.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

A nucleic acid-grafted semiconducting polymer (SP-g-DNA) comprising recurring units a1 as shown below:

wherein the nucleic acid sequence of S1 is SEQ ID NO. 1: 5'-TAGCTTATCAGACTG-3' are provided.

The preparation method of the nucleic acid grafted semiconductor polymer comprises the following steps:

(1) azide-functionalized poly ((9,9-bis (6-bromohexyl) -2,7-diyl) -co- (1, 4-benzol- (2,1',3) -thiadazole) SP-N which is a semiconductor polymer modified by azide₃(repeating Unit)In a molar amount of 5. mu. mol, SP-N₃With a molecular weight of 4341Da), CuBr (1.0. mu. mol), tris ((1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl) amine (TBTA, 1.0. mu. mol), well glass beads CPG (50mg, 1.5. mu. mol) loaded with alkynyl-modified S1, and a magnetic stir bar were placed in a flask in this order, degassed three times, and after adding a mixed solution (total of 1mL) of DCM, DMF, and DMSO (volume ratio 1:1:1) via syringe, the mixture was stirred at 40 ℃ for 12H. After the reaction was complete, the product-loaded CPG beads were collected and washed with DCM and DMSO to remove unreacted SP and other impurities;

(2) using concentrated ammonia solution (28% NH) at 55 deg.C₃) After the CPG beads obtained in the step (1) are treated for 16 hours, removing ammonia through vacuum until the odor of ammonia disappears; purifying the crude SP-g-DNA product by a centrifugal filter to remove unbound free DNA; the purified SP-g-DNA was lyophilized to give a yellow powder.

The purity of the SP-g-DNA provided in this example was confirmed by agarose gel electrophoresis analysis, the result of which is shown in FIG. 1.

Example 2

A nucleic acid assembly (S-SNA) prepared by self-assembly of the nucleic acid-grafted semiconducting polymer (SP-g-DNA) provided in example 1 was prepared as follows:

the SP-g-DNA provided in example 1 was mixed with water to obtain an aqueous solution (yellow aqueous solution) having a concentration of 10. mu.g/mL, and the SP-g-DNA was self-assembled into spherical nanoparticles, i.e., the nucleic acid assemblies, due to hydrophobic interaction and pi-. pi.stacking force, and the DNA was densely arranged on the surfaces of the nanoparticles (S1), and the self-assembly scheme is shown in FIG. 2.

The morphology of the S-SNA in this example was measured by a transmission electron microscope (TEM, Hitachi HT7700), and the transmission electron micrograph obtained is shown in FIG. 3.

The particle size of the S-SNA in this example was measured by a dynamic light scattering particle sizer (DLS, Brookhaven Instruments Corporation, USA), and the resulting particle size distribution graph is shown in FIG. 4, from which it can be seen that the hydration radius of the S-SNA is about 28 nm.

Example 3

A nucleic acid probe comprises a nucleic acid assembly and a dye Cy5 modified DNA single strand S2(Cy5-cDNA) hybridized on the nucleic acid assembly, wherein the nucleic acid sequence of S2 is SEQ ID NO. 4: 5'-TCAACATCAGTCTGATAAGCTA-3', respectively; the nucleic acid assembly was self-assembled from the SP-g-DNA provided in example 1 (same as example 2); the preparation method comprises the following steps:

mixing the SP-g-DNA provided in example 1 with water to obtain an aqueous solution with a concentration of 10. mu.g/mL, wherein the SP-g-DNA self-assembles into spherical nanoparticles due to hydrophobic interaction force and pi-pi stacking force, and the surfaces of the nanoparticles are densely packed with DNA (S1), namely S-SNA in example 2; a nucleic acid probe was obtained by adding 1nM, 2nM, 5nM, 8nM, and 10nM Cy5-cDNA to a 1. mu.g/mL S-SNA solution and subjecting the S-SNA and Cy5-cDNA to molecular hybridization.

The nucleic acid probe in this example was subjected to fluorescence detection using an Agilent Cary Eclipse (excitation wavelength of 450nM), and the fluorescence spectrum obtained without Cy5-cDNA (i.e., 0nM) as a control was shown in FIG. 5. As can be seen from FIG. 5, the distance between SP and Cy5 was shortened by hybridization complementation of nucleic acids, and the energy of S-SNA was efficiently transferred to the receptor Cy5-cDNA to form Fluorescence Energy Resonance Transfer (FERT); meanwhile, as the concentration of Cy5-cDNA increased, the fluorescence energy resonance transfer efficiency gradually increased, and the fluorescence intensity at 670nm increased.

