Patent Application
20230002769
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.
Among all biomacromolecules, RNA exhibits the most diverse biological functions. In the Central Dogma of biology, RNA serves as the transmitter of genetic material (messenger RNA), template for protein synthesis (ribosomal RNA), and amino acid transporter (transfer RNA), constitutes a series of physiological processes, and finally realizes gene transcription and expression. In the past few decades, scientists have gradually discovered the crucial functions of RNA in a variety of vital activities, including many RNA-protein complexes, such as telomerase, splicing enzyme, ribozyme, and riboswitch or the like. In addition, some non-coding RNAs, such as short interfering RNA (siRNA), microRNA (microRNA), and long non-coding RNA (lncRNA), have played an irreplaceable role in the regulation of gene expression at the post-transcriptional level for the past few years. Real-time monitoring of RNA transport and metabolic processes in cells is critical for studying the relationship between RNA localization and gene expression, as well as cell regulation. At present, scientists have identified several mechanisms that can lead to different subcellular localizations of RNA, such as active transport, passive diffusion, and anchoring and so on. In many polar cells, especially nerve cells, the spatial specific expression of mRNA is closely related to the plasticity, learning and memory of neurons. Therefore, once these regulation processes of RNA are damaged, it will cause neuronal dysfunction and neurological diseases.
RNA fluorescence in situ hybridization technology is a method that has been widely used for a long time in the study of the level and distribution of RNA in cells, and uses molecular hybridization in the fluorescent labeling and imaging of specific RNA molecules. However, its operation is relatively complicated and contains an elution step, so it can only be used for the study of immobilized cells, i.e. dead cells, rather than for real-time monitoring of the dynamic changes of RNA in living cells. Molecular Beacon is the first developed RNA imaging technology of living cells. It is a stem-loop double-labeled oligonucleotide probe using an auto hairpin structure formed at the 5′ and 3′ ends, and, when it binds to the target RNA, the quenching effect of a quenched group labeled at one end on the fluorescence group is eliminated and the fluorescent group produces fluorescence, or the FRET of the fluorescent groups at both ends disappears. However, Molecular Beacons have disadvantages such as low fluorescence signals, difficulty in entering cells, being easily degradable, serious non-specific aggregation in the nucleus, being susceptible to RNA secondary structure and a custom oligonucleotide probes being needed for each RNA, etc., and these shortcomings limit the wide application of this technology.
The current methods of RNA imaging for living cells mainly use the MCP-FPs system, which can specifically recognize mRNA molecules fused with multiple-copies of MS2 sequences, and monitor mRNA synthesis and distribution in real time by detecting fluorescent protein signals (Ozawa et al., Nature Methods, 2007. 4: 413-419). However, due to the high background fluorescence generated by MCP-FPs not bound to mRNA molecules, the signal-to-noise ratio of this method is very low. Then, scientists added nuclear localization signals to MCP-FPs fusion protein so that GFP-MS2 not bound to mRNA molecules were localized in the nucleus, thereby reducing the non-specific fluorescence in cytoplasm to a certain extent and increasing the signal-to-noise ratio of the detection.
In addition to cellular RNA detection by means of the RNA-binding protein-fluorescent protein technology, scientists have been looking for RNA fluorescent label, which is similar to GFP, for RNA imaging. Scientists constructed a fluorophore-quencher combination, wherein the quencher cannot quench the fluorescent signal of the fluorophore when the fluorophore aptamer (Aptamer) binds to the fluorophore, and the complex formed of aptamer-fluorophore-quencher is fluorescent at this moment. When the aptamer of the fluorophore is not present, the fluorescence signal of the fluorophore will be quenched by the quencher. Based on this principle, scientists have realized the imaging of mRNA in bacteria (Arora et al., Nucleic Acids Research, 2015. 21: e144). In addition, a label named IMAGE (intracellular multi aptamer genetic) has also been developed, which is formed of two different aptamer-micromolecule complexes. When the micromolecules bind to the aptamer in RNA sequence, the fluorophore carried by two adjacent micromolecules will undergo fluorescence resonance energy transfer (FRET), and RNA in cells can be detected by the detection of changes in the fluorescence signal. However, neither of these two methods has achieved real-time monitoring of RNA in mammalian cells. In 2011, S. Jaffrey's research group obtained a nucleic acid aptamer called “Spinach”, which can specifically bind to a fluorophore (3,5-difluoro-4-hydroxybenzyli-dene imidazolinone, DFHBI) and thus significantly increases its fluorescence (Paige et al., Science, 2011. 