APTAMER NUCLEIC ACID MOLECULE, AND COMPLEX AND APPLICATION THEREOF

Abstract

The present application relates to an aptamer nucleic acid molecule, a complex containing the aptamer nucleic acid molecules, a method of detecting intracellular or extracellular RNA, DNA or other target molecules, and a kit containing the aptamer. The aptamer of the present application is capable of specifically binding a kind of fluorophore micromolecules, and can significantly enhance fluorescence intensity under excitation light of appropriate wavelength.

Claims

1. A nucleic acid aptamer molecule containing following nucleotide sequences (a), (b) or (c), wherein (a): a nucleotide sequence N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32, wherein N.sub.1, N.sub.19, N.sub.20, N.sub.21 and N.sub.32 represent nucleotide fragments greater than or equal to 1 in length, and at least one base pair in N.sub.1 and N.sub.32 nucleotide sequences forms a complementary pair, and at least one base pair in N.sub.19 and N.sub.21 nucleotide sequences forms a complementary pair; (b): a nucleotide sequence with an identity of at least 70% to the nucleotide sequence defined by (a); and (c): a nucleic acid aptamer molecule derived from (a) at a position not including N.sub.1, N.sub.19, N.sub.20, N.sub.21 and N.sub.32 in the nucleotide sequence defined by (a), with substitution, missing and/or addition of one or several nucleotides, and having an aptamer function.

2. The nucleic acid aptamer molecule according to claim 1, wherein, when N.sub.1 and N.sub.32 in the nucleotide sequence (a) form a complementary pair, a direction of N.sub.1 nucleotide sequence is 5′-3′, and a direction of N.sub.32 nucleotide sequence is 3′-5′; and when N.sub.19 and N.sub.21 form a complementary pair, a direction of N.sub.19 nucleotide sequence is 5′-3′, and a direction of N.sub.21 nucleotide sequence is 3′-5′.

3. The nucleic acid aptamer molecule according to claim 2, wherein, when at least one fragment of N.sub.1 and N.sub.32 is greater than or equal to 5 nucleotide bases in length, at least two base pairs in N.sub.1 and N.sub.32 nucleotide sequences form complementary pairs; when at least one fragment of N.sub.19 and N.sub.21 is greater than or equal to 5 nucleotide bases in length, at least two base pairs in N.sub.19 and N.sub.21 nucleotide sequences form complementary pairs.

4. The nucleic acid aptamer molecule according to claim 1, wherein the nucleotide substitution in the nucleotide sequence (a) is selected from one of the following groups: C3A, C3U, A4U, A4G, A4C, A5G, A5C, U6A, U6G, U6C, C7A, C7U, G8C, U9A, G11A, G11U, C12G, C12A, C12U, G13C, U14A, U14G, C17U, G18U, G18C, C27G, C27U, G28U, C29G, C29U, C30A, C30U, C2G/G31C, C2U/G31A, C2A/G3 1U, G 10A/C30U, G 10C/C30G, G 10U/C30A, C2G/G3 1C/C3A, C2G/G3 1C/A4C, C2G/G3 1C/A5C, C2G/G3 1C/G8C, C2G/G31C/C12U, C2G/G31C/U14G, C2G/G31C/C27U, C2G/G31C/C29G, C2G/G31C/C30U, C2G/G31C/ G10A/C30U, C2G/G31C/G10C/C30G, C2G/G31C/G10U/C30A, C2U/G31A/G10A/C30U, C2U/G31A/G10C/C30G, C2U/G31A/G10U/C30A, C2A/G31U/G10A/C30U, C2A/G31U/G10C/C30G, C2A/G31U/G10U/C30A, C2G/G31C/G10C/C30G/C3A, C2G/G31C/G10C/C30G/A4C, C2G/G31C/G10C/C30G/A5C, C2G/G31C/G10C/C30G/G8C, C2G/G31C/G10C/C30G/C12U, C2G/G31C/G10C/C30G/U14G, C2G/G31C/G10C/C30G/C27U, C2G/G31C/G10C/C30G/C29G, C2G/G31C/G10A/C30U/U6G/C27U, C2G/G31C/G10C/C30G/U6G/C27U, C2G/G31C/G10U/C30A/U9A/U14G/C27U and C2A/G31U/G10U/C30A/U9A/U14G/C27U.

5. The nucleic acid aptamer molecule according to claim 1, wherein nucleotide sequences at N.sub.1 and N.sub.32 in the nucleotide sequence (a) are F30 or tRNA scaffold RNA sequences.

6. The nucleic acid aptamer molecule according to claim 1, wherein the nucleic acid aptamer molecules are RNA molecules or RNA base-modified molecules.

7. The nucleic acid aptamer molecule according to claim 1, wherein the nucleic acid aptamer molecules are DNA-RNA hybrid molecules or DNA-RNA base-modified molecules.

8. The nucleic acid aptamer molecule according to claim 1, wherein N.sub.19-N.sub.20-N.sub.21 in the nucleotide sequence (a) contains a nucleotide sequence capable of identifying target molecules.

9. The nucleic acid aptamer molecule according to claim 8, wherein the target molecules is at least one of proteins, nucleic acid, lipid molecules, carbohydrates, hormones, cytokines, chemokines, and metabolite metal ions.

10. The nucleic acid aptamer molecule according to claim 8 or 9, wherein N.sub.19-N.sub.2o-N.sub.21 in the nucleotide sequence (a) is a nucleotide sequence capable of identifying GTP and adenosine molecules.

11. The nucleic acid aptamer molecule according to claim 1, wherein the aptamer function refers to that the nucleic acid aptamer can enhance fluorescence intensity of fluorophore molecules under excitation light of appropriate wavelength by at least two times, at least 5 to 10 times, at least 20 to 50 times, at least 100 to 200 times or at least 500 to 1,000 times.

12. The nucleic acid aptamer molecule according to claim 1, further containing: concatemers that can bind multiple fluorophore molecules, and the concatemers are connected by spacer sequences of appropriate length having 2, 3, 4, 5, 6, 7, 8 or more nucleotide fragments. Nucleotides of the concatemers can be selected from but are not limited to a sequence SEQ ID No: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19.

13. The nucleic acid aptamer molecule according to claim 1, having a sequence SEQ ID No: 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22 or 23.

14. A complex of nucleic acid aptamer molecules and fluorophore molecules, wherein the nucleic acid aptamer molecules are the nucleic acid aptamer molecules according to claim 1, the fluorophore molecules having a structure shown in Formula (I) below: ##STR00041## wherein: D- is X.sub.1O— or N(X.sub.2)(X.sub.3)—; X.sub.1, X.sub.2, X.sub.3 are respectively and independently selected from hydrogen, straight or branched alkyl and modified alkyl with 1 to 10 carbons, and X.sub.2 and X.sub.3 are optionally interconnected to be a satured or unsatured ring; R— is selected from hydrogen, cyano group, carboxyl, amide group, ester group, hydrixy, and straight or branched alkyl or modified alkyl with 1 to 10 carbons; Ar.sub.i and Ar.sub.2 are respectively and independently selected from monocyclic aryl subunits, monocyclic heteraryl subunits, or aromatic subunits with 2 to 3 ring structures condensed from one or both of monocyclic aryl group and monocyclic heteroaryl group; wherein: hydrogen atoms in Ar.sub.1, and Ar.sub.2 can be independently substituted by F, Cl, Br, I, hydroxyl group, nitro group, aldehyde group, carboxyl group, cyano group, sulfonic acid group, sulfuric acid group, phosphoric acid group, amino group, primary amino group, secondary amino group, straight or branched alkyl and modified alkyl with 1 to 10 carbons; wherein: the modified alkyl is a group obtained by replacing any carbon atom of the alkyl with at least one group selected from F, Cl, Br, I, —O—, —OH, —CO, —NO.sub.2, —CN, —S—, —SO.sub.2—, —(S═O)—, azide group, phenylene, primary amino group, secondary amino group, tertiary amino group, quaternary ammonium group, ethylene oxide, succinate, isocyanate, isothiocyanate, acyl chloride, sulfonyl chloride, saturated or unsaturated monocyclic or dicyclic subcyclic hydroxyl, bridged ester heterocycle, and the modified alkyl has 1 to 10 carbon atoms, wherein the carbon-carbon single bond is optionally and independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond; and wherein the nucleic acid aptamer molecules and the fluorophore molecules in the complex respectively exist in an individual solution, or the nucleic acid aptamer molecules and the fluorophore molecules are in the same solution.

15. The complex according to claim 14, wherein the modified alkyl contains at least one group selected from —OH, —O—, glycol unit, monosaccharide unit, disaccharide unit, —O—CO—, —NH—CO—, —SO.sub.2—O—, —SO—, Me.sub.2N—, Et.sub.2N , —S—S—, —CH═CH—, F, Cl, Br, I, —NO.sub.2 and cyano group; the aromatic rings contained in the fluorophore molecules being selected from structures represented by the following formulae (□-1) to (□-15): ##STR00042## ##STR00043##

16. The complex according to claim 14, wherein fluorophore molecules are selected from the following compounds represented by the following formulae: ##STR00044## ##STR00045## ##STR00046##

17. The complex according to claim 14, wherein the aptamer molecules in the complex contain nucleotide sequence SEQ ID No: 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

18. A kit containing: the nucleic acid aptamer molecules according to claim 1, at least one of the complexes, the expression vectors or the host cells according to claim 14, wherein the expression vector contains DNA molecules for transcribing the nucleic acid aptamer molecules according to claim 1; and the host cell contains the expression vector.

19. Application of the complex according to claim 14 to detection or labeling of complex in vitro or in vivo target nucleic acid molecules, detection or labeling of in vitro or in vivo target molecules, genomic DNA imaging, detection of mRNA and protein contents in cells, detection of genomic DNA, or extraction or purification of RNA.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0054] FIG. 1 Secondary structure prediction of nucleic acid aptamer molecules. (A) is the predicted general structure of Pepper, comprising N.sub.1 and N.sub.32 that can form a stem structure, and N.sub.19, N.sub.20 and N.sub.21 that can form a stem-loop structure. (B) is the predicted general structure of Pepper-1, wherein the base sequence of N.sub.1 and N.sub.32 is shown in the dotted box corresponding to Stem 1 in the drawing, and the base sequence of N.sub.19, N.sub.20 and N.sub.21 is shown in the dotted box corresponding to the stem-loop.

[0055] FIG. 2 Secondary structure prediction of F30-Pepper-1.

[0056] FIG. 3 Secondary structure prediction of tRNA-Pepper-2.

[0057] FIG. 4 Nature identification of F30-Pepper-1-III-3 complex. (A) Fluorescence excitation spectrum and emission spectrum of F30-Pepper-1-III-3 complex; (B) absorption spectra of F30-Pepper-1-III-3 complex and III-3; (C) oligomerization identification of F30-Pepper-1-III-3 complex; the “ruler” is a single-stranded DNA standard used for calibrating size of the aptamer; (D) dissociation constant determination of the binding of F30-Pepper-1 to III-3; (E) temperature stability determination of F30-Pepper-1-III-3 complex; (F) pH stability determination of F30-Pepper-1-III-3 complex; (G) determination of dependence of F30-Pepper-1-III-3 complex on K+.

[0058] FIG. 5 Activation effect of Pepper modified with different bases on III-3. (A) The secondary structure diagram of Pepper-3 aptamers containing deoxyribonucleotides (dark color in the drawing); (B) the secondary structure diagram of Pepper-4 aptamers with 2′F modification (dark color in the drawing); (C) activation effects of Pepper with different modifications on III-3. “Control” is to replace Pepper-3 or Pepper-4 aptamer with buffer.

[0059] FIG. 6 Activation effects of different Pepper concatemers on III-3. (A) Obtaining Pepper concatemers in a way of “Tandem 1”; (B) obtaining Pepper concatemers in a way of “Tandem 2”; (C) obtaining Pepper concatemers in a way of “Tandem 3”; (D) activation effects of different Pepper concatemers obtained in the way of “Tandem 1” on III-3; (E) activation effects of different Pepper concatemers obtained in the way of “Tandem 2” on III-3; (F) activation effects of different Pepper concatemers obtained in the way of “Tandem 3” on III-3.

[0060] FIG. 7 The labeling effect of F30-Pepper-1-III-3 complex on RNA in bacteria.

[0061] FIG. 8 The labeling effect of F30-Pepper-1-III-3 complex on RNA in yeast cells.

[0062] FIG. 9 The labeling effects of Pepper, III-3 and analogue thereof in labeling RNA in mammalian cells. (A) Comparison of the effects of F30-Pepper-1-III-3, F30-Broccoli-DFHBI-1T and tRNA-Corn-DFHO on RNA labeling in mammalian cell; (B) statistical results of fluorescence in Fig. (A); (C) effects of F30-8Pepper-5 and III-3 analogs on RNA labeling in mammalian cells.

[0063] FIG. 10 Probe construction based on Pepper-1. (A) Diagram of probe construction, wherein the stem-loop structure can identify adenosine or GTP; (B) detection effect of adenosine probe; (C) detection effect of GTP probe.

[0064] FIG. 11 RNA localization in tracer cells by using Pepper. (A) Detection of GAPDH mRNA localization by using Pepper; (B) detection of Pepper TMED2 mRNA localization by using Pepper.

[0065] FIG. 12 Exploration of the relationship between mRNA and protein in cells by using Pepper. (A) Flow cytometry analysis result of BFP protein and RNA expression thereof; (B) flow cytometry analysis result of mCherry protein and RNA expression thereof.

[0066] FIG. 13 Detection of genomic DNA by using Pepper. (A) Diagram of dCas9 and different chimeric sgRNA; (B) imaging results of genomic DNA by dCas9 and different chimeric sgRNA; (C) statistical results of bright spot particles in each cell in (B).

[0067] FIG. 14 Super-resolution imaging of RNA by using Pepper. (A) Co-localization of 4Pepper-9-MS2 RNA and tdMCP-BFP-H2B protein; (B) imaging results of wide field and SIM in nuclear middle layer; (C) imaging results of wide field and SIM in nuclear top layer.

[0068] FIG. 15 Pepper serving as labels for RNA extraction and purification. The “ruler” is a single-stranded DNA standard used for calibrating size of the aptamer.

EXAMPLES

[0069] The following definitions and embodiments cited in the present application will be described in details here. The contents of all patents and published literature referred to herein, including all sequences disclosed in these patents and published literature, are expressly incorporated herein by reference. Hereinafter, “nucleotides” and “nucleotide bases” are used interchangeably and stand for the same meaning.

[0070] Following are detailed explanations of some terms used in the present application.

Nucleic Acid Aptamer Molecules

[0071] The “nucleic acid aptamer molecules” of the present application are also referred to as “aptamer molecules”. The nucleic acid aptamer molecule contains (a) a nucleotide sequence N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 (corresponding to the General Formula Pepper structure in FIG. 1A); or (b) which is a nucleotide sequence with an identity of at least 70% to the nucleotide sequence of (a); wherein at least one base pair in N.sub.1 and N.sub.32 nucleotide sequences forms a reverse complementary pair, namely, the direction of N.sub.1 nucleotide sequence is 5′-3′, and the direction of N.sub.32 nucleotide sequence is 3′-5′ . When the length of at least one nucleotide base of N.sub.1 and N.sub.32 is smaller than or equal to 4, at least one base pair is needed for forming the complementary pair; and when the length of at least one nucleotide base of N.sub.1 and N.sub.32 is greater than or equal to 5, at least two base pairs are needed for forming the complementary pair. Wherein, at least one base pair in N.sub.19 and N.sub.21 nucleotide sequences forms a reverse complementary pair, namely, the direction of N.sub.19 nucleotide sequence is 5′-3′, and the direction of N.sub.21 nucleotide sequence is 3′-5′. When the length of at least one nucleotide base of N.sub.19 and N.sub.21 is smaller than or equal to 4, at least one base pair is needed for forming the complementary pair; and when the length of at least one nucleotide base of N.sub.19 and N.sub.21 is greater than or equal to 5, at least two base pairs are needed for forming the complementary pair. N.sub.20 therein is a nucleotide base of any length or composition; or (c) which is at any position in the nucleotide sequence (a) with the substitution, missing and/or addition of 1 to 7 nucleotides.