Example 4

A method of nucleic acid detection comprising the steps of:

adding target nucleic acids (MicroRNA-21 with the sequence of SEQ ID NO. 5: 5'-UAGCUUAUCAGACUGAUGUUGA-3') with the concentrations of 2nM, 5nM, 8nM, 9nM and 10nM to PBS solution of nucleic acid probes (example 3, with the concentration of receptor Cy5-cDNA of 10nM) respectively to obtain a solution to be detected, and taking the solution without the target nucleic acid (namely 0nM) as a control group; the fluorescence detection (fluorescence spectrophotometer Agilent Cary Eclipse, excitation wavelength 450nm) was performed on the solution to be tested, and the obtained fluorescence spectrum is shown in FIG. 7.

In the nucleic acid detection provided in this example, due to competitive hybridization displacement of the nucleic acid molecules, the target nucleic acid and Cy5-cDNA form a double-stranded complex and are dispersed in the solution, so that the distance between the energy acceptor Cy5 and S-SNA is increased, FRET disappears, the principle schematic diagram of the detection process is shown in FIG. 6, the fluorescence spectrum is shown in FIG. 7, and the fluorescence ratio (the ratio of the fluorescence intensity at 670nm to the fluorescence intensity at 550nm, I) is expressed as the fluorescence ratio₆₇₀/I₅₅₀) The ordinate and the concentration of the target nucleic acid were plotted on the abscissa, and the obtained graph showing the relationship between the fluorescence ratio and the concentration of the target nucleic acid is shown in FIG. 8.

As is clear from the combination of FIG. 7 and FIG. 8, the target nucleic acid and the nucleic acid probe undergo a hybridization substitution of the nucleic acid molecule, and the target nucleic acid and Cy5-cDNA form a double-stranded complex, so that the distance between Cy5 and S-SNA increases, FRET disappears, and the fluorescence intensity decreases; as the concentration of target nucleic acid increases, more Cy5-cDNA is detached from S-SNA and the overall FRET efficiency continues to become lower. The fluorescence ratio has a good linear relationship with the concentration of the target nucleic acid, and the target nucleic acid can be sensitively detected by calculating the relationship between the FERT efficiency (fluorescence ratio) and the concentration of the target nucleic acid.

The detection method in the embodiment is adopted to carry out specific characterization on the target nucleic acid of the microRNA21 series, and the specific method is as follows: the same detection was performed for miR21A and miR21B at the same concentrations. The sequences of the two sequences are 5'-UAGCUUAUCAGACUGAGGUUGA-3' (miR21A, SEQ ID NO.6) and 5'-UAGCUUAUGAGACUGAUGUUGA-3' (miR21B, SEQ ID NO.7), the obtained specificity characterization maps are shown in FIG. 9, and as can be seen from FIG. 9, neither miR21A nor miR21B can respond to the nucleic acid probe, while miR21 (SEQ ID NO. 5: 5'-UAGCUUAUCAGACUGAUGUUGA-3') can respond well, which indicates the excellent sequence specificity of the nucleic acid probe.

Example 5

A nucleic acid-grafted semiconducting polymer (PFPV-g-DNA) comprising recurring unit a4 as shown below:

wherein the nucleic acid sequence of S1 is SEQ ID NO. 2: 5'-TCCCTGAGACCCTAA-3' are provided.

The preparation method of the nucleic acid grafted semiconductor polymer comprises the following steps:

(1) azide-functionalized poly ((9,9-bis (6-bromohexyl) -2,7-diyl) -co- (2-methoxy-5- (2-ethylhexyloxy) -1, 4-phenylene)) PFPV-N is prepared by reacting azide-modified semiconductor polymer₃(repeating Unit)In a molar amount of 5. mu. mol, PFPV-N₃Molecular weight of 6765Da), CuBr (1.0. mu. mol), TBTA (1.0. mu. mol), CPG (50mg, 1.5. mu. mol) as well as a magnetic stir bar loaded with alkynyl-modified S1 were placed in a flask in this order, degassed three times, and after adding a mixed solution (total of 1mL) of DCM, DMF and DMSO (volume ratio 1:1:1) via syringe, the mixture was stirred at 40 ℃ for 12 h. After completion of the reaction, the product-loaded CPG beads were collected and washed with DCM and DMSO to remove unreacted PFPV and other impurities;