333: 642-646; Strack et al., Nature Methods, 2013. 10: 1219-1224). “Spinach2”, which is a “Spinach” mutant, is more stable and provides an excellent tool for labeling RNA in living cells by means of genetic coding. The research group replaced a stem-loop structure in “Spinach” with a nucleic acid aptamer which can specifically bind to cell metabolites, and developed a tool that can detect cell metabolites based on Spinach-DFHBI complex (Paige et al., Science, 2012. 335: 1194). So far, this method has been successfully used to monitor and analyze the dynamic changes of RNA in bacteria, yeast and mammalian cells. Subsequently, the research group also developed a Corn-DFHO complex for the activity detection of RNA polymerase III promoter in mammalian cells (Song et al., Nature Chemical
Biology, 2017. 13: 1187-1194). However, the wide application of this method has been greatly limited by the following shortcomings: (1) the binding ability of the aptamer-fluorophore complex is weak with a dissociation constant (kd) of tens to hundreds nM; (2) the fluorescence signal of the aptamer-fluorophore complex is unstable and can be easily quenched, making its fluorescence signal undetectable (Han et al., Journal of the American Chemical Society, 2013. 135: 19033-19038); (3) so far, the spectrum is only green and yellow, lacking longer wavelengths for the imaging of RNA in living animals (Song et al., Journal of the American Chemical Society, 2014. 136: 1198-1201); (4) Corn is a dimer, which may interfere with the functions of target RNA; (5) currently, there are no other aptamer-fluorophore complexes capable of simultaneously monitoring multiple RNAs in cells.
To sum up, the currently used RNA labeling technologies have their own obvious shortcomings. MCP-FPs labeling technology has strong unbound background fluorescence and low signal-to-noise ratio. The RNA labeling technology based on the complex formed of aptamer-fluorophore-quencher currently realizes the labeling of RNA only in bacteria, and has not yet realized the labeling of RNA in mammalian cells. RNA labeling technology based on single fluorophore-nucleic acid aptamer seems to be a perfect RNA labeling technology, but it has not been widely used due to the non-ideal nature of the complex formed by the current fluorophore (DFHBI, DFHBI-1T, DFHO) and nucleic acid aptamer. Therefore, more effective fluorophore-nucleic acid aptamer complexes, which can overcome the shortcomings of the previous fluorophore-nucleic acid aptamer complexes in real-time labeling of RNA or DNA in living cells, have always been needed in the scientific and industrial communities.
The present application provides a nucleic acid aptamer molecule, a DNA molecule encoding the nucleic acid aptamer molecule, a complex of nucleic acid aptamer molecules and fluorophore molecules, and uses of the complex.
Provided by the present application
The present application provides a nucleic acid aptamer molecule containing following nucleotide sequences (a), (b) or (c):
(a): a nucleotide sequence N1CCAAUCGUGGCGUGUCGN19-N20-N21ACUGGCGCCGN32 (called as General Formula Pepper structure), wherein N1, N19, N20, N21 and N32 represent nucleotide fragments greater than or equal to 1 in length, and at least one base pair in N1 and N32 nucleotide sequences forms a complementary pair, and at least one base pair in N19 and N21 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 N1, N19, N20, N21 and N32 in the nucleotide sequence defined by (a), with substitution, missing and/or addition of one or several nucleotides, and having an aptamer function.
In some embodiments, the nucleotide sequence (b) has an identity of at least 75%, 76%, 78%, 80%, 82%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% to the General Formula Pepper structure nucleotide sequence defined by the nucleotide sequence (a). In some embodiments, the nucleotide sequence (c) is nucleic acid aptamer molecules obtained with substitution, missing and/or addition of 10, 9, 8, 7, 6, 5, 4, 3, 2 or one nucleotide at a position in the General Formula Pepper structure nucleotide sequence defined by the nucleotide sequence (a) and not including N1, N19, N20, N21 and N32. In some embodiments, the nucleotide sequence (c) is nucleic acid aptamer molecules obtained with substitution, missing and/or addition of 7, 6, 5, 4, 3, 2 or one nucleotide at a position in the General Formula Pepper structure nucleotide sequence defined by the nucleotide sequence (a) and not including N1, N19, N20, N21 and N32.
In some embodiments, when N1 and N32 in the nucleotide sequence (a) form a complementary pair, a direction of N1 nucleotide sequence is 5′-3′, and a direction of N32 nucleotide sequence is 3′-5′; and when N19 and N21 form a complementary pair, a direction of N19 nucleotide sequence is 5′-3′, and a direction of N21 nucleotide sequence is 3′-5′.
In some embodiments, when at least one fragment of N1 and N32 in the nucleotide sequence (a) is greater than or equal to 5 nucleotide bases in length, at least two base pairs in N1 and N32 nucleotide sequences form complementary pairs; when at least one fragment of N19 and N21 is greater than or equal to 5 nucleotide bases in length, at least two base pairs in N19 and N21 nucleotide sequences form complementary pairs.
In some embodiments, the nucleotide substitution in the General Formula Pepper structure is selected from one of the following groups: C3A, C3U, A4U, A4G, A4C, ASG, ASC, 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, GlOU/C30A, C2G/G31C/C3A, C2G/G31C/A4C, C2G/G31C/ASC, 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.
In some embodiments, the nucleotide substitution in the General Formula Pepper structure is selected from one of the following groups: C3A, C3U, A4C, A5C, C7U, G8C, U9A, C12G, C12U, U14G, C27U, C29G, 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/C 12U, C2G/G31C/G10C/C30G/U14G, C2G/G31C/G10C/C30G/C27U, C2G/G31C/G10C/C30G/C29G, C2G/G31C/G10A/C30U/U6G/C27U and C2G/G31C/G10C/C30G/U6G/C27U.
In some embodiments, the nucleotide substitution in the General Formula Pepper structure is selected from one of the following groups: C3A, C3U, A4C, A5C, C7U, G8C, U9A, C 12G, C 12U, U14G, C27U, C29G, 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/C3OG and C2A/G31U/G10U/C30A.
In some embodiments, nucleotide sequences at N1 and N32 in the nucleotide sequence (a) are F30 or tRNA scaffold RNA sequences.
In some embodiments, the nucleic acid aptamer molecules are RNA molecules or RNA base-modified molecules.
In some embodiments, the nucleic acid aptamer molecules are DNA-RNA hybrid molecules or DNA-RNA base-modified molecules.
In some embodiments, N19-N20-N21 in the nucleotide sequence (a) contains a nucleotide sequence capable of identifying target molecules.
In some embodiments, the target molecules include but are not limited to: proteins, nucleic acid, lipid molecules, carbohydrates, hormones, cytokines, chemokines, and metabolite metal ions.
In some embodiments, N19-N20-N21 in the nucleotide sequence (a) is a nucleotide sequence capable of identifying GTP and adenosine molecules.
In some embodiments, 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.
In some embodiments, the nucleic acid aptamer molecules may further include 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.
In some embodiments, the nucleic acid aptamer molecules have 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.
The present application further provides a complex of nucleic acid aptamer molecules and fluorophore molecules, wherein the nucleic acid aptamer molecules are any kind of the afore-mentioned nucleic acid aptamer molecules, and the fluorophore molecules have a structure shown in Formula (I) below:
wherein: D- is X1O— or N(X2)(X3)—; X1, X2, X3 are respectively and independently selected from hydrogen, straight or branched alkyl and modified alkyl with 1 to 10 carbons, and X2 and X3 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; Ar1 and Ar2 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 Ar1, and Ar2 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—, —NO2, —CN, —S—, —SO2—, —(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.
In some embodiments, the modified alkyl contains at least one group selected from —OH—, —O—, glycol unit, monosaccharide unit, disaccharide unit, —O—CO—, —NH—CO—, —SO2—O—, —SO—, Me2N—, Et2N—, —S—S—, —CH═CH—, F, Cl, Br, I, —NO2 and cyano group.
In some embodiments, aromatic rings contained in the fluorophore molecules are selected from structures represented by the following formulae (□-1) to (□-15):
In some embodiments, fluorophore molecules are selected from the following compounds represented by the following formulae:
In some embodiments, the fluorophore molecules in the complex are selected from III-1, III-2, III-3, III-4, III-5, III-6, III-7, III-8, III-9, III-10, III-11, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III-19, III-20 and III-21.
In some embodiments, 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.
The present application further provides any of the afore-mentioned complexes used for detection or labeling of target nucleic acid molecules in vitro or in vivo.
The present application further provides any of the afore-mentioned complexes used for detection or labeling of extracellular or intracellular target molecules.
The present application further provides any of the afore-mentioned complexes used for imaging of genomic DNA.
The present application further provides any of the afore-mentioned complexes used for detecting a relationship between mRNA and protein content in cells.
The present application further provides a DNA molecule which transcribes any of the nucleic acid aptamer molecules.
The present application further provides an expression vector containing the DNA molecules.
The present application further provides a host cell containing the expression vector.
The present application further provides a kit containing any of the nucleic acid aptamer molecules and/or any of the expression vectors and/or any of the host cells and/or any of the complexes.