[0072] The nucleic acid aptamer molecules contain substitution of the nucleotides in General Formula Pepper structure, the substitution being selected from one of the following groups: C3A, C3U, A4U, A4G, A4C, A5G, A5C, U6A, U6G, U6C, C7A, C7U, G8C, U9A, G11A, G11U, C12G, C12A, C12U, G13C, U14A, U14G, C17U, G18U, G18C, C27G, C27U, G28U, C29G, C29U, C30A, C30U, C2G/G31C, C2U/G31A, C2A/G31U, G10A/C30U, G10C/C30G, G10U/C30A, C2G/G31C/C3A, C2G/G31C/A4C, C2G/G31C/A5C, C2G/G31C/G8C, C2G/G31C/C12U, C2G/G31C/U14G, C2G/G31C/C27U, C2G/G31C/C29G, C2G/G31C/C30U, C2G/G31C/ G10A/C30U, C2G/G31C/G10C/C30G, C2G/G31C/G10U/C30A, C2U/G31A/G10A/C30U, C2U/G31A/G10C/C30G, C2U/G31A/G10U/C30A, C2A/G31U/G10A/C30U, C2A/G31U/G10C/C30G, C2A/G31U/G10U/C30A, C2G/G31C/G10C/C30G/C3A, C2G/G31C/G10C/C30G/A4C, C2G/G31C/G10C/C30G/A5C, C2G/G31C/G10C/C30G/G8C, C2G/G31C/G10C/C30G/C12U, C2G/G31C/G10C/C30G/U14G, C2G/G31C/G10C/C30G/C27U, C2G/G31C/G10C/C30G/C29G, C2G/G31C/G10A/C30U/U6G/C27U, C2G/G31C/G10C/C30G/U6G/C27U, C2G/G31C/G10U/C30A/U9A/U14G/C27U and C2A/G31U/G10U/C30A/U9A/U14G/C27U (which are the aptamer molecule structures in Table 1). These mutants can specifically bind fluorophore molecules, and can significantly increase fluorescence intensity of fluorophore molecules under excitation light of appropriate wavelength after binding. The nucleotide position sequence corresponds to the position shown in FIG. 1A.

[0073] The afore-mentioned mutants indicate that nucleotide substitution occurs at the corresponding sites of the aptamer nucleotide sequence of the General Formula Pepper structure. For example, C3A indicates that the cytosine nucleotide C at the third position of Pepper is substituted by adenine nucleotide A, i.e. Pepper (C3A) in Table 1; C2G/G3 1C indicates that C at the second position of Pepper is substituted by G, and G at the 31.sup.st position is substituted by C, i.e. Pepper (C2G/G31C) in Table 1.

TABLE-US-00001 TABLE 1 Aptamer structure of Pepper general formula structure after substitution of 7, 6, 5, 4, 3, 2 or one nucleotide Substitutions of Pepper general Aptamer structure general formula after substitution formula structure (underlined are bases after substitution) Pepper (C3A) N.sub.1CAAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (C3U) N.sub.1CUAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (A4U) N.sub.1CCUAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (A4G) N.sub.1CCGAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (A4C) N.sub.1CCCAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (A5G) N.sub.1CCAGUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (A5C) N.sub.1CCACUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (U6A) N.sub.1CCAAACGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (U6G) N.sub.1CCAAGCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (U6C) N.sub.1CCAACCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (C7A) N.sub.1CCAAUAGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (C7U) N.sub.1CCAAUUGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (G8C) N.sub.1CCAAUCCUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (U9A) N.sub.1CCAAUCGAGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (G11A) N.sub.1CCAAUCGUGACGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (G11U) N.sub.1CCAAUCGUGUCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (C12G) N.sub.1CCAAUCGUGGGGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (Cl2A) N.sub.1CCAAUCGUGGAGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (C12U) N.sub.1CCAAUCGUGGUGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (G13C) N.sub.1CCAAUCGUGGCCUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (U14A) N.sub.1CCAAUCGUGGCGAGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (U14G) N.sub.1CCAAUCGUGGCGGGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (C17U) N.sub.1CCAAUCGUGGCGUGUUGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (G18U) N.sub.1CCAAUCGUGGCGUGUCUN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (G18C) N.sub.1CCAAUCGUGGCGUGUCCN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32 Pepper (C27G) N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGGGCCGN.sub.32 Pepper (C27U) N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGUGCCGN.sub.32 Pepper (G28U) N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCUCCGN.sub.32 Pepper (C29G) N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGGCGN.sub.32 Pepper (C29U) N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGUCGN.sub.32 Pepper (C30A) N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCAGN.sub.32 Pepper (C30U) N.sub.1CCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCUGN.sub.32 Pepper (C2G/G31C) N.sub.1GCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCCN.sub.32 Pepper (C2U/G31A) N.sub.1UCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCAN.sub.32 Pepper (C2A/G31U) N.sub.1ACAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCUN.sub.32 Pepper (C10A/C30U) N.sub.1CCAAUCGUAGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCUGN.sub.32 Pepper (C10C/C30G) N.sub.1CCAAUCGUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGGN.sub.32 Pepper (C10U/C30A) N.sub.1CCAAUCGUUGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCAGN.sub.32 Pepper (C2G/G31/C3A) N.sub.1GAAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCCN.sub.32 Pepper (C2G/G31C/A4C) N.sub.1GCCAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCCN.sub.32 Pepper (C2G/G31C/A5C) N.sub.1GCACUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCCN.sub.32 Pepper (C2G/G31C/G8C) N.sub.1GCAAUCCUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCCN.sub.32 Pepper (C2G/G31C/C12U) N.sub.1GCAAUCGUGGUGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCCN.sub.32 Pepper (C2G/G31C/U14G) N.sub.1GCAAUCGUGGCGGGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCCN.sub.32 Pepper (C2G/G31C/C27U) N.sub.1GCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGUGCCCN.sub.32 Pepper (C2G/G31C/C29G) N.sub.1GCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGGCCN.sub.32 Pepper (C2G/G31C/C30U) N.sub.1GCAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCUCN.sub.32 Pepper (C2G/G31C/G10A/ N.sub.1GCAAUCGUAGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCUCN.sub.32 C30U) Pepper (C2G/G31C/G10C/ N.sub.1GCAAUCGUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGCN.sub.32 C30G) Pepper (C2G/G31C/G10U/ N.sub.1GCAAUCGUUGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCACN.sub.32 C30A) Pepper (C2U/G31A/G10C/ N.sub.1UCAAUCGUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGAN.sub.32 C30G) Pepper (C2U/G31A/G10U/ N.sub.1UCAAUCGUUGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCAAN.sub.32 C30A) Pepper (C2A/G31U/G10A/ N.sub.1ACAAUCGUAGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCUUN.sub.32 C30U) Pepper (C2A/G31U/G10C/ N.sub.1ACAAUCGUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGUN.sub.32 C30G) Pepper (C2A/G31U/G10U/ N.sub.1ACAAUCGUUGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCAUN.sub.32 C30A) Pepper (C2G/G31C/G10C/ N.sub.1GAAAUCGUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGCN.sub.32 C30G/C3A) Pepper (C2G/G31C/G10C/ N.sub.1GCCAUCGUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGCN.sub.32 C30G/A4C) Pepper (C2G/G31C/G10C/ N.sub.1GCACUCGUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGCN.sub.32 C30G/A5C) Pepper (C2G/G31C/G10C/ N.sub.1GCAAUCCUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGCN.sub.32 C30G/G8C) Pepper (C2G/G31C/G10C/ N.sub.1GCAAUCGUCGUGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGCN.sub.32 C30G/C12U) Pepper (C2G/G31C/G10C/ N.sub.1GCAAUCGUCGCGGGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCGCN.sub.32 C30G/U14G) Pepper (C2G/G31C/G10C/ N.sub.1GCAAUCGUCGCGGGUCGN.sub.19-N.sub.20-N.sub.21ACUGGUGCGCN.sub.32 C30G/C27U) Pepper (C2G/G31C/G10C/ N.sub.1GCAAUCGUCGCGGGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGGGCN.sub.32 C30G/C29G) Pepper (C2G/G31C/G10C/ N.sub.1GCAAUCGUCGCGGGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGGGCN.sub.32 C30G/C29G) Pepper (C2G/G31C/G10A/ N.sub.1GCAAGCGUAGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGUGCUCN.sub.32 C30U/U6G/C27U) Pepper (C2G/G31C/G10C/ N.sub.1GCAAGCGUCGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGUGCGCN.sub.32 C30G/U6G/C27U) Pepper (C2G/G31C/G10U/ N.sub.1GCAAUCGAUGCGGGUCGN.sub.19-N.sub.20-N.sub.21ACUGGUGCACN.sub.32 C30A/U9A/U14G/C27U) Pepper (C2A/G31U/G10U/ N.sub.1ACAAUCGAUGCGGGUCGN.sub.19-N.sub.20-N.sub.21ACUGGUGCAUN.sub.32 C30A/U9A/U14G/C27U)

[0074] Aptamer molecules are single-stranded nucleic acid molecules that have a secondary structure (FIG. 1) consisting of one or more base pairing regions (stem) and one or more unpaired regions (loop). The nucleic acid molecules of the present application contain a secondary structure as predicted in FIG. 1. The secondary structure comprises two loop structures, two stem structures and a stem-loop structure, wherein Stem 1 plays the role of stabilizing the entire nucleic acid aptamer molecule structure, and can be substituted by other nucleotide base pairs of any length and any composition that can form stem structures. The 5′ end or 3′ end of Stem 1 structure can be fused with any objective RNA molecule for extracellular or intracellular detection of the target RNA molecules. In a preferable embodiment of the present application, the 5′ end of the nucleic acid aptamer molecule is fused with a 5S RNA sequence (Genebank: NR 023377.1); in another preferable embodiment of the present application, the 5′ end of the nucleic acid aptamer molecule is fused with a GAPDH RNA sequence (Genebank: BC009081).

[0075] The stem-loop structure in FIG. 1 plays the role of stabilizing the entire nucleic acid aptamer molecule structure, and can be replaced with other nucleotide base pairs of any length and any composition that can form stem-loop structures. The aptamer molecules of the present application may also contain other nucleotide sequences that can be inserted into the position of N.sub.19-N.sub.20-N.sub.21, wherein the inserted nucleotide sequence replaces the stem-loop structure in FIG. 1A. The nucleotide sequence can specifically identify/bind target molecules. In the absence of target molecules, the binding of aptamer molecules and fluorophore molecules is weak, as a result of which the fluorophore molecules show weak fluorescence light; in the presence of target molecules, the binding of target molecules and the aptamer will promote binding of the aptamer and fluorophore molecules, and thus can significantly enhance the fluorescence of fluorophore molecules under excitation light of appropriate wavelength. The target molecules can be small molecules, and signal molecules on the cell surface, etc. These nucleic acid aptamers bind to specific target molecules through non-covalent binding, which mainly depends on intermolecular ionic forces, dipole force, hydrogen bonds, Van der Waals forces, positron and negative electron interactions, stacking or the combination of the above forces. The stem-loop structure can be replaced with an RNA sequence that identifies the target molecules for extracellular or intracellular detection of the target molecules. In a preferable embodiment of the present application, the stem-loop structure of aptamer molecules can bind GTP molecules; in another preferable embodiment of the present application, the stem-loop structures can bind adenosine molecules.

[0076] In a preferable embodiment of the present application, the nucleic acid aptamer molecules are preferably SEQ ID NO: 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22 or 23, or their mutation sequence which can bind fluorophore molecules so as to significantly enhance the fluorescence of fluorophore molecules under excitation light of appropriate wavelength.

[0077] The nucleic acid aptamer molecules of the present application can also comprise a fragment of nucleotide sequence that increases its stability. In a preferable embodiment of the present application, F30 scaffold RNA (Sequence 2) was adopted, and its connection mode with the nucleic acid aptamer molecules is shown in FIG. 2; in another preferable embodiment of the present invention, tRNA scaffold RNA (Sequence 3) was adopted, and its connection mode with the nucleic acid aptamer molecules is shown in FIG. 3.

[0078] The “nucleic acid aptamer molecules” in the present application are RNA molecules, or DNA-RNA hybrid molecules with part of nucleotides being replaced with deoxyribonucleotides, wherein the nucleotides can be in a form of D and L enantiomers thereof and also contain derivatives thereof, including but not limited to 2′-F, 2′-amino, 2′-methoxyl, 5′-iodo, 5′-bromine-modified polynucleotide. Nucleic acids contain various modified nucleotides.

Identity

[0079] “Identity” describes the correlation between two nucleotide sequences in the present application. The calculation of identity of two aptamer nucleotide sequences in the present application does not include N.sub.1, N.sub.19, N.sub.20, N.sub.21, N.sub.32 in Sequence (a). As for the present application, identity of two aptamer nucleotide sequences is determined by using, for instance, Needle program, preferably Needleman-Wunsch Algprithm (Needleman and Wunsch, 1970, J.Mol.Bio1.48: 443-453) executed in 3.0.0 version or later, of EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends in Genetics 16: 276-277). Optional parameters in use are gap penalty 10, gap extension penalty 0.5 and EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). Output result marked by Needle as “longest identity” (obtained by using the “-nobrief” option) serves as the percentage identity, and is calculated in a way as follows:

(Identical residue×100)/(Alignment length−Total number of gaps in alignment).

[0080] For instance, the sequence of Pepper (C3A) in Table 1 of the present application is N.sub.1CAAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32, and the sequence of Pepper (C3U) is N.sub.1CUAAUCGUGGCGUGUCGN.sub.19-N.sub.20-N.sub.21ACUGGCGCCGN.sub.32, and, according to the definition of the present application, their identity alignment should not include the nucleotide bases of N.sub.1 N.sub.19-N.sub.20-N.sub.21 and N.sub.32, so the alignment result of their sequence identity is 96.3% (the difference being one nucleotide).

Fluorophore Molecules

[0081] The “fluorophore molecules” in the present application are also called as “fluorophore” or “fluorescence molecules”. “Fluorophore molecules” in the present application are a kind of fluorophore molecules that can be conditionally activated, and show a relatively low quantum yield in the absence of nucleic acid aptamers. In specific embodiments, when a fluorophore is not bound to specific aptamers, its quantum yield is lower than 0.1, preferably lower than 0.01, and optimally lower than 0.001; when the fluorophore is bound to specific aptamers, its quantum yield will be enhanced by more than two times, preferably by more than 10 times, and optimally by more than 100 times. Fluorophore molecules are preferably water-soluble, non-toxic to cells and easy to penetrate membranes. Fluorophore of the present application can preferably enter cytoplasm or pericytoplasm through membrane or cell wall by means of active transport or passive diffusion. In the embodiments of the present application, the fluorophore can penetrate outer and inner membranes of Gram-negative bacteria, cell walls and membranes of plant cells, cell walls and membranes of fungi, membranes of animal cells, and GI and endothelial membranes of living animals.

[0082] The nucleic acid aptamer molecules in the present application can specifically bind a fluorophore and significantly increase its fluorescence value under excitation light of specific wavelength. The expressions such as “improving fluorescence signals”, “fluorescence increase”, “enhancing fluorescence intensity”, “improving fluorescence intensity” in the present application refers to the increase of the quantum yield of the fluorophore or the migration (relative to emission peaks of fluorophore itself in ethanol or aqueous solution) of the maximum emission peak of fluorescence signals under the excitation light of appropriate wavelength, or an increase of molar extinction coefficient, or two or more of the above. In a preferable embodiment of the present application, the quantum yield is increased by at least two times; in another preferable embodiment of the present application, the quantum yield is increased by at least 5 to 10 times; in another more preferable embodiment of the present application, the quantum yield is increased by at least 20 to 50 times; in another more preferable embodiment of the present application, the quantum yield is increased by at least 100 to 200 times; in another more preferable embodiment of the present application, the quantum yield is increased by at least 500 to 1,000 times; in another more preferable embodiment of the present application, the quantum yield is increased by at least 1,000 to 10,000 times; in another more preferable embodiment of the present application, the quantum yield is increased by more than 10,000 times; the light source used for exciting the fluorophore to produce fluorescence signals can be any appropriate lighting device, such as LED lamp, incandescent lamp, fluorescent lamp and laser; excitation light can be either emitted directly from these devices or obtained indirectly by means of other fluorophores, such as donor fluorophores of FERT, or donor luminophors of BRET.

Target Molecules

[0083] The target molecules of the present application can be any biomaterial or micromolecules, including but not limited to: proteins, nucleic acid (RNA or DNA), lipid molecules, carbohydrates, hormones, cytokines, chemokines, and metabolite metal ions and so on. Target molecules can be molecules associated with diseases or pathogen infection.

[0084] In the structure shown in FIG. 1, the inserted nucleotide sequence replaced the stem-loop structures of N.sub.19, N.sub.20, N.sub.21 in FIG. 1 by means of the aptamer molecules in the present application, wherein the nucleotide sequence can specifically indentify/bind target molecules. In the absence of target molecules, aptamer molecules do not or weakly bind fluorophore molecules, and thus cannot significantly improve the fluorescence of fluorophore molecules under excitation light of appropriate wavelength; in the presence of target molecules, the binding of target molecules and the nucleotide sequence will promote binding of the aptamer molecules and fluorophore molecules, and thus can significantly improve the fluorescence of fluorophore molecules under excitation light of appropriate wavelength, thereby realizing detection, imaging and quantitative analysis of target molecules.

[0085] Target molecules can also be whole cells or molecules expressed on the entire cell surface. Typical cells include but are not limited to cancer cells, bacterial cells, fungal cells and normal animal cells. The target molecules can also be virus particles. At present, many aptamers of the afore-mentioned target molecules have been identified, and can be integrated into the polyvalent nucleic acid aptamers of the present application. RNA aptamers that have been reported to bind target molecules include but are not limited to: T4 RNA polymerase aptamer, HIV reverse transcriptase aptamer, and phage R17 capsid protein aptamer.

[0086] In a preferable embodiment of the present application, the target molecule is adenosine, and the corresponding probe sequence for identifying the target molecule is SEQ ID NO: 21 (as shown in FIG. 10A); in a preferable embodiment of the present application, the target molecule is GTP, and the corresponding probe sequence for identifying the target molecule is SEQ ID NO: 22 (as shown in FIG. 10A).

Objective Nucleic Acid Molecules

[0087] “Objective nucleic acid molecules”, also called as “target nucleic acid molecules”, refer to the nucleic acid molecules to be detected, which can be either intracellular or extracellular; objective nucleic acid molecules include objective RNA molecules and objective DNA molecules. Objective nucleic acid molecules are connected with the nucleic acid aptamer molecules, and are bound to the nucleic acid aptamer molecules via fluorophore molecules so as to significantly improve the fluorescence value of fluorophore molecules under excitation light of appropriate wavelength, thereby detecting the content and distribution of objective nucleic acid molecules.

[0088] “Objective RNA molecules” in the present application include any RNA molecule, including but not limited to pre-mRNA, mRNA, pre-rRNA, rRNA, tRNA, hnRNA, snRNA, miRNA, siRNA, shRNA, sgRNA, crRNA, and long non-coding RNA of coding cells per se or exogenous expression products thereof, wherein phage capsid protein MCP identifies the binding sequence MS2RNA, phage capsid protein PCP identifies the binding sequence PP7RNA, λ phage transcription termination protein N identifies the binding sequence boxB RNA or the like. Target RNA can be fused at 5′ end or 3′ end or the position of N.sub.19-N.sub.20-N.sub.21 of the RNA aptamer molecules of the present application.

[0089] “sgRNA” in the present application refers to single guide RNA (single guide RNA, sgRNA) formed by modifying tracrRNA and crRNA in the CRISPR/Cas9 system, wherein the sequence of about 20 nt at the 5′ end of the systemtargets DNA site via base pair complementation, and promotes the Cas9 protein to induce DNA double-strand break at this site.

Concatemers of Nucleic Acid Aptamer

[0090] The nucleic acid aptamer molecules of the present application may further include concatemers that can bind multiple fluorophore molecules. The concatemers are connected by spacer sequences of appropriate length, and the number of Pepper structures in series may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. The concatermers may be in many forms. In a preferable embodiment of the present application, the series form is “Series 1”, as shown in FIG. 6A, and a preferable nucleotide sequence is SEQ ID NO: 8, 9, 10, 11 or 12, wherein 2Pepper-5 indicates Concatemer 1 with 2 Pepper-5 structures; in another preferable embodiment of the present application, the series form is “Series 2”, as shown in FIG. 6B, and a preferable nucleotide sequence is SEQ ID NO: 13, 14, 15 or 16, wherein 2xPepper-6 indicates Concatemer 2 with 2 Pepper-6 structures; in another preferable embodiment of the present application, the series form is “Series 3”, as shown in FIG. 6C, and a preferable nucleotide sequence is SEQ ID NO: 17, 18 or 19, wherein 2x2Pepper-5 indicates Concatemer 3 with 4 Pepper-5 structures; in any form, the spacer sequences between the concatemers can be changed.

[0091] The monomer aptamers of the present application refer to aptamers containing only one Pepper structure, that is, the aptamers containing two stem structures, two loop structures and one stem-loop structure (FIG. 1A).

[0092] The polymer aptamers refer to aptamers containing more than one Pepper structure, including but not limited to the aptamer composed of several series forms as shown in FIG. 6.

Aptamer-fluorophore Complex

[0093] The aptamer-fluorophore complex of the present application contains one nucleic acid aptamer molecule and one or more fluorophore molecules. In an embodiment of the present application, the molecule complex containing one nucleic acid molecule and one fluorophore molecule is F30-Pepper-2-III-3, F30-Pepper-2-III-7, F30-Pepper-2-III-6, F30-Pepper-2-III-8, F30-Pepper-2-III-4, F30-Pepper-2-III-15, F30-Pepper-2-III-18 and F30-Pepper-2-III-21.

[0094] In another embodiment of the present application, nucleic acid molecules of the concatemer and a plurality of fluorophore molecules forms a complex, for instance, a complex 8Pepper-5-8×(III-3) formed in the way of “Series 1” by F30-8Pepper-5 containing 8 aptamer units and 8 fluorophore molecules, namely, 111-3, 8Pepper-5-8×(III-7), 8Pepper-5-8×(III-6), 8Pepper-5-8×(III-8), 8Pepper-5-8×(III-4), 8Pepper-5-8×(III- 15), 8Pepper-5-8×(III- 18) and 8Pepper-5-8×(III-21). The molecule complexes may exist in vitro in the form of two separate solutions, or in the same solution, or in cells.

[0095] Nucleic Acid Aptamer Function

[0096] The aptamer function of the present application means to significantly enhance fluorescence intensity of fluorophore molecules under excitation light of appropriate wavelength, and aptamers can be detected by function detection of nucleic acid aptamer as shown in common Experimental Method (V) in the embodiments. In a preferable embodiment of the present application, the fluorescence intensity is increased by at least two times (the fluorescence intensity is detected according to the Experimental Method (V)); in another preferable embodiment of the present application, the fluorescence intensity is increased by at least 5 to 10 times; in another more preferable embodiment of the present application, the fluorescence intensity is increased by at least 20 to 50 times; in another more preferable embodiment of the present application, the fluorescence intensity is increased by at least 100 to 200 times; in another more preferable embodiment of the present application, the fluorescence intensity is increased by at least 500 to 1,000 times; in another more preferable embodiment of the present application, the fluorescence intensity is increased by at least 1,000 to 10,000 times; in another more preferable embodiment of the present application, the fluorescence intensity is increased by more than 10,000 times.

Secondary Structure of Nucleic Acid Aptamers

[0097] In the present application, the secondary structure of nucleic acid aptamers is obtained by simulation and prediction using mFold online analysis software (http://unafold.rna.albany.edu/?q=mfold). The stem structure in the secondary structure refers to a local double-strand structure formed by complementary pairing of hydrogen bonds in some regions of the single strand of nucleic acid aptamer molecules. In general, the formation of the double-strand structure does not require complementary pairing of all nucleotides in this region; in general, the stem structure will be formed when complementary pairing occurs between at least 50% of the nucleotides in a fragment of sequences N.sub.1 and N.sub.32, as well as N.sub.19 and N.sub.21 and the other fragment. If N.sub.1 and N.sub.32 are single nucleotides, the stem structure can be formed with complete complement of N.sub.1 and N.sub.32 (as shown in FIG. 1).

DNA Molecules Expressing Nucleic Acid Aptamers

[0098] The DNA molecules contain a DNA sequence which can encode the nucleic acid aptamer molecules of the present application. The DNA molecules contain a nucleotide sequence R.sub.1CCAATCGTGGCGTGTCGR.sub.19-R.sub.20-R.sub.21ACTGGCGCCGN.sub.32, as well as a nucleotide sequence with identity of at least 70%, wherein R.sub.i encodes N.sub.1 in the General Formula Pepper structure, R.sub.19 encodes N.sub.19 in the General Formula Pepper structure, R.sub.20 encodes N.sub.20 in the General Formula Pepper structure, R.sub.21 encodes N.sub.21 in the General Formula Pepper structure, and R.sub.32 encodes N.sub.32 in the General Formula Pepper structure. The DNA molecules may also contain a promoter which controls DNA transcription, wherein the promoter is in operable connection to the DNA sequence encoding the nucleic acid aptamer. In an embodiment of the present application, the DNA molecule contains an U6 promoter; in another embodiment of the present application, the DNA molecules contain a CMV promoter. The DNA molecules may further contain a DNA sequence which encodes any objective nucleic acid molecule. In an embodiment of the present application, the DNA molecules encoding the objective RNA contain a DNA sequence (sequences for embedding RNA are respectively SEQ ID No: 24, 25) encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transmembrane emp24 domain-containing protein 2 (TMED2). In another embodiment of the present application, the DNA molecules encoding the objective RNA contain a DNA sequence (sequences for embedding RNA are respectively SEQ ID No: 26, 27) encoding mCherry and TagBFP.

Promoters

[0099] “Promoters” in the present application include promoters of eukaryotic and prokaryotic cells. Promoter sequences of eukaryotic cells are totally different from those of prokaryotic cells. Generally, eukaryotic promoters cannot be identified by RNA polymerases in prokaryotic cells or mediate RNA transcription. Similarly, prokaryotic promoters cannot be identified by RNA polymerases in eukaryotic cells or mediate RNA transcription either. The strength of different promoters varies greatly (strength refers to the ability to mediate transcription). According to actual application, strong promoters can be used for achieving high level transcription. For instance, high level expression is better for labeling, and, for evaluation of transcription behavior, lower level transcription will allow cells to process transcription in a timely manner. One or more suitable promoters can be selected for different host cells. For instance, being used in Escherichia coli cells, T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosome RNA promoter, PR and PL promoters in λ phage, and other promoters, but not limited to: lacUV5 promoter, ompF promoter, bla promoter, 1pp promoter etc. Moreover, a hybrid trp-lacUV5 promoter (tac promoter) or other Escherichia coli cells obtained through recombinant or synthetic DNA technology can all be used for transcribing the RNA aptamers of the present application. Some of the operator sequences in bacteria per se can combine with promoter sequences to form inducible promoters, and specific inducers need to be added at this moment to induce transcription of DNA molecules. For instance, the expression of lac operator needs to be induced by the addition of lactose or lactose analogues (IPTG), other operators including trp, pro or the like.

[0100] As mentioned above, the regulating sequence of 5′ end of DNA molecule decoding sequence is promoters. Suitable promoters need to be selected according to the promoter intensity either to obtain RNA apatmers via in vitro transcription or to express aptamers in cultured cells or tissues. Since the expression of aptamers in vivo can be genetically manipulated, another type of promoters is inducible promoters that induce DNA transcription in response to a specific environment, such as in a specific tissue, at a specific time, and in a specific developmental stage. These different promoters can be identified by RNA polymerase I, II or III.

[0101] Promotion of transcription in eukaryotic cells also needs suitable promoters, including but not limited to β-globulin promoter, CAG promoter, GAPDH promoter, β-actin promoter, actin promoter, Cstf2t promoter, SV40 promoter, PGK promoter, MMTV promoter, adenovirus Ela promoter, CMV promoter and so on. Termination of transcription in eukaryotic cells depends on the specific cleavage site in RNA sequence. Similarly, since the transcription genes of RNA polymerase are different, transcriptional terminators also vary significantly. However, those skilled in the art can realize screening of suitable 3′ transcriptional terminator sub-regions by means of routine experimental skills.

Expression System

[0102] The “expression system” of the present application, also called as “expression vector”, contains and is integrated with DNA molecules expressing nucleic acid aptamer. The expression system of the present application can be a plasmid or a virus particle.

[0103] Recombinant virus of “expression vector” can be obtained by transfection of plasmids into viral-infected cells. Suitable vectors include but are not limited to virus vectors such as λ vector system gt11, gt WES.tB, Charon 4, and plasmid vectors include pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG399, pR290, pKC37, pKC101, pBluescript II SK+/− or KS+/− (see Stratagene cloning system), pET28 series, pACYCDuet1, pCDFDuet 1, pRSET series, pBAD series, pQE, pIH821, pGEX, pIIIEx426 RPR and so on.

[0104] A large number of host expression systems can be used for expressing the DNA molecules of the present application. Mostly, the vector system has to be compatible to the host cells in use, wherein the host vector system includes but is not limited to: transformed phage DNA, or plasmid DNA, or bacteria with cosmid DNA; yeast containing yeast vector; mammalian cells infected with a virus (e.g. adenovirus, adeno-associated virus, retrovirus); insect cells infected with a virus (e.g. baculovirus); and plant cells infected with bacteria or transformed by means of particle bombardment. Expression elements in the vectors are significantly different in strength and characteristics. Any one or more suitable transcription elements can be selected according to the host-vector system in use.

[0105] Once the constructed DNA molecules are cloned into the vector system, it will be easy to transfer them into host cells. Based on different vector or host cell systems, the method includes but is not limited to transformation, transduction, conjugation, fixation, electrical transfer or the like.

[0106] An embodiment of the present application provides expression plasmids pET28a-T7-F30-Pepper-2, pLKO.1-F30-Pepper-2 and pYES2.1-F30-Pepper-2 containing DNA molecules for encoding F30-Pepper-2 RNA. Another embodiment of the present application provides expression plasmid pLK0.1-F30-8Pepper-5 containing DNA molecules for encoding F30-8Pepper-5 RNA. Another embodiment of the present application provides expression plasmids pCDNA3.1 hygro(+)-BFP-4Pepper-7, pCDNA3.1 hygro(+)-mCherry-4Pepper-7, pCDNA3.1 hygro(+)-GAPDH-4Pepper-7 and pCDNA3.1 hygro(+)-TMED2-4Pepper-7 containing DNA molecules for encoding BFP-4Pepper-7, mCherry-4Pepper-7, GAPDH-4Pepper-7 and TMED2-4Pepper-7. Another embodiment of the present application provides expression plasmids psgRNA-Pepper-8 (loop 1), psgRNA-Pepper-8 (tetraloop) and psgRNA-Pepper-8 (loop 1 and tetraloop) containing DNA molecules for encoding sgRNA-Pepper-8 (loop 1), sgRNA-Pepper-8 (tetraloop), sgRNA-Pepper-8 (loop 1 and tetraloop). Another embodiment of the present application provides an expression plasmid pLKO.1-4Pepper-9-MS2 containing DNA molecules for encoding 4Pepper-9-MS2.

[0107] The present application further provides expression vectors integrated with DNA molecules for encoding nucleic acid aptamers, but with vacant encoding DNA sequences of objective RNA molecules, wherein the vacancy of encoding DNA sequences of objective RNA molecules allows the users to choose DNA sequences of objective RNA molecules to be detected, for instance, corresponding encoding DNA sequence of GAPDH mRNA inserts the DNA sequence into the expression vector of the present application by means of standard recombination DNA technology, and guides the obtained expression vector into the host cells of (transfection, transform, infection and so on), thereby detecting the content and distribution of objective RNA.

Host Cells

[0108] “Host cells” in the present application include but are not limited to bacteria, yeast, mammalian cells, insect cells, plant cells, zebra fish cells, fruit fly cells, and nematode cells. Host cells preferably are cultured cells in vitro or whole in vivo living tissue. Mammalian cells contained in the host cells of the present application include but are not limited to 297T, COS-7, BHK, CHO, HEK293, HeLa, H1299, stem cells of fertilized eggs, inducible totipotent stem cell, and primary cells isolated directly from mammalian tissues and so on; escherichia coli cells contained therein include but are not limited to BL21 (DE3), BL21 (DE3, Star), TOP10, Mach1, and DH5a; and yeast cells contained therein include but are not limited to BY4741, BY4742, and AH109.

Detection Array

[0109] The detection array of the present application includes one or more nucleic acid aptamer molecules of the present application, wherein the nucleic acid aptamer molecules are anchored at discrete locations on the array surface composed of solid supports, including but not limited to glass, metals, and ceramic and so on. The nucleic acid aptamer molecules of the present application can be anchored to the array surface by, but not limited to, the following methods: (1) labeling the 5′ end or 3′ end of the nucleic acid aptamer molecule with biotin, coating the array surface with streptavidin, and anchoring the nucleic acid aptamer molecule by specific binding of biotin and streptavidin; (2) identifying the binding sequence MS2 by using the phage capsid protein MCP, identifying the biding sequence PP7 by using the phage capsid protein PCP or identifying the binding sequence boxB by using the λ phase transcription terminating protein N, fusing the RNA sequence at the 5′, 3′ or stem-loop structure of the nucleic acid aptamer molecules, coating the array surface with protein MCP, PP7 or λ.sub.N protein identified and bound thereby, and anchoring the nucleic acid aptamer molecules through the specific effects of MS2 with MCP protein, PP7 with PCP protein or boxB RNA with λ.sub.N protein; (3) fusing a fragment of RNA or DNA sequence at the 5′ end or 3′ end of the nucleic acid aptamer molecules, anchoring an RNA sequence in complementary pairing with the RNA sequence segment or an DNA sequence in complementary pairing with the DNA sequence segment on the array surface, and anchoring the nucleic acid aptamer molecules on the array surface by means of the molecular hybridization principle. The detection array can be used for detecting the presence or absence of the target molecule as well as the concentration level, as a result, the nucleic acid aptamer molecules be bound with the fluorophore molecules and significantly improve the fluorescence intensity under excitation light of appropriate wavelength only with the presence of target molecules; and, within a certain range, the higher the concentration of the target molecules, the higher the fluorescence intensity.

Kit

[0110] Kit of the present application includes the nucleic acid aptamer molecules and/or the fluorophore molecules of the present application, and corresponding instructions; or includes an expression system for expressing the nucleic acid aptamer molecules and/or the fluorophore molecules, and corresponding instructions; or includes host cells expressing the aptamer molecular expression system and/or the fluorophore molecules, and corresponding instructions. The nucleic acid aptamer molecules and the fluorophore molecules in the kits respectively exist in individual solutions, or exist in the same solution.

[0111] The present application will be further elaborated in the following examples, which are merely used for giving examples, rather than limiting the scope of the present application. The examples mainly adopt conventional cloning methods of molecular biology in genetic engineering, which are well known to ordinary technicians in this field, for instance, relevant chapters from Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench by Jane Roskams et al, and Molecular Cloning—A Laboratory Manual (Third Edition, Aug. 2002, Science Press, Beijing) written by Sambrook. J, D. W. Russell and translated by Peitang HUANG et al. Based on the following examples, it easy for one skilled in the art to successfully implement the present application after slight amendment and change made according to actual situations.

[0112] In the examples, the pCDNA3.1 hygro (+) plasmid vector was purchased from Invitrogen Company, pLKO.1-puro plasmid vector was purchased from Sigma Company, pET28a plasmid vector was purchased from Novagen Company, pYES2.1 TOPO TA plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified correct via mass spectrometry by Shanghai Generay Biotech Co., Ltd. Expression plasmids constructed in the examples all went through the sequence determination accomplished by JIE LI BIOLOGY. Taq DNA polymerase used in all examples was purchased from Yeasen Biotechnology (Shanghai) Co., Ltd., PrimeSTAR DNA polymerase was purchased from TaKaRa Company, and corresponding polymerase buffers and dNTP were included during purchasing of these three kinds of polymerases. EcoRI, BamHI, BglII, HindIII, NdeI, XhoI, SacI, Xbal, SpeI and other restriction endonuclease, T4 ligase, T4 phosphorylase (T4 PNK), and T7 RNA polymerase were purchased from Fermentas company, and corresponding polymerase buffers and so on were included during purchasing. Hieff CloneTM One Step cloning kits used in the examples were purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. Unless otherwise stated, chemical reagents such as inorganic salts were all purchased from Shanghai Chemical Reagent Company of Sinopharm. Kanamycin was purchased from Ameresco Company; Amp was purchased from Ameresco Company; and 384-well and 96-well fluorescence detection blackboard was purchased from Grenier Company. DFHBI-1T and DFHO were purchased form Lucerna Company. GTP and SAM were purchased from Sigma Company.

[0113] DNA purification kits used in the examples were purchased from BBI Company (Canada), ordinary plasmid micro extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. BL21 (DE3, Star) bacterial strains were purchased from Invitrogen Company. 293T/17 cells and COS-7 cells were purchased from Cell Bank of Committee of Typical Culture Collection, Chinese Academy of Sciences. BY4741 yeast strain was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0114] Main instruments used in the examples: Synergy Neo2 Multiscan Spectrum (Bio-Tek Company of America), X-15R high speed freezing centrifuge (Beckman Company of America), Microfuge22R tabletop high speed freezing centrifuge (Beckman Company of America), PCR amplifier (Biometra Company of German), in vivo imaging system (Kodak Company of America), photometer (Wako Company of Japan), nucleic acid electrophoresis apparatus (Shenneng Bocai Company).

[0115] Meanings of abbreviations are as follows: “h” refers to hours, “min” refers to minutes, “s” refers to seconds, “d” refers to days, “pL” refers to micro-liters, “ml” refers to milliliters, “L” refers to liters, “bp” refers to base pairs, “mM” refers to millimoles, and “μM” refers to micromoles.

Commonly Used Experimental Methods and Materials in the Examples

1.Preparation of Nucleic Acid Aptamer

[0116] The cDNA corresponding to the RNA to be detected was amplified using primers containing T7 promoter, and the RNA was transcribed using T7 RNA polymerase (purchased from Fermentas) using the double-stranded cDNA as the template. 10 μL 3 M NaAc, 115 μL DEPC water were added into the 20 tit transcription system and mixed well. Then 150 μL phenol-chloroform-isopropanol mixture (phenol: chloroform: isopropano1=25:24:1) was added and shaken to mix well. The mixture was centrifugated at 10,000 rpm for 5 min, and the supernatant was taken. An equal volume of chloroform solution was added into the supernatant, shaken, mixed, centrifugated at 10,000 rpm for 5 min. The supernatant was taken and the same procedure was repeated once again. 2.5 times the volume of absolute ethanol was added into the supernatant and mixed. The mixture was placed in a refrigerator at −20 ° C. for 30 min, and centrifugated at 12000 rpm at 4° C. for 5 min. The supernatant was discarded and the precipitate was washed twice with pre-cooled 75% ethanol. After the ethanol was evaporated, an appropriate amount of screening buffer was added to resuspend the pellet. The solution was treated at 75° C. for 5 minutes and placed at room temperature for more than 10 minutes for subsequent experiments.

2.Cell Culture and Transfection

[0117] All the cells in this example were cultured in DMEM (high glucose) supplemented with 10% FBS, streptomycin and penicillin in a CO2 incubator. All cell lines were split at a confluence of 80-90%. The FuGENE®HD (purchased from Promega) was used for transfection according to the instructions.

3.Fluorescence Imaging

[0118] The main imaging experiments in the examples were carried out using a Leica SP8 confocal laser scanning microscope equipped with a HCXPL APO 63.0×1.47 OIL objective, and a HyD detector. A 488 nm laser was used to image the fluorescence of Pepepr-III-3 complex. A 405 nm and a 561 nm laser were used to image the fluorescence of BFP and mCherry, respectively. A 458 nm laser, a 458 nm laser, a 488 nm laser, a 488 nm laser, a 488 nm laser, a 561 nm laser and a 561 nm laser were used to image the fluorescence of epper-III-7, Pepper-III-6, Pepper-III-8, Pepper-III-4, Pepper-III-15, Pepper-III-18 and Pepper-III-21, respectively. A 448 nm laser was used to image the fluorescence of Broccoli-DFHB I-1T and Corn-DFHO.

4.Homologous Recombination-based construction of Recombinant Plasmids

[0119] (1) Preparation of linearized vector: select a suitable cloning site and linearize the vector by restriction enzyme-based digestion or inverse PCR amplification.

[0120] (2) PCR amplification of the insertion fragments: use the forward and reverse PCR primers containing 15-25 bp (excluding restriction site) homologous sequence of the linearized vector to amplify the template to obtain insertion fragment harboring the identical sequences corresponding to the two ends of the linearized vector.

[0121] (3) Determination of the concentrations of linearized vector and insertion fragment: dilute the linearized vector and insertion fragment, take 1 λL of the original solution and diluted solution to perform agarose gel electrophoresis, determine the concentration of the fragments by comparing the intensities of the bands to those of the DNA molecular weight standard (DNA maker).

[0122] (4) Recombination reaction: the optimal amount of vector used in the recombination reaction system is 0.03 pmol; the optimal molar ratio of linearized vector to insertion fragment is 1:2-1:3, i.e., the optimal amount of insertion fragment is 0.06-0.09 pmol.

TABLE-US-00002 Component Recombination reaction ddH.sub.2O Maximum to 20 μL 2 × Hieff Clone Enzyme Premix 10 μL (from Yeasen) linearized vector X μL insertion fragment Y μL

[0123] X and Y are calculated according to the formula to obtain the linearized vector and insert fragment. After the preparation of the system, mix the components and place at 50° C. for 20 min. When the insertion fragment >5 kb, the incubation temperature can be extended to 25 min. After the reaction is complete, it is recommended to cool the reaction tube on ice for 5 min. The reaction product can be transformed directly, or stored at −20° C. before transformation needed.

5.Characterization of the Aptamers

[0124] Preparation of Pepper or Pepper mutant aptamer according to the commonly used experimental method (1). 5 μM aptamer and 1 μM fluorophore were incubated in the detection buffer (40 mM HEPES, pH 7.4, 125 mM KCI, 5 mM MgCl2, 5% DMSO). The maximal excitation and emission peaks of the aptamer-fluorophore complexes were determined using a Synergy Neo2 multifunctional microplate reader. The fluorescence intensities of the aptamer-fluorophore complexes at the maximal excitation and emission peaks were determined using a Synergy Neo2 multifunctional microplate reader. The control sample (1 μM fluorophore without aptamer) was also measured under the same conditions. Then the ratios of fluorescence intensity were calculated. For example, the fluorescence maximal excitation and emission peaks of the complex of 5 μM F30-Pepper-2 and 1 μM III-3 fluorophore are 485 nm and 530 nm, respectively. The fluorescence intensity of the complex at 485±10 nm excitation and 530 nm±10 nm emission was 36,000, the fluorescence intensity of the control sample (1 μM III-3 fluorophore only) under the same detection conditions was 10. Thus, the activation ratio of F30-Pepper-2 aptamer on 111-3 fluorophore was 3,600-fold.

Example 1. The Secondary Structure of Pepper Aptamer

[0125] The secondary structure of Pepper aptamer was analyzed using the mFold online RNA structure analysis software. Pepper contains two stems, two loops and one stem-loop structures (FIG. 1A). For one of the stem 1 and stem-loop, the predicted secondary structure of Pepper-1 (SEQ ID NO: 1) was shown in FIG. 1B.

Example 2. Characterization of Pepper-III-3 Complex

[0126] In order to detect the spectral properties of the Pepper-III-3 complex, F30-Pepper-1 (SEQ ID NO: 2) RNA was prepared according to the commonly used experimental method (1). 1 μM III-3 with 5 μM F30-Pepper-1 was incubated. The results showed that the maximum excitation and emission of the F30-Pepper-1 -III-3 complex were 485 nm, and 530 nm, respectively (FIG. 4A). In order to detect the absorption difference between F30-Pepper- 1 -III-3 complex and III-3 fluorophore itself, the absorption of F30-Pepper-1 and III-3 complex (5 μM III-3 and 25 μM F30-Pepper-1) or 5 μM alone III-3 was detected. The results showed that the maximum absorption of F30-Pepper- 1 -III-3 complex has a large red shift relative to III-3 itself, and the maximum light absorption is 484 nm (FIG. 4B).

[0127] In order to detect whether Pepper binds to 111-3 in the form of monomer or polymer, F30-Pepper-1 was identified by the Native PAGE using the monomeric F30-Broccoli (SEQ ID NO: 4) and F30-2dBroccoli (SEQ ID NO: 5) (Filonov et al. Journal of the American Chemical Society 2014. 136: 16299-16308; Filonov et al. Chemistry & biology 2015. 22: 649-660) as the controls. The fluorescence imaging result was compared with the staining result by SYBR Gold (universal nucleic acid dye that was purchased from Invitrogen). The results showed that F30-Pepper-1 was located at about 100 bp similar to F30-Broccoli, which was consistent with its actual size of 103 bp. Therefore, the results indicate that F30-Pepper-1 binds to III-3 in the form of monomer (FIG. 4C).

[0128] In order to detect the binding constant of Pepper and III-3, 2 nM F30-Pepper-1 was incubated with different concentrations of III-3 and their fluorescence was determined. The results showed that the binding constant of F30-Pepper-1 and III-3 was 3.5 nM (FIG. 4D).

[0129] In order to test the thermostability of Pepper, 10 μM III-3 was incubated with 1 tiM F30-Pepper-1, and then was placed at different temperatures for 5 minutes before the fluorescence was determined. 10 μM DFHBI-1T was incubated with 1 μM F30-Broccoli to be as the control. The results showed that the T.sub.m of F30-Pepper-1 is 55° C., which is significantly higher than the 48° C. of F30-Broccoli (FIG. 4E), indicating that F30-Pepper-1 has better thermostability.

[0130] In order to test the stability of Pepper-III-3 complex at different pH, F30-Pepper-1-III-3 complex was incubated in different pH environment for 60 min before the fluorescence was detected. The F30-Broccoli-DFHBI-1T complex was used as the control. The results showed that the F30-Pepper-1-III-3 complex maintained a high fluorescence in the range of pH 5-9, while the fluorescence of F30-Broccoli-DFHBI-1T decreased rapidly with the decrease of pH (FIG. 4F), indicating that the F30-Pepper-1-III-3 complex has better pH stability.

[0131] In order to detect the dependence of Pepper-III-3 complex on K.sup.+, 1 μM F30-Pepper-1 and 5 μM III-3 were incubated in buffer containing 100 mM KC1 or 100 mM LiC1, respectively. The solution was treated at 70 ° C5 for 5 min and placed at room temperature for more than 15 min before the fluorescence was detected. The F30-Broccoli-DFHBI-1T complex was used as the control. Previous studies have shown that the structure of Broccoli contains a G-quadruplex, and the stability of the G-quadruplex structure is highly dependent on K.sup.+, which was consistent with the results. The fluorescence of F30-Broccoli-DFHBI-1T complex in LiC1 buffer was a few percent of that in KC1 buffer (FIG. 4G). In comparison, the fluorescence of the F30-Pepper-1 -III-3 complex did not depend on K.sup.+ (FIG. 4G), indicating that Pepper does not contain G-quadruplex on its structure.

Example 3. Fluorescence Activation of III-3 Fluorophore by Different Pepper Mutants

[0132] In order to detect the fluorescence activation of III-3 fluorophore by different Pepper mutant, the Pepper-1 sequence in F30-Pepper- 1 was mutated according to Table 1. The Pepper RNA containing different mutations were prepared according to the commonly used experimental method (1). 1 μM III -3 was incubated with 5 μM RNA and their fluorescence activation folds were determined according to the commonly used experimental method (5). The results showed that most of the F30-Pepper- 1 mutants containing single mutation retained a strong fluorescence activation of III-3 (>10 times) (Table 2). several F30-Pepper-1 mutants containing 2-7 mutations still retained strong fluorescence activation of III-3 (>100 times) (Table 3). In summary, many Pepper mutants containing single and multiple mutants still retain the ability to activate the fluorescence of III-3 fluorophore.

TABLE-US-00003 TABLE 2 Activation of III-3 by Pepper mutants with single mutation Activation Activation Activation Mutant folds Mutant folds Mutant folds F30-Pepper-1 3600 G10A 847 G18C 1028 C3G 360 G10C 856 A22U 87 C3A 2484 G11U 1512 A22G 687 C3U 2016 G11A 1526 A22C 147 A4U 1836 G11C 325 C23G 65 A4G 2160 C12G 2125 C23A 547 A4C 2772 C12A 458 G26U 532 A5G 1800 C12U 2268 C27G 1875 A5C 2628 G13U 587 C27A 186 U6A 1872 G13A 792 C27U 3158 U6G 1980 G13C 1758 G28U 873 U6C 2088 U14A 1524 G28A 42 C7A 1044 U14G 3152 G28C 145 C7U 2268 G15C 15 C29G 2145 G8C 3168 U16A 28 C29U 1437 G8A 324 U16G 125 C29A 18 U9A 2124 C17A 52 C30G 145 U9C 72 C17U 1268 C30U 2587 G10U 900 G18U 1024 C30A 1596

[0133] Note: F30-Pepper-1 in Table 2 is an aptamer with the sequence of SEQ ID NO: 2. Other aptamers are generated by mutating the corresponding nucleotide in Figure 1A within the Pepper-1 sequence in F30-Pepper-1.

TABLE-US-00004 TABLE 3 Activation of III-3 by Pepper mutants with multiple mutations Mutant Activation folds Mutant Activation folds Mutant Activation folds F30-Pepper-1 3600 U9A/C27U 478 C17U/C29U 125 C2G/G31C 3124 U9A/C27G 178 C17U/C30U 578 C2U/G31A 3256 U9A/C27A 17 C17U/C30A 268 C2A/G31U 3365 G13C/C17U 87 C17U/C27U 689 G10A/C30U 3147 U14A/C17U 256 C17U/C27G 876 G10C/C30G 3514 U14G/C17U 145 G18U/C29G 148 G10U/C30A 3254 G11U/C17U 478 G18U/C27G 547 A5G/U6C 500 C12G/C30U 445 G18U/C27U 789 A5C/C7U 368 C12A/C29G 14 G18U/C30U 698 U6A/C27G 457 G11A/C29U 456 G18U/C30A 789 U6G/C27U 880 G11C/C29U 15 A22G/C30U 157 U6C/C27U 758 C12U/C30U 568 C23A/C29G 78 C7A/C29G 245 G13U/C17U 125 G26U/C27U 89 C7U/C29G 356 G13C/C17U 87 C27G/C29G 1025 G8C/C29G 268 U14A/C17U 256 C27G/C30U 1278 U9A/C30U 248 U13G/C16U 145 C27G/C30A 785 U9A/C30A 356 C17U/C29G 457 C27U/C29G 1457 C27U/C30U 786 C29G/C30U 1203 C29U/C30A 536 G28U/C29G 99 C29G/C30A 986 U6C/G8C/C2 256 G28U/C30U 125 C29U/C30U 487 U9A/U14G/C 258 C2G/G31C/ 256 C21U/C30U/ 324 C2G/G31C/ 652 A5G/U6C C17U/C27G G10U/C30A/ U6G/C27U G10A/C30U/ 68 C2U/G31A/ 126 C2U/G31A/ 458 C17U/C27U C27G/C29G G10A/C30U/ U6G/C27U G10A/C30U/ 126 C2U/G31A/ 245 C2U/G31A/ 796 C17U/C27U C12U/C30U G10C/C30G/ U6G/C27U G10C/C30G/ 234 C2A/G31U/ 158 C2U/G31A/ 564 U9A/C27U U6G/C27U G10U/C30A/ U6G/C27U G10C/C30G/ 257 C2A/G31U/ 59 C2A/G31U/ 358 U6G/C27U C12U/C30U G10A/C30U/ U6G/C27U G10U/C30A/ 156 C2U/G31A/ 2583 C2A/G31U/ 498 C17U/C27U G10U/C30A G10C/C30G/ U6G/C27U G10U/C30A/ 87 C2A/G31U/ 3105 C2A/G31U/ 786 U6G/C27U G10A/C30U G10U/C30A/ U6G/C27U C2G/G31C/ 2365 C2A/G31U/ 3475 C2G/G31C/ 236 G10A/C30U G10C/C30G G10A/C30U/ U9A/U14G/C C2G/G31C/ 2684 C2A/G31U/ 3024 C2G/G31C/ 245 G10C/C30G G10U/C30A G10C/C30G/ U9A/U14G/C C2G/G31C/ 3125 C2G/G31C/ 758 C2G/G31C/ 198 G10U/C30A G10A/C30U/ G10U/C30A/ U6G/C27U U9A/U14G/C C2U/G31A/ 2578 C2G/G31C/ 736 C2U/G31A/ 178 G10A/C30U G10C/C30G/ G10A/C30U/ U6G/C27U U9A/U14G/C C2U/G31A/ 2986 C2G/G31C/ 652 C2U/G31A/ 196 G10C/C30G G10U/C30A/ G10C/C30G/ U6G/C27U U9A/U14G/C C2A/G31U/ 216 C2A/G31U/ 103 C2A/G31U/ 156 G10A/C30U/ G10C/C30G/ G10U/C30A/ U9A/U14G/C U9A/U14G/C U9A/U14G/C

Example 4. Base-modified Pepper's Activating Effect on III-3

[0134] In order to detect the activation of III-3 by modified Pepper, base-modified Pepper-3 was synthesized (SEQ ID NO: 6, the underlined nucleotides in GGCCCCCAAUCGUGGCGUGUCGGCCUGCUUCGGCAGGCACUGGCGCCGGGGCC contain deoxyribonucleotide bases) and Pepper-4 (SEQ ID NO: 7, the underlined nucleotides in GCCCCCCAAUCGUGGCGUGUCGGCCUGCUUCGGCAGGCACUGGCGCCGGGGGC C contain 2′-F modification) (synthesized by Shanghai GenePharma Co.,Ltd), which contain deoxyribonucleotides replacement (shaded bases in FIG. 5A) or 2′-F modification (shaded bases in FIG. 5B) in the stem-loop region of Pepper. Detection of the fluorescence activation of III-3 fluorophore by these modified Peppers was carried out according to the commonly used experimental method (5). The results showed that the modified Pepper-3 and Pepper-4 could still significantly activate the fluorescence of III-3 fluorophore (FIG. 5C).

Example 5. Pepper Tandem Arrays

[0135] In order to detect the fluorescence activation of III-3 by different Pepper arrays, Pepper is connected to form tandem arrays in different forms, including the following three types:

[0136] (1) “tandem array 1” (FIG. 6A), different copies of Pepper are connected via “head-to-tail” to generate nPepper (where n represents any copy number of Pepper). In this example, the cDNA encoding F30-2Pepper-5, F30-4Pepper-5, F30-8Pepper-5, F30-16Pepper-5 and F30-32 Pepper-2 (The sequences of the RNA aptamer are SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, respectively). After PCR amplification, the RNA aptamers were prepared according to the commonly used experimental methods (1). 0.1 μM RNA aptamer was incubated with 10 μM III-3 and fluorescence was measured according to the commonly used experimental method (5). The results showed that the fluorescence of nPepper-III-3 increased along with the increasing copy number of Pepper (n) (FIG. 6D). When n>8, with the increase of n, the fluorescence of nPepper-III-3 did not increase along with the increasing n, but was still much higher than that of Pepper-III-3 (FIG. 6D), indicating that the “ tandem array 1” can be used to increase the fluorescence intensity of Pepper-III-3 complex.

[0137] (2) “tandem array 2” (FIG. 6B), Pepper serves as a structural unit and is connected to generate nxPepper (where n represents any copy number of Pepper). In this example, the cDNAs encoding 2×Pepper-6, 4×Pepper-6, 8×Pepper-6 and 16×Pepper-6 (The sequences of the RNA aptamer are SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15 and SEQ ID NO:16 respectively) were commercial synthesized. The RNA aptamers were prepared according to the commonly used experimental methods (1). 0.1 μM RNA aptamer was incubated with 10 μM 111-3 and fluorescence was measured according to the commonly used experimental method (5). The results showed that that the fluorescence of nxPepper-III-3 also increased along with the increasing copy number of Pepper (n) (FIG. 6E), indicating that the fluorescence intensity of Pepper-III-3 complex can be increased through the form of “tandem array 2”.

[0138] (3) “tandem array 3” (FIG. 6C), which is a combination of “tandem array 1” and “ tandem array 2”. nPepper generated from “tandem array 1” serves as a structural unit and is connected to generate n1×n2Pepper according to the form of “tandem array 2” (where n1 and n2 represent any copy number of Pepper). In this example, the cDNAs encoding 2×2Pepper-5, 4×2Pepper-5, and 8×2Pepper-5 (The sequences of the RNA aptamer are SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively) were commercial synthesized. The RNA aptamers were prepared according to the commonly used experimental methods (1). 0.1 μM RNA aptamer was incubated with 20 μM III-3 and fluorescence was measured according to the commonly used experimental method (5). The results showed that the fluorescence intensity of Pepper-III-3 obtained by the form of “tandem array 3” was significantly higher than that of Pepper-III-3 (FIG. 6F), indicating that the fluorescence of Pepper-III complex can be improved by the form of “tandem array 3”.

Example 6. Characterization of III-3 Analogues

[0139] F30-Pepper-1 RNA aptamer was prepared according to the commonly used experimental methods (1), and was used to detect the properties of III-3 analogues upon Pepper binding, including the fluorescence spectrum, extinction coefficient, quantum yield, fluorescence activation fold and the binding constant (Kd). The results were shown in Table 4. From the data shown in the table, F30-Pepper-1 still could activate the fluorescence of III-3 analogues.

TABLE-US-00005 TABLE 4 The properties of F30-Pepper-1 RNA aptamer with different fluorophores Extinction Ex.sub.max Em.sub.max coefficient Quantum Activation K.sub.d (nm) (nm) (M.sup.−1 cm.sup.−1) yield fold (nM) F30-Pepper-1-III-7 443 485 49100 0.42 691 8.0 F30-Pepper-1-III-6 435 497 54700 0.57 16601 6.7 F30-Pepper-1-III-8 458 508 42500 0.30 9091 27.0 F30-Pepper-1-III-4 458 514 44100 0.45 4748 12.0 F30-Pepper-1-III-15 491 525 74100 0.70 585 3.8 F30-Pepper-1-III-3 485 530 65300 0.66 3595 3.5 F30-Pepper-1-III-18 515 599 54400 0.43 708 18.0 F30-Pepper-1-III-21 577 620 100000 0.58 12600 6.1 F30-Pepper-1-III-1 482 528 59600 0.62 1256 6.3 F30-Pepper-1-III-2 481 527 62300 0.52 1563 12.3 F30-Pepper-1-III-5 462 501 23000 0.19 697 45.3 F30-Pepper-1-III-9 455 502 39600 0.23 1360 23.2 F30-Pepper-1-III-10 459 506 40200 0.26 2365 18.3 F30-Pepper-1-III-11 453 492 42000 0.15 596 53.3 F30-Pepper-1-III-12 452 486 39600 0.16 650 63.2 F30-Pepper-1-III-13 476 512 52300 0.23 853 35.1 F30-Pepper-1-III-14 465 512 45600 0.18 453 64.3 F30-Pepper-1-III-16 492 534 78600 0.76 698 6.1 F30-Pepper-1-III-17 508 596 52300 0.51 584 23.2 F30-Pepper-1-III-19 498 586 42500 0.36 542 36.5 F30-Pepper-1-III-20 501 590 39600 0.31 365 67.3

Example 7. Labeling of Bacterial RNA Using Pepper-III-3 Complex

[0140] In order to test the effect of Pepper-III-3 in bacteria, a bacterial expression plasmid expressing F30-Pepper-1 was constructed. Primers were used to amplify F30-Pepper-1 in Example 2. Primers were used to amplify pET28a to remove the promoter and multiple cloning site regions. The obtained F30-Pepper-1 fragment and the linearized pET28a were ligated according to the commonly used experimental method (4). The obtained recombinant plasmid was named pET28a-T7-F30-Pepper-1.

[0141] The primers used to amplify the F30-Pepper-1 fragment are:

TABLE-US-00006 Forward primer (P1): 5’-GATCCCGCGAAATTAATACGACTCACTATAGGGTTGCCATGTGTA TGTGGG-3’ Reverse primer (P2): 5’-CAAGGGGTTATGCTATTGCCATGAATGATCC-3’

[0142] The primers used to amplify and linearize the pET28a vector are:

TABLE-US-00007 Forward primer (P3) 5′-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG-3′ Reverse primer (P4):  5′-ATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACG-3′

[0143] The recombinant plasmid pET28a-T7-F30-Pepper- 1 was transformed into

[0144] BL21(DE3, Star) E. coli strain. A single clone was picked and cultured at 37° C. to an OD.sub.600 around 0.2 before addition of 1 mM IPTG to induce the expression of F30-Pepper-1. 4 hours after induction, the bacteria were harvested and resuspended in PBS solution containing 2 μM III-3. BL21 (DE3, Star) E. coli transformed with pET28a empty vector was used as the control. The results showed that bacteria exhibited bright yellow-green fluorescence only when F30-Pepper-1 was expressed and in the presence of III-3 (FIG. 7), indicating that Pepper-III-3 complex can be used for fluorescent labeling of RNA in bacteria.

Example 8. Labeling of Yeast RNA Using Pepper-III-3 Complex

[0145] In order to test the effect of Pepper-III-3 in yeast, a yeast expression plasmid expressing F30-Pepper-1 was constructed. The F30-Pepper-1 DNA fragment in Example 2 was amplified using primers, and the amplified F30-Pepper-1 fragment was inserted into the pYES2.1TOPO TA vector according to the commonly used experimental method (4). The obtained recombinant plasmid was named pYES2.1-F30-Pepper-1.

[0146] The primers used to amplify the F30-Pepper-1 fragment are:

TABLE-US-00008 Forward primer (P5) 5’-GGAATATTAAGCTCGCCCTTTTGCCATGTGTATGTGGG-3’ Reverse primer (P6) 5’-TGACCTCGAAGCTCGCCCTTGTTGCCATGAATGATCC-3’

[0147] The recombinant plasmid pYES2.1-F30-Pepper-1 was transformed into BY4741 strain, and a single clone was picked and cultured at 30° C. to an OD600=0.1 before addition of 1 mM galactose to induce the expression of F30-Pepper-1. 10 hours after induction, the yeast cells were harvested and resuspended in PBS containing 2 μM III-3. The untreated BY4741 strain was used as the control. The results showed that yeast cells exhibited bright yellow-green fluorescence only when F30-Pepper-1 was expressed and in the presence of III-3 (FIG. 8), indicating that Pepper-III-3 complex can be used for RNA labeling in yeast cells.

Example 9. RNA Labeling in Mammalian Cells Using Pepper and III-3 and its Analogs.

[0148] In order to use Pepper and III3 for RNA labeling in mammalian cells, the reported Broccoli and Corn aptamer (binding to DFHBI-1T and DFHO fluorophores, respectively) were used as the controls (Filonov et al. Journal of the American Chemical Society 2014. 136: 16299-16308; Song et al. Nature chemical biology 2017. 13: 1187-1194). The mammalian cell expression plasmids expressing the RNA aptamers were constructed. The primers P7 and P8 were used to amplify F30-Pepper-1 and F30-Broccoli in Example 2, and the primers P9 and P10 were used to amplify the synthesized tRNA-Corn cDNA fragment (the RNA sequence was SEQ ID No: 20). The obtained fragments were ligated into the pLK0.1 puro vector according to the commonly used experimental method (4). The obtained expression vectors were named pLK0.1-F30-Pepper-1, pLK0.1-F30-Broccoli and pLKO.1-tRNA-Corn, which express F30-Pepper-1, F30-Broccoli and tRNA-Corn RNA, respectively.

[0149] The primers used to amplify F30-Pepper-1 and F30-Broccoli are:

TABLE-US-00009 Forward primer (P7): 5′-GGAAAGGACGAAACTCTAGATTGCCATGTGTATGTGGG-3′ Reverse primer (P8): 5′-TGTCTCGAGGTCGAGAATTCAAAAAAAGTTGCCATGAAT GATCC-3′

[0150] The primers used to amplify tRNA-Corn are:

TABLE-US-00010 Forward primer (P9): 5′-GGAAAGGACGAAACTCTAGAGCCCGGATAGCTCAGTCGG-3′ Reverse primer (P10): 5′-TGTCTCGAGGTCGAGAATTCAAAAAAATGGCGCCCGAA CAGGGACTTGCGAGCTCAGGATCCTTCCGTTTCGCACTGG-3′

[0151] In order to use Pepper and III-3 analogues for RNA labeling in mammalian cells, a mammalian expression plasmid expressing F30-8Pepper-5 was constructed. The primers P7 and P8 in this example were used to amplify the F30-8Pepper-5 fragment in Example 5, and the fragments was inserted into the pLKO.1 puro vector using the commonly used experimental method (4). The obtained expression vector was named pLK0.1-F30-8Pepper-5.

[0152] The pLKO.1-F30-8Pepper-5 plasmid was transfected into 293T/17 cells. Different III-3 analogues were added into the culture for labeling 24 hours after transfection. Fluorescence imaging was performed according to the commonly used experimental method (3). The results showed that different III-3 analogs could be used to specifically label cells expressing F30-8Pepper-5, but not the control cells without expression of F30-8Pepper-5 (FIG. 9C), indicating that Pepper and III-3 and its analogs can be used to label RNA in mammalian cells.

Example 10. Construction of Pepper-based Sensors

[0153] In order to construct Pepper-based sensors for detecting analytes, the nucleotides in the stem-loop region in the Pepper-1 (SEQ ID No: 2) structure were replaced with the aptamers that can specifically recognize and bind adenosine and guanosine (GTP), respectively. The aptamers Pepper-1 were fused by linkage with different lengths and different nucleotides (FIG. 10A). The RNA sensors were prepared according to the commonly used experimental method (1). The RNA sensors were incubated with III-3 fluorophore, and the fluorescence was measured in the presence or absence of adenosine or GTP using a multifunctional microplate reader. The results showed that, for the adenosine sensor, the activation fold could reach 88 folds when the linkage between the adenosine aptamer and Pepper-1 was linkage 2 in FIG. 10B. The corresponding sequence of the RNA sensor was SEQ ID No:21; for the GTP sensor, the activation fold could reach 10 folds when the linkage between the adenosine aptamer and Pepper-1 was linkage 3 in FIG. 10B. The corresponding sequence of the RNA sensor was SEQ ID No:22.

Example 11. Tracking of RNA Localization in Cells by Pepper

[0154] In order to use Pepper to track RNA localization in cells, chimeric RNA expression plasmids in which Pepper was fused to different RNAs were constructed. cDNA encoding 4Pepper-7 (the sequence of the RNA aptamer is SEQ ID No: 23) was synthesized and amplified using primers. The obtained fragment was inserted into the Hindlll and XhoI sites of pCDNA3.1 hygro(+) vector to generate pCDNA3.1 hygro(+)-4Pepper-7 plasmid. The GAPDH and TMED2 gene fragments (the gene sequences of GAPDH and TMED2 are shown in Genebank: BC009081 and BCO25957, respectively) were synthesized and amplified using primers. The obtained GAPDH and TMED2 gene fragments were inserted into the Nhel and Hindlll sites of pCDNA3.1 hygro(+)-4Pepper-7 vector to generate pCDNA3.1 hygro(+)-GAPDH-4Pepper-7 and pCDNA3.1 hygro(+)-TMED2-4Pepper-7 plasmids that express GAPDH-4Pepper-7 and TMED2-4Pepper-7 chimeric RNAs, respectively. The sequences of the chimeric RNAs are SEQ ID Nos: 24 and 25, respectively.

[0155] The primers used to amplify 4Pepper-7 are:

TABLE-US-00011 Forward primer (P11): 5′-TAGCGTTTAAACTTAAGCTTCCCACGGAGGATCCCCAATC-3′ Reverse primer (P12): 5′-ACGGGCCCTCTAGACTCGAGCCCACGGAGGATCCCGGCGCC-3′

[0156] The primers used to amplify GAPDH are:

TABLE-US-00012 Forward primer (P13): 5′-GGAGACCCAAGCTGGCTAGCATGGGGAAGGTGAAGGTCGG-3′ Reverse primer (P14): 5′-GGATCCTCCGTGGGAAGCTTAACCATGCTCTAGCGAGTGTTA CTCCTTGGAGGCCATGT-3′

[0157] The primers used to amplify TMED2 are:

TABLE-US-00013 Forward primer (P15): 5′-GGAGACCCAAGCTGGCTAGCATGGTGACGCTTGCTGAACT-3′ Reverse primer (P16): 5′-GGATCCTCCGTGGGAAGCTTAACCATGCTCTAGCGAGTTAAA CAACTCTCCGGACTTC-3′

[0158] After construction of above plasmids, the inserted sequences were validated by sequencing to ensure correct insertion. The plasmids were extracted using a transfection-grade plasmid extraction kit for subsequent transfection experiments.

[0159] The pCDNA3.1 hygro(+)-GAPDH-4Pepper-7 and pCDNA3.1 hygro(+)-TMED2-4Pepper-7 plasmids constructed in this example were co-transfected with pCDNA3.1 hygro(+)-BFP into COS-7 cells, respectively. 24 hours after transfection, the cells were imaged according to the fluorescence imaging method described in the commonly used experimental method (3). The imaging results showed that the fluorescence of GAPDH-4Pepper-7-III-3 was mainly localized in the cytoplasm, while the fluorescence of TMED2-4Pepper-7-III-3 exhibited endoplasmic reticulum enrichment, which was consistent with previous studies and the results obtained by fluorescent-labeled in situ hybridization (FISH) (FIG. 11). These results indicate that Pepper can be used to track RNA location.

Example 12. Detection of the Correlation Between mRNA and Protein by Pepper

[0160] In order to use Pepper to detect mRNA translation in cells, it was necessary to construct plasmids expressing different Pepper-fused mRNA. Primers were used to amplify mCherry and TagBFP gene fragments using mCherry2-N1 (Addgene: 54517) and EasyFusion T2A-H2B-TagBFP (Addgene: 113086) as the templates. The obtained gene fragments were inserted into the Nhel and HindIll sites of pCDNA3.1 hygro(+)-GAPDH-4Pepper-7 vector to generate pCDNA3.1 hygro(+)-mCherry-4Pepper-7 and pCDNA3.1 hygro(+)-TagBFP-4Pepper-7 plasmids that encode mCherry-4Pepper- 7 and TagBFP-4Pepper-7, respectively. The sequences of the chimeric RNAs are SEQ ID Nos: 26 and 27, respectively.

[0161] The primers used to amplify mCherry are:

TABLE-US-00014 Forward primer (P17): 5′-GGAGACCCAAGCTGGCTAGCATGGTGAGCAAGGGCGAGGAGG-3′ Reverse primer (P18): 5′-GGATCCTCCGTGGGAAGCTTAACCATGCTCTAGCGAGTTACTTG TACAGCTCGTCCATG-3′

[0162] The primers used to amplify TagBFP are:

TABLE-US-00015 Forward primer (P19): 5′-GGAGACCCAAGCTGGCTAGCATGAGCGAGCTGATTAAGGA-3′ Reverse primer (P20): 5′-GGATCCTCCGTGGGAAGCTTCTCCCAAACCATGCTCTA GCGAGTGTTAATTGAGCTTGTGCCCCA-3′

[0163] The recombinant plasmids pCDNA3.1 hygro(+)-BFP-4Pepper-7 and pCDNA3.1 hygro(+)-mCherry-4Pepper-7 were transfected into COS-7 cells, respectively. 24 hours after transfection, the transfected cells were labeled with 0.2 μM III-3 fluorophore. The fluorescence of mRNA (4Pepper-7-III-3) and fluorescence proteins (BFP and mCherry) was analyzed using flow cytometry. The fluorescence of mRNA and protein was fitted using the Michaelis equation to obtain R.sup.2. The results showed that the translation efficiencies of different mRNAs varied significantly different (FIG. 12), indicating that Pepper can be used to detect the correlation between mRNA and protein.

Example 13. Detection of Genomic DNA by Pepper

[0164] In order to use Pepper to detect genomic DNA, a recombinant plasmid expressing chimeric RNA of Pepper-8 and sgRNA was constructed. cDNAs encoding sgRNA-Pepper-8 (loopl), sgRNA-Pepper-8 (tetraloop) and sgRNA-Pepper-8 (loopl and tetraloop) containing centromere targeting sequence were synthesized. The encoded RNA sequences are SEQ ID No: 28, 29 and 30, respectively. Primers P21 and P22 were used to amplify above cDNAs, primers P23 and P24 were used to amplify the psgRNA plasmid (Shao et al. Nucleic acids research 2016. 44: e86). The obtained cDNAs were inserted into the linearized psgRNA vector according to the commonly used experimental method (4) to generate psgRNA-Pepper-8 (loopl), psgRNA-Pepper-8 (loop2) and psgRNA-Pepper-8 (loop1 and tetraloop), respectively (FIG. 13A). Primers P25 and P26 were used to amplify the dCas9-GFP gene fragment using pSLQ1645(dCas9-GFP) (Shao et al. Nucleic acids research 2016. 44: e86) as the template. The obtained gene fragment was inserted into the HindIII and Xhol sites of pCDNA3.1 hygro(+) vector to generate pCDNA3.1 hygro(+)-dCas9-GFP according to the commonly used experimental method (4).

[0165] The primers used to amplify the cDNA encoding Pepper and sgRNA chimeric RNA are:

TABLE-US-00016 Forward primer (P21): 5′-AAAGGACGAAACACCGAATCTGCAAGTGGATATTGTTTGAG-3′ Reverse primer (P22) 5′-TGATCTAGAAAAAAAGC ACCGACTCGGTGCCAC-3′

[0166] The primers used to amplify the psgRNA plasmid to linearize it are:

TABLE-US-00017 Forward primer (P23): 5′-TTTTTTTCTAGATCATAATCAGCCATACC-3′ Reverse primer (P24): 5′-GGTGTTTCGTCCTTTCCACAAG-3′

[0167] The primers used to amplify SpdCas9-GFP are:

TABLE-US-00018 Forward primer (P25): 5′-TAGCGTTTAAACTTAAGCTTGTGCAGGCTGGCGCCAC CATGGCCCC-3′ Reverse primer (P26): 5′-ACGGGCCCTCTAGACTCGAGTTACTTGTACAGCTCGTC CATGC-3′

[0168] pCDNA3.1 hygro(+)-dCas9-GFP and psgRNA-Pepper-6 (loop1), psgRNA-Pepper-6 (loop2) and psgRNA-Pepper-6 (loop1 and tetraloop) were co-transfected into COS-7 cells, respectively. 24 hours after transfection, the cells were labeled with 1 μM III-21 and Hoechst, and the fluorescence of Pepper-8-III-21, GFP and Hoechst were imaged using a fluorescence microscope. The imaging results showed that the fluorescence of Pepper-8-III-21 was mainly localized in the nucleus to exhibit aggregates in dots (centromeres), which was almost completely overlayed with the fluorescence of dCas9-GFP (FIG. 13B). Furthermore, the number of dots was consistent with the sgRNA alone (FIG. 13C), indicating that Pepper can be used to image genetic DNA.

Example 14. Super-Resolution Imaging of RNA by Pepper

[0169] In order to use Pepper for super-resolution imaging of RNA, a plasmid that tethered RNA to the nucleus was constructed. The 4Pepper-9-MS2 DNA fragment (SEQ ID No: 31) was synthesized and amplified using primers P27 and P28. The obtained fragment was inserted into Xbal and EcoRI sites of pLKO.1 vector to generate pLKO.1-4Pepper-9-MS2 according to the commonly used experimental method (4). Primers were used to amplify H2B gene fragment using pCS-H2B-EGFP (Addgene: 53744) as the template, primers were used to amplify tdMCP gene fragments using pHAGE-Ubc-NLS-HA-tdMCP-GFP (Addgene: 40649) as the template, primers were used to amplify tagBFP gene fragment, the overlap PCR was used to fuse tdMCP, tagBFP and H2B gene fragments to obtain the tdMCP-tagBFP-H2B fusion fragment. The obtained tdMCP-tagBFP-H2B fragment was inserted into pmTurquoise2-Golgi (Addgene: 36205) to generate pH2B-tdMCP-tagBFP that encodes a nuclear-localized tdMCP-tagBFP according to the commonly used experimental method (4).

[0170] The primers used to amplify the 4Pepper-9-MS2 DNA fragment are:

TABLE-US-00019 Forward primer (P27): 5′-GGAAAGGACGAAACTCTAGAGGGGCCCCCCAATCGTGG-3′ Reverse primer (P28): 5′-TGTCTCGAGGTCGAGAATTCAAAAAAAGGGGCCCCCGGCG CCAGTG-3′

[0171] The primers used to amplify the tdMCP gene fragment are:

TABLE-US-00020 Forward primer (P29): 5′-GAACCGTCAGATCCGCTAGCCACCATGGGCTACCCC TACGACGTGCCCG-3′ Reverse primer (P30): 5′-TCCAGAATCCGCGTAGATGCCGG-3′

[0172] The primers used to amplify the tagBFP gene fragment are:

TABLE-US-00021 Forward primer (P31): 5′-CTACGCGGATTCTGGAGGCGGTGGATCCATGAGCGA GCTGATTAAGGAG-3′ Reverse primer (P32): 5′-AGATCTATTGAGCTTGTGCCCCAGTTTG-3′

[0173] The primers used to amplify H2B gene fragment are:

TABLE-US-00022 Forward primer(P33) 5′-CAAGCTCAATAGATCTATGCCTGAACCGGCAAAATCC-3′ Reverse primer (P34): 5′-GACTGCAGAATTCGAAGCTTACTTGGAGCTGGTGT ACTTG-3′

[0174] The pLKO.1-4Pepper-9-MS2 and pH2B-tdPP7-tagBFP plasmids were co-transfected into COS-7 cells. 24 hours after transfection, the transfected cells were labeled with III-21 fluorophore, and the fluorescence distribution of Pepper-III-21 complex was imaged using a Zeiss Elyra PS.1 super-resolution fluorescence microscope using an excitation of a 561 nm long-pass filter equipped with a Zeiss Plan-Apochromat 63×(NA, 1.4) Oil DIC M27 objective and a CMOS size of 1024×1024 pixels. The imaged were processed using a ZEN 2011 Black (Zeiss) software. The imaging results showed that the cells co-transfected with pLKO.1-4Pepper-9-MS2 and pH2B-tdMCP-tagBFP showed an obvious nucleopore structure (FIG. 14). The results indicate that Pepper can be used for super-resolution imaging of RNA.

Example 15. RNA Extraction and Purification by Pepper

[0175] In order to use Pepper for RNA extraction and purification, the pCDNA3.1 hygro(+)-TagBFP-4Pepper-7 and pCDNA3.1 hygro(+)-mCherry-4Pepper-7 plasmids in Example 12 were transfected into COS-7 cells, respectively. 24 hours after transfection, the cells were collected and the total RNA of the cells was extracted using the Easyp Super Total RNA Extraction Kit (Promega). The extracted total RNA was dissolved in buffer containing 40 mM HEPES, pH 7.4, 125 mM KC1, 5 mM MgCl.sub.2. The RNA was incubated at 70° C. for 10 min and placed at room temperature for more than 30 min.

[0176] 500 uL activated Thiol Sepharose 4B (GE Healthcare) was washed twice with 500 μL PBS, and then was incubated with PBS containing 10 mM TCEP (Sigma) for 1 h at room temperature. After washing twice with 500 μL PBS, maleamide conjugated 111-3 fluorophore (Mal-III-3) was added to react for 30 min at room temperature, and was washed three times with 500 μL PBS. The treated total RNA was incubated with the treated beads at room temperature. After 30 minutes, the mixture was centrifuged at 4000 rpm for 2 minutes, and the supernatant was discarded. The agarose beads were washed with buffer containing 40 mM HEPES, pH 7.4, 125 mM KC1, and 5 mM MgCl.sub.2 for 6 times, and the supernatant was removed by centrifugation each time. The beads were resuspended with DEPC water, treated at 70° C. for 10 min, and centrifuged at 4000 rpm for 2 min. Then the supernatant was collected. 1/10 volume of NaAc, 2.5 times volume of absolute ethanol was added into the collected supernatant and placed in a refrigerator at -80° C. for 20 min. The mixture was centrifuged at 14000 rpm at 4° C. for 10 min. The precipitate was collected and the supernatant was discarded. The pre-cooled 70% ethanol solution was used to wash the precipitate. The mixture was then centrifuged at 14000 rpm for 10 min at 4° C. The precipitate was collected and the supernatant was discarded. Such procedure was repeated once again. The precipitate was placed at room temperature for 5 minutes, and then a small volume of DEPC water was used to resuspend the precipitate after the alcohol was evaporated.

[0177] The recovered RNA was validated by electrophoresis. The gel was incubated with buffer containing 5 μM III-3 in 40 mM HEPES, pH 7.4, 125 mM KC1, and 5 mM MgCl.sub.2 for 30 min, and the fluorescence of 4Pepper-III-3 in the gel was detected. The imaging results showed that two RNA bands in the gel exhibited bright Pepper-III-3 fluorescence signals, which were TagBFP-4Pepper and mCherry-4Pepper, respectively (FIG. 15), indicating that Pepper can be used as a tag for RNA extraction and purification.

Example 16: The Synthesis of Compound III-1 and its analogues Compound III-1:

[0178] ##STR00007##

[0179] To a stirring solution of p-dimethylaminobenzaidehyde (0.35 g, 2.3 mmol) and 4-cyano-benzeneacetonitrile (0.4 g, 2.8 mmol) in 20 mL methanol, 2 drops of piperidine were added. After stirring at ambient temperature for 2 h, the mixture was cool to room temperature, A large amount of precipitate was appeared, Then the precipitate was obtained by filtration and washed with cold EtOH three times. The orange solid was obtained after dried under vacuum (0.60 g, yield 95%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=3.05 (s, 6 H), 6.83 (d, J=9.2 Hz, 2 H,), 7.84-7.94 (m, 6H), 8.02 ppm (s, 1H). HRMS (ESI-TOF): Caled. For C.sub.18H.sub.16O.sub.3 [M+H].sup.+: 274.1344. Found: 274.1345.

Compound III-2

[0180] ##STR00008##

[0181] With reference to the synthetic method of compound III-1(0.34, yield 89%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=1.23 (t, J=7.60 Hz. 6H), 3.05 (t, J=7.60 Hz, 4H), 6.84 (d, J=9.2 Hz, 2 H,), 7.84-7.95 (m, 6H), 8.09 ppm (s, 1H), HRMS (ESI-TOF): Caled. For C.sub.20H.sub.20O.sub.3 [M+H].sup.+: 302.1657. Found: 302.1658.

Compound H1-3

[0182] ##STR00009##

[0183] With reference to the synthetic method of compound III-1 (0.33 g, yield 95%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=7.96 (s, 1H), 7.85 (d, J=16.0 Hz, 6H), 6.81 (d, J=8.0 Hz, 2H), 4.77 (s, 1H), 3.55 (d, J=28.0 Hz, 4H), 3.04 (s, 1H), HRMS (ESI-TOF): Caled. For C.sub.19H.sub.18N.sub.3O [M+H].sup.+: 304.1450. Found: 304.1451.

Compound III-4

[0184] ##STR00010##

[0185] To stirring solution of compound III-3 (0.61 g, 2.0 mmol) and TEA (0.25 g, 2.2 mmol) in 40 mL dried DCM. 4-tosyl chloride (0.38 g, 2.0 mrnol in 10 DCM was added slowly under 0 C. The resulting mixture was stirred under Ar atomo and was permitted to warm to room temperature. After complete the reaction, the mixture was quenched by 2 mL of water. The reaction mixture was extracted three times and the organic phase was dried with anhydrous Na.sub.2SO.sub.4 and evaporation under reduced pressure, the residue was used in the next step without purified.

[0186] To a stirring solution of the residue in 20 mL CH.sub.3CN, 1 ml MeNH2 was added under Ar atmosphere. The mixture was heated to refluxed overnight. Upon completing the reaction, the reaction mixture was cooled to room temperature and the organic liquid was removed under reduce pressure, Then the residue was dissolved in 50 mL DCM and the organic phase was washed with water and brine (2×100 ml). Upon drying over anhydrous Na.sub.2SO.sub.4 and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford orangered solid. (0.54g, 82%). .sup.1H MIR (400 MHz, CDCl.sub.3): δ=7.88 (d, J=9.0 Hz, 2H), 7.74-7.65 (m. 4H), 7.48 (s, 1H), 6.73 (d, J=9.1 Hz, 2H), 3.60-3,55 (m, 2H), 3.08 (s, 3H), 2.57-2.52 (m, 2H). 2.34 (s, 6H). HRMS (ESI-TOF): Calcd. For C.sub.2 [M+H].sup.+: 331,1923, Found: 331.1925.

Compound III-5

[0187] ##STR00011##

[0188] To a stirring solution of 3,5-difluoro-4-hydroxybenzaldehyde (0.32 g, 2.0 mmol) and 4-cyano-benzeneacetonitrile (0.35 g, 2.4 mmol) in 40 mL anhydrous EtOH, 2 drops of piperidine were added. After stirring at ambient temperature for 2 h, the mixture was cool to room temperature. A large amount of precipitate was appeared. Then the precipitate was obtained by filtration and washed with cold EtOH three times. The orange solid was obtained after dried under vacuum. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=7.80 (d, J=9.0 Hz, 2H), 7.74-7.66 (m, 4H), 7.48 (s, 1H), HRMS (ESI-TOF): Calcd. For C.sub.16H.sub.9F.sub.2N.sub.2O [M+H].sup.+: 283.0683. Found: 283.0684.

Compound Compound III-6

[0189] ##STR00012##

[0190] Wherein compound 5-(N-methyl-N-(2-hydroxyethyl)amino) pyrazine-2-carbakiehyde

##STR00013##

[0191] To a stirring solution of N-methyl-N-(2-hydroxyethyl)amino (2.6 g, 35 mmol) and 5-chloro-pyrazine-2-carbaldehyde (0.50 g, 3.5 mmol) in 20 mL dry CH.sub.3CN, K.sub.2CO.sub.3 (0.71 g, 5.3 trimol) was added in one portion. The mixture was heated to reflux under Ar atmosphere, The mixture was heated. to refluxed. for 24 h. Upon completing the reaction, the reaction mixture was cooled to room temperature and the organic liquid was removed under reduce pressure. Then the residue was dissolved in 100 mL DCM and the organic phase was washed with water and brine (2 ×100 ml). Upon drying over anhydrous Na.sub.2SO.sub.4 and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford target compound. (0.48 g, 76%). .sup.1H MIR (400 MHz, CDCl.sub.3): δ 9.88 (s, 1H), 8.62 (d, J=1.2 Hz, 1H), 8.14 (d, J=1.1 Hz, 1H), 3.92 (m, 2H), 3.88-3.83 (m, 2H), 3.28 (s, 3H). HRMS (ESI-TOF): Calcd. For C.sub.8H.sub.12N.sub.3O.sub.2 [M+H].sup.+: 182.1.Found: 182.1.

[0192] Compound III-6 was synthesized with reference to the synthetic method of compound III-1 (0.36 g, 96%), .sup.1H NMR (400 MHz, CDCI.sub.3): δ 8.39 (s, 1H), 8.30 (s, 1H), 7.80 (d, J=8.5 Hz, 2H), 7.72 (d, J=8.4 Hz, 2H), 7.51 (s, 1H), 3.93 (t, J=4.9 Hz, 2H), 3.88-3.83 (m, 2H), 3.29 (s, 3H). HRMS (ESI-TOF): Calcd. For C.sub.17H.sub.16N.sub.5O [M+H].sup.+:306.1355. Found: 306.1357.

Compound III-7

[0193] ##STR00014##

[0194] With reference to the synthetic method of compound III-4 (0.21 g , 67%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.37 (d, J=5.2 Hz, 2H), 8.06 (s, 1H) 8.00-7.85 (m, 4H). 3.77 ,t, J=6.5 Hz, 2H), 3.20 (s, 3H), 2.56 (m, 2H), 2.23 (s, 6H). HRMS (ESI-TOF); Calcd. For C.sub.19H.sub.21N.sub.6 [M+H].sup.+: 333.1828. Found: 333.1829.

Compound III-8

[0195] ##STR00015##

[0196] Wherein, compound 6-(N-methyl-N-(2-hydroxyethyl)amino) pyridine -2-carbaldehyde

##STR00016##

[0197] With reference to the synthetic method of Compound 5-(N-)methyl-N-(2-hydroxyethyl)amino) pyrazine-2-carbaldehyde: (0.45 g, 68% .sup.1H NMR (400 MHz, CDCl.sub.3): δ=9.69 (s, 1H), 8.43 (d, J=2.1 Hz, 1H), 7.86 (dd, J=9.0, 2.3 Hz, 1H), 6.56 (d, J=9.1 Hz, 1H), 3.86-3.79 (m 4H ), 3.15 (s, 3H). HRMS (ESI-TOF): Calcd. For C.sub.9H.sub.13O.sub.2N.sub.2 [M+H].sup.+: 181.1 Found: 181.1.

[0198] Compound III-8 was synthesized with reference to the synthetic method of compound III-1, (0.39 g 89%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.54 (d, J =4.0 Hz. 1H), 8.30 (dd, J=9.3, 2.5 Hz, 1H), 8.03 (s, 1H), 7.92 (d, J=8.0 Hz. 2H), 7.85 (d, J =8.0 Hz, 2H), 6.84 (d, J=8.0 Hz, 1H), 4.77 (t, J=5.4 Hz, 1H), 3.67 (t, J=5.3 Hz, 2H), 3.60 (q, J=5.4 Hz, 2H), 3.15 (s, 3H). HRMS (ESI-TOF): Calcd. For C.sub.18CC.sub.18H.sub.27N.sub.4O [M+H].sup.+: 305.1402. Found: 305.1401.

Compound III-9

[0199] ##STR00017##

[0200] With reference to the synthetic method of compound III -4, (0.31 g, 92%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.55 (d, J=4.0 Hz, 1H), 8.31 (dd, J=9.3, 2.5 Hz, 1H), 8.05 (s, 1H), 7.93 (d, 8.0 Hz, 2H), 7.84 (d, J=8.0 Hz, 2H), 6.85 (d, J-32 8.0 Hz, 1H), 4.78 (t, J=5.4 Hz, 1H), 3.67 t, J=5.3 Hz, 2H). 3.60 (q, J=5,4 Hz, 2H) 3.17 (t, J=8.0 Hz, 4H) , 1.17 (t, J=8.0 Hz, 6H). HRMS (ESI-TOF): Calcd. For C.sub.22H.sub.26N.sub.5 [M+H].sup.+: 360.2188. Found: 360.2187.

Compound III-10

[0201] ##STR00018##

[0202] Wherein, Compound 4-(N,N-dimethylamino)- pyrazine-6-carbaldehyde

##STR00019##

[0203] With reference to the synthetic method of compound III-4, (0.31 g, 49%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=9.86 (d, J=0.6 Hz, 1H), 8.17 (d, J=2.9 Hz, 1H), 7.83 (d, J=8.9 Hz. 1H), 6.94 (dd, J=8.8, 2.9 Hz, 1H), 3.10 (s, 6H1, HRMS (ESI-TOF): Calcd. For C.sub.8H.sub.11N.sub.2O [M+H]: 151.1. Found: 151.1.

[0204] Compound III-10 was synthesised with reference to the synthetic method of compound III-1, (0.36 g, 96%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=9.86 (d, J=0.6 Hz, 1H), 8.26 (s, 11-1), 8.17 (d, J=2.9 Hz, 1H), 7.83 (d, 8.9 Hz, 1H), 7.46 (m, 4H), 6.94 (dd, J=8.8, 2.9 Hz, 1H), 3.10 (s, 6H). HRMS (ESI-TOF): Calcd. For C.sub.17H.sub.15N.sub.4 [M+H].sup.+: 275.1297. Found: 275.1298.

Compound 11

Compound III-11

[0205] ##STR00020##

[0206] Wherein, compound 2-N-methyl-N-(2--hydroxyethyl)amino) pyrimidine-5-carhaidehyde

##STR00021##

[0207] With reference to the synthetic method of compound III-4, (0.42 g, 72%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=9.89 (s, 1H), 8.73 (s, 2H), 3.64 (t, J=8.9 Hz, 2H), 3.45 (t, J=8.8 Hz, 2H), 3.10 (s, 3H). HRMS (ESI-TOF): Calcd. For C.sub.8H.sub.12N.sub.3O [M+H].sup.+: 182.1. Found: 182.1.

[0208] Compound III-11 was synthesised with reference to the synthetic method of compound III-1, (0.36 g, 96%). .sup.1H NMR (400 MHz DMSO-d.sub.6): δ=8.26 (s, 1H), 8.73 (s, 2H), 7.64 (m, 4H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz. 2H1, 3.11 (s, 3H). HRMS (ESI.TOF): Calcd. For C.sub.17H.sub.16N.sub.5O [M+H].sup.+: 306.1355. Found: 306.1356.

Compound 12

Compound III-12

[0209] ##STR00022##

[0210] Wherein, compound 5-(N-methyl-N-(2-hydroxyethyl)amino) pyrimidine-2-carbaldehyde

##STR00023##

[0211] With reference to the synthetic method of compound III-4Y (0.42 g, 72%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=9.98 (s, 1H), 8.21 (s, 2H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz, 2H). 3.12 (s, 3H). HRMS (ESI-TOF): Calcd. For C.sub.8H.sub.12N.sub.3O.sub.2 [M+H].sup.+: 182.1. Found: 182.1.

4-(1-cyano-2-(5-((2-hydroxyethyll)(methyl)amino)pyrimidin-2-yl)vinyl)be nzonitrile1

[0212] ##STR00024##

[0213] With reference to the synthetic method of compound III-1, (0.5g, 89%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.21 (s, 2H), 7.99 (s, 4H), 7.64 (s, 4H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz, 2H), 3.12 (s, 3H). HRMS (ESI-TOE): Calcd. For C.sub.17H.sub.16N.sub.5O [M+H].sup.+: 306.1. Found: 306.1.

[0214] Compound III-12 was synthesised with reference to the synthetic method of compound III-4, (0.36 g, 96%). .sup.1H NMR (400 MHz. DMSO d.sub.6): δ=8.21 (s, 2H), 7.99 (s, 1H), 7.64 (s, 4H), 3.77 (t, J=6.5 Hz, 2H), 3.20 (s, 3H), 2.56 (m, 2H), 2.23 (s, 6H). HRMS (ESI-TOF): Calcd. For C.sub.19H.sub.21N.sub.6 [M+H].sup.+: 333.1828. Found: 333.1829.

Compound 13

Compound III- i 3

[0215] ##STR00025##

[0216] Wherein, 5-cyano-2- acetonitrile-pyridine:

##STR00026##

[0217] To a stirring solution of 2-(brornornethyl)-benzonitrile (0.50 g, 2.5 mmol) in 50 mL THF, 10 ml NaCN aqueous solution (2 M) was added. The mixture was reflexed for 12 h under Ar atmosphere. Upon cooling to room temperature, the reaction mixture was extracted with DCM (3×100 ml). The organic phase was washed with water and brine (2×100 ml). Upon drying over anhydrous Na.sub.2SO.sub.4 and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford target compound.(0.19 g, 56%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.78 (s, 1H), 7.95 (m, 1H), 7.56 (m, 1H), 4.01 (s, 2H). HRMS (ESI-TOF): Calcd. For C.sub.8H.sub.6 N.sub.3 [M+H].sup.+: 144.1, Found: 144.1.

[0218] Compound III-13 was synthesised with reference to the synthetic method of compound III-1, (0.45 g, 95%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.78 (s, 1H), 8.21 (s, 1H), 7.94 (m, 1H), 7.86 (d, J=8.0 Hz, 2H ), 7.57 (m, 1H), 6.80 (d, J=8.0 Hz, 2H), 3.64 (t, J=8.9 Hz, 2H), 3.44 (t, J=8.8 Hz, 2H), 3.12 (s, 3H). HRMS (ESI-TOF): Calcd. For C.sub.18H .sub.17 N.sub.4O [M+H].sup.+: 305.1402. Found: 305.1403.

Compound 14

Compound III-14

[0219] ##STR00027##

[0220] Wherein, 5-cyano-2-acetonitrile-pyrazine:

##STR00028##

[0221] To a stirring solution of 2-(5-chloropyrazin-2-yl)acetonitrile (0.32 g, 2.0 mmol) in dry 30 mL DMSO CuCN (0,93 g, 10.0 ramol) was added in one portation. The mixture was heated for 12 h under Ar atmosphere. Upon cooling to room temperature. the reaction mixture was poured into 100 mL water, then extracted with DCM (4×50 ml). The organic phase was washed with water and brine (2×100 ml). Upon drying over anhydrous Na.sub.2SO.sub.4 and evaporation under reduced pressure the residue was purified by column chromatography on silica gel to afford target compound (0.20 g, 69%). .sup.1H NMR (400 MHz, DIVISO-d.sub.6): δ=8.60 (s, 1H), 8.48 (s, 1H), 3.92 (s, 2H). HRMS (ESI-TOF): Calcd, For C.sub.7H.sub.5N.sub.4 [M+H].sup.+: 145.1. Found: 145.1.

[0222] Compound III-14 was synthesised with reference to the synthetic method of compound III-1, (0.25 g, 91%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.60 (s, 1H), 8.48 (s, 1H), 8.11 (s, 1H), 7.81 (d, J=8.2 Hz, 2H), 6.84 (d, J=8.2 Hz, 2H), 3.60 (t, J=9.2 Hz, 2H), 3.46 (t, J=9.2 Hz, 2H), 3.12 (s,3H). HRMS (ESI-TOF): Calcd. For C.sub.17H.sub.16N.sub.5O [M+H].sup.+: 306.1355. Found: 306.1354.

Compound 15

Compound III-15:1145

[0223] ##STR00029##

[0224] With reference to the synthetic method of compound III-1, (0.25 g, 91%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.22 (s, 1H), 8.00 (d, J=9.1 Hz, 1H), 7.77-7.69 (m, 1H), 7.43-7.34 (m, 1H), 6.88 (d, J=9.1 Hz, 1H), 4.81 (t, J=5.2 Hz, 1LH), 3.64-3.52 m, 3H), 3.09 (s, 1H). LR-HRMS (ESI-TOF): Calcd. For C.sub.1pH.sub.18N.sub.3O.sub.2 [M+H].sup.+: 320.1399. Found: 320.1397.

Compound III-16

[0225] ##STR00030##

[0226] With reference to the synthetic method of compound III-1, (0.29 g, 94%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.11 (2H, d, J=10.4 Hz), 7.99 (3H, dd, J=8.6, 3.0 Hz), 7.54 (1H, dd, J=8.0, 8.0 Hz), 7.44 (1H, dd, J=8.0. 8.0 Hz),6.88 (2H, d, J=9.2 Hz), 4.82 (1H, bt, t, J=5.2 Hz), 3.60 (2H, t, J=5.2 Hz,), 3,56 (2H, t, J=5.2 Hz), 3.09 (3H, s), LR-HRMS (ESI-TOF): Calcd. For C.sub.19H.sub.18N.sub.3OS [M+H].sup.+: 336.1171. Found: 336.1170.

Compound III-17

Compound III-17

[0227] ##STR00031##

[0228] Wherein, 6-(methylamino)benzo[b]thiophene-2-carbaldehyde

##STR00032##

[0229] 6-(methylamino)benzo[b]thiophene-2-carbaldehyde (0.42 g, 1.7 mmol), 40% aqueous N,N-Dimethylethylamin solution (1 g, 8.9 mmol.), Cul (13.9 mg, 0.073 mmol), K.sub.3PO.sub.4.H.sub.2O (155.4 mg, 0.73 mmol), 1 mL 33% aqueous methylamine solution and stirring bar was sealed in a screwed tube and stirred at 60°C. for 12 h. upon cooling to room temperature, the mixture was poured into 50 mL water. The organic layer was separated and the aqueous layer was extracted with DCM (3×100 ml). Combined the organic phase and dried over anhydrous Na.sub.2SO.sub.4 and evaporation under reduced pressure, the residue was purified by column chromatography on silica. gel to afford target compound (0.23 g, 68%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=9.92 (1H, s), 8.14 (1H, s), 7.82 (1H, d, J=9.1Hz), 7.18 (1H, d, J=2.1 Hz), 7.01 (1H, dd, J=9.1, 2.3 Hz), 3.05 (3H, s), HRMS (ESI-TOF): Calcd. For C.sub.10H.sub.10NOS [M+H].sup.+: 192.0, Found: 192.0.

[0230] Compound III -17 was synthesised with reference to the synthetic method of compound III-1, (0.29 g, 94%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.45 (s, 1H), 7.92 (d, J=8.6 Hz, 2H), 7.85 (d, J=8.3 Hz, 3H), 7.73 (dd, J=8.6, 3.9 Hz, 1H), 7.21 (d, J=1.9 Hz, 1H), 7.21 (d, J=1.9 Hz, 1H), 6.96 (dd, J=9.1, 2.3 Hz, 1H), 3.05 (s, 3H). HRMS (ESI-TOF): Calcd, For C.sub.19H.sub.14N.sub.3S [M+H].sup.+: 360.1171. Found: 360.1173.

Compound III-8

[0231] ##STR00033##

[0232] Wherein, 64(((2-hydroxyethyl)(methyl)arnino)benzo[b]thlophene-2-carbaldehyde:

##STR00034##

[0233] With reference to the synthetic method of compound 6-(methylamino)benzo[b]thiophene-2-carbaldehyde, (0.54 g 79%3). .sup.1NMR (400 MHz, DMSO-d.sub.6): δ=9.91 (s, 1H), 8.14(s, 1H), 7.81 (d, J=5.2 Hz, 1H), 7.17 (d, J=2.0 Hz, 1H), 7.01 (dd, J=2.0, 8.8 Hz, 1H), 4.76 (t, J=5.6 Hz, 1H), 3.58 (t, J=4.2 Hz, 2H), 3.52 (t, J=4.2 Hz, 2H), 3.04 (s, 3H) HRMS (ESI-TOF):m/z Calcd. For C.sub.12H.sub.14NO.sub.2S, [M+H].sup.+: 235.1. Found 236.1.

[0234] Compound III-18 was synthesised with reference to the synthetic method of compound III-1, (0.21 g, 95%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.45 (s, 1H), 7.92 (d, J=8.6 Hz, 2H), 7.85 (d, J=8.3 Hz, 3H), 7.73 (dd, J=8.6, 3.9 Hz, 1H), 7.21 (d, J=1.9 Hz, 1H), 7.21 (d, J=1.9 Hz, 1H), 6.96 (dd, J =9.1, 2.3 Hz, 1H), 3.69-3.57 (m, 2H), 3.52 (1, J=5.7 Hz, 2H), 3.05 (s, 3H), HRMS (ESI-TOF): Calcd. For C.sub.21H.sub.19N.sub.3OS [M+H].sup.+: 360.1171. Found: 360.1173.

Compound III-19

[0235] ##STR00035##

[0236] Wherein, 5-(N, N-dimethylamino)-thieno[3,2-b]thiophene-2-carbaldehyde:

##STR00036##

[0237] With reference to the synthetic method of compound 6-((2-hydroxyethyl)(methyl)amino)benzo[b]thiophene-2-carbaldehyde, (0.54 g, 79%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=9.66 (s, 1H), 8.05 (s, 1H), 6.30 (s, 1H), 4.88 (bt, 1H), 3.07 (s, 6H), HRMS (EST-TOF): m/z Calcd. For C.sub.9H.sub.12NOS.sub.2 [M+H].sup.+: 214.0. found 214.0.

[0238] Compound III-19 was Synthesised with reference to the synthetic method of compound III-1(0.31 g,90%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.34 (s,1H), 7.86 (d, J=8.0 Hz, 2H), 7.81 (s, 1H), 7.77 (d, J=8.0 Hz.,2H), 6.32 (s, 1H), 4.88 (t, J=4.0 Hz, 1H), 3.08 (s, 6H). HRMS (ESI-TOF): Calcd. For C.sub.18H.sub.14N.sub.3S.sub.2 [M+H].sup.30 : 336.0629. Found: 336.0630.

Compound III-20

[0239] ##STR00037##

[0240] Wherein, 5-(N,N-diethylamino)-thieno[3,2-b]thiophene-2-carbaldehyde:

##STR00038##

[0241] With reference to the synthetic method of compound 5-(N,N-dimethylamino)- thieno[3,2- b]thiophene-2-carbaldehyde, (0.44 g, 75%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=9.78 (s, 1H), 8.09 (s, 1H), 6.30 (s, 1H), 4.87 (bt, 1H), 3.27 (t, J=8.4 Hz, 4H), 1.26 (t, J=8.4 Hz, 4H). HRMS (ESI-TOF): m/z Calcd. For C.sub.9H.sub.12NOS.sub.2 [M+H].sup.+: 214.0; found 214.0.

[0242] Compound III-20 was synthesised with reference to the synthetic method of compound III-1, 0.31 g, 90%), .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.34 (s, 1H), 7.86 (d, J=8.0 Hz, 2H), 7.81 (s, 1H), 7.77 (d, J 8.0Hz, 2H), 6.32 (s, 1H), 4.88 (t, J=4.0 Hz, 1H), 3.27 (t, J=8.4 Hz, 4 H), 1.26 (t, J=8.4 Hz, 4 H). HRMS (ESI-TOF): Calcd. For C.sub.20H.sub.18N.sub.3S.sub.2 [M+H].sup.+: 364.0942. Found: 364.0943.

Compound III-21

[0243] ##STR00039##

[0244] Wherein,:

[0245] 5-(2-hydroxyethyl)(methyl)amino)-thieno[3,2-b]thiophene-2-carbaldehyde:

##STR00040##

[0246] With reference to the synthetic method of compound 6-((2-hydroxyethyl)(methylamino)benzo[b]thiophene-2-carbaldehyde (0.44 g, 75%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=9.66 (s,1H), 8.05 (s, 1H), 6.30 (s, 1H), 4.88 (bt, 1H), 3.64 (t, J=5.6 Hz. 2H), 3.44 (t, J=5.6 Hz, 2H), 3.07 (s, 3H), HRMS (ESI-TOF): m/z Calcd. For C.sub.10H.sub.12NO.sub.2S.sub.2 [M+H].sup.+: 241.0; found 242.0.

[0247] Compound III-21 was synthesised with reference to the synthetic method of compound III-1, (0.31 g, 90%). .sup.1H NMR (400 MHz. DMSO-d.sub.6): δ8.34 (s, 1H), 7.86 (d, J=8.0 Hz, 2H), 7.81 (s, 1H), 7.77 (d, J=8.0 Hz, 2H), 6.32 (s, 1H), 4.88 (t, J=4.0 Hz, 1H), 3.65 (q, J=5.5 Hz, 2H), 3.44 (t, J=5.5 Hz, 2H), :3.34 (s, 1H), 3.08 (s, 3H). HRMS (ESI-TOF): Calcd. For C.sub.19H.sub.16N.sub.3OS.sub.2 [M+H].sup.+: 366.0735. Found: 366.0736.

[0248] It will be understood that the dosages, reaction conditions, etc in the examples are approximate values unless noted otherwise, and they can be exactly changed base on the situations to obtain similar results. AB of the professional terms used in the Description, except those specially defined, have identical meanings to those known by persons skilled in the art. Ail the references referred to are incorporated into the application as a whole. The preferable embodiments are only exemplified for the illustration of the invention. Those skilled in the art can adopt similar methods or materials to obtain similar results. All the changes and modifications are within the scope of the attached claims.