(2) using concentrated ammonia solution (28% NH) at 50 deg.C₃) After the CPG beads obtained in the step (1) are treated for 16 hours, removing ammonia through vacuum until the odor of ammonia disappears; purifying the resulting crude PFPV-g-DNA product by a centrifugal filter to remove unbound free DNA; the purified PFPV-g-DNA was lyophilized to give a green powder.

The PFPV-g-DNA provided in this example was mixed with water to obtain an aqueous solution having a concentration of 10. mu.g/mL, and the PFPV-g-DNA was self-assembled into spherical nanoparticles, i.e., nucleic acid assemblies, due to hydrophobic interaction and pi-. pi.stacking force, and the DNA was densely arranged on the surfaces of the nanoparticles (S1: 5'-TCCCTGAGACCCTAA-3').

The S-SNA in this example was subjected to a morphology confirmation test by a transmission electron microscope (TEM, Hitachi HT 7700); the particle size of the S-SNA in this example was measured by a dynamic light scattering particle sizer (DLS, Brookhaven Instruments Corporation) and had a hydration radius of about 35 nm.

The nucleic acid assembly can be hybridized with a fluorescent dye Cy3 modified DNA single strand S2(Cy3-cDNA) to form a nucleic acid probe.

Example 6

A nucleic acid-grafted semiconducting polymer (PFO-g-DNA) comprising the repeating unit A6 shown below:

wherein the nucleic acid sequence of S1 is SEQ ID NO. 3: 5'-TGAGGTAGTAGGTTG-3' are provided.

The preparation method of the nucleic acid grafted semiconductor polymer comprises the following steps:

(1) azide-functionalized poly (9, 9-dihexylfluoronyl-2, 7-diyl) PFO-N of semiconductor polymer modified by azide₃(repeating Unit)In a molar amount of 5. mu. mol, PFO-N₃Was placed in a flask in this order with a molecular weight of 8634Da), CuBr (1.0. mu. mol), TBTA (1.0. mu. mol), CPG (50mg, 1.5. mu. mol) as well as a magnetic stir bar loaded with alkynyl-modified S1, degassed three times, and after adding a mixed solution (total 1mL) of DCM, DMF and DMSO (volume ratio 1:1:1) via syringe, the mixture was stirred at 42 ℃ for 12 h. After the reaction was completed, the CPG beads loaded with the product were collected and washed with DCM and DMSO to remove unreacted PFO and other impurities;

(2) using concentrated ammonia solution (28% NH) at 60 deg.C₃) After processing the CPG beads obtained in the step (1) for 15 hours, removing ammonia through vacuum until the odor of ammonia disappears; purifying the resulting crude PFO-g-DNA by a centrifugal filter to remove unbound free DNA; the purified PFO-g-DNA was lyophilized to obtain a powder.

PFO-g-DNA provided in this example was mixed with water to obtain an aqueous solution having a concentration of 10. mu.g/mL, and PFO-g-DNA was self-assembled into spherical nanoparticles, i.e., nucleic acid assemblies, due to hydrophobic interaction force and pi-. pi.stacking force, and DNA was densely arranged on the surfaces of the nanoparticles (S1: 5'-TGAGGTAGTAGGTTG-3').

The S-SNA in this example was subjected to a morphology confirmation test by a transmission electron microscope (TEM, Hitachi HT 7700); the particle size of the S-SNA in this example was measured by a dynamic light scattering particle sizer (DLS, Brookhaven Instruments Corporation) and had a hydration radius of about 48 nm.

The nucleic acid assembly can be hybridized with a fluorescent dye Alexa 430 modified DNA single strand S2(Alexa 430-cDNA) to form a nucleic acid probe.

The applicant states that the present invention is illustrated by the above examples to a nucleic acid grafted semiconducting polymer, a nucleic acid probe, a preparation method and applications thereof, but the present invention is not limited to the above process steps, i.e., it does not mean that the present invention must rely on the above process steps to be implemented. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.