The present application further provides a method of detecting target molecules, including:
adding any of the complexes to a solution containing the target molecules;
exciting the complex with light of appropriate wavelength; and
detecting fluorescence of the complex.
The present application further provides a method of detecting genome DNA, including imaging genome DNA with any of the complexes.
The present application further provides a method of extracting and purifying RNA, including extracting and purifying RNA with any of the complexes.
The inventor designed brand new nucleic acid aptamer molecules, and synthesized brand new fluorophore molecules, so as to form a brand new fluorophore-nucleic acid aptamer complex. After binding of the aptamer molecules to the fluorophore molecules, the fluorescence intensity of the fluorophore molecules can be significantly increased under the excitation light of appropriate wavelength. They overcome the shortcomings of previous fluorophore-nucleic acid aptamer complexes, and can be used for effective real-time RNA/DNA labeling in living cells. The nucleic acid aptamer of the present application have a strong affinity for fluorophore molecules, and shows different fluorescence spectra and fine light and temperature stability. These nucleic acid aptamer-fluorophore molecule complexes can be used for real-time labeling and imaging of RNA/DNA in prokaryotic and eukaryotic cells, detecting protein-RNA interactions, exploring the relationship between mRNA content and protein in cells, or as labels for RNA extraction and purification.
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.
Following are detailed explanations of some terms used in the present application.
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 N1CCAAUCGUGGCGUGUCGN19-N20-N21ACUGGCGCCGN32 (corresponding to the General Formula Pepper structure in
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
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 31st position is substituted by C, i.e. Pepper (C2G/G31C) in Table 1.
Aptamer molecules are single-stranded nucleic acid molecules that have a secondary structure (
The stem-loop structure in
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.
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
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” 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 N1, N19, N20, N21, N32 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).
For instance, the sequence of Pepper (C3A) in Table 1 of the present application is N1CAAAUCGUGGCGUGUCGN19-N20-N21ACUGGCGCCGN32, and the sequence of Pepper (C3U) is N1CUAAUCGUGGCGUGUCGN19-N20-N21ACUGGCGCCGN32, and, according to the definition of the present application, their identity alignment should not include the nucleotide bases of N1 N19-N20-N21 and N32, so the alignment result of their sequence identity is 96.3% (the difference being one nucleotide).
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.
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.
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.
In the structure shown in
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.
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
“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.
“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 N19-N20-N21 of the RNA aptamer molecules of the present application.
“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.
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
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 (
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
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.
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.
Nucleic Acid Aptamer Function
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.
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 N1 and N32, as well as N19 and N21 and the other fragment. If N1 and N32 are single nucleotides, the stem structure can be formed with complete complement of N1 and N32 (as shown in
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 R1CCAATCGTGGCGTGTCGR19-R20-R21ACTGGCGCCGN32, as well as a nucleotide sequence with identity of at least 70%, wherein Ri encodes N1 in the General Formula Pepper structure, R19 encodes N19 in the General Formula Pepper structure, R20 encodes N20 in the General Formula Pepper structure, R21 encodes N21 in the General Formula Pepper structure, and R32 encodes N32 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” 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.
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.
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.
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.
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.
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.
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.
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.
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” 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.
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 λN 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 λN 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 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.
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.
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.
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.
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).
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.
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.
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.
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.
(1) Preparation of linearized vector: select a suitable cloning site and linearize the vector by restriction enzyme-based digestion or inverse PCR amplification.
(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.
(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).
(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.
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.
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.
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 (
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 (
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 (
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 (
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 Tm of F30-Pepper-1 is 55° C., which is significantly higher than the 48° C. of F30-Broccoli (
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 (
In order to detect the dependence of Pepper-III-3 complex on K+, 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+, 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 (
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.
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
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
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:
(1) “tandem array 1” (
(2) “tandem array 2” (
(3) “tandem array 3” (
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.
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.
The primers used to amplify the F30-Pepper-1 fragment are:
The primers used to amplify and linearize the pET28a vector are:
The recombinant plasmid pET28a-T7-F30-Pepper- 1 was transformed into
BL21(DE3, Star) E. coli strain. A single clone was picked and cultured at 37° C. to an OD600 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 (
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.
The primers used to amplify the F30-Pepper-1 fragment are:
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 (
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.
The primers used to amplify F30-Pepper-1 and F30-Broccoli are:
The primers used to amplify tRNA-Corn are:
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.
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 (
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 (
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.
The primers used to amplify 4Pepper-7 are:
The primers used to amplify GAPDH are:
The primers used to amplify TMED2 are:
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.
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) (
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.
The primers used to amplify mCherry are:
The primers used to amplify TagBFP are:
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 R2. The results showed that the translation efficiencies of different mRNAs varied significantly different (
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 (
The primers used to amplify the cDNA encoding Pepper and sgRNA chimeric RNA are:
The primers used to amplify the psgRNA plasmid to linearize it are:
The primers used to amplify SpdCas9-GFP are:
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 (
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).
The primers used to amplify the 4Pepper-9-MS2 DNA fragment are:
The primers used to amplify the tdMCP gene fragment are:
The primers used to amplify the tagBFP gene fragment are:
The primers used to amplify H2B gene fragment are:
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 (
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 MgCl2. The RNA was incubated at 70° C. for 10 min and placed at room temperature for more than 30 min.
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 MgCl2 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.
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 MgCl2 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 (
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%). 1H NMR (400 MHz, DMSO-d6): 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 C18H16O3 [M+H]+: 274.1344. Found: 274.1345.
With reference to the synthetic method of compound III-1(0.34, yield 89%). 1H NMR (400 MHz, DMSO-d6): 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 C20H20O3 [M+H]+: 302.1657. Found: 302.1658.
With reference to the synthetic method of compound III-1 (0.33 g, yield 95%). 1H NMR (400 MHz, DMSO-d6): 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 C19H18N3O [M+H]+: 304.1450. Found: 304.1451.
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 Na2SO4 and evaporation under reduced pressure, the residue was used in the next step without purified.
To a stirring solution of the residue in 20 mL CH3CN, 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 Na2SO4 and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford orangered solid. (0.54g, 82%). 1H MIR (400 MHz, CDCl3): 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 C2 [M+H]+: 331,1923, Found: 331.1925.
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. 1H NMR (400 MHz, CDCl3): δ=7.80 (d, J=9.0 Hz, 2H), 7.74-7.66 (m, 4H), 7.48 (s, 1H), HRMS (ESI-TOF): Calcd. For C16H9F2N2O [M+H]+: 283.0683. Found: 283.0684.
Wherein compound 5-(N-methyl-N-(2-hydroxyethyl)amino) pyrazine-2-carbakiehyde
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 CH3CN, K2CO3 (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 Na2SO4 and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford target compound. (0.48 g, 76%). 1H MIR (400 MHz, CDCl3): 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 C8H12N3O2 [M+H]+: 182.1.Found: 182.1.
Compound III-6 was synthesized with reference to the synthetic method of compound III-1 (0.36 g, 96%), 1H NMR (400 MHz, CDCI3): 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 C17H16N5O [M+H]+:306.1355. Found: 306.1357.
With reference to the synthetic method of compound III-4 (0.21 g , 67%). 1H NMR (400 MHz, DMSO-d6): 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 C19H21N6 [M+H]+: 333.1828. Found: 333.1829.
Wherein, compound 6-(N-methyl-N-(2-hydroxyethyl)amino) pyridine -2-carbaldehyde
With reference to the synthetic method of Compound 5-(N-)methyl-N-(2-hydroxyethyl)amino) pyrazine-2-carbaldehyde: (0.45 g, 68% 1H NMR (400 MHz, CDCl3): 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 C9H13O2N2 [M+H]+: 181.1 Found: 181.1.
Compound III-8 was synthesized with reference to the synthetic method of compound III-1, (0.39 g 89%). 1H NMR (400 MHz, DMSO-d6): 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 C18CC18H27N4O [M+H]+: 305.1402. Found: 305.1401.
With reference to the synthetic method of compound III -4, (0.31 g, 92%). 1H NMR (400 MHz, DMSO-d6): 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 C22H26N5 [M+H]+: 360.2188. Found: 360.2187.
Wherein, Compound 4-(N,N-dimethylamino)- pyrazine-6-carbaldehyde
With reference to the synthetic method of compound III-4, (0.31 g, 49%). 1H NMR (400 MHz, DMSO-d6): 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 C8H11N2O [M+H]: 151.1. Found: 151.1.
Compound III-10 was synthesised with reference to the synthetic method of compound III-1, (0.36 g, 96%). 1H NMR (400 MHz, DMSO-d6): 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 C17H15N4 [M+H]+: 275.1297. Found: 275.1298.
Wherein, compound 2-N-methyl-N-(2--hydroxyethyl)amino) pyrimidine-5-carhaidehyde
With reference to the synthetic method of compound III-4, (0.42 g, 72%). 1H NMR (400 MHz, DMSO-d6): 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 C8H12N3O [M+H]+: 182.1. Found: 182.1.
Compound III-11 was synthesised with reference to the synthetic method of compound III-1, (0.36 g, 96%). 1H NMR (400 MHz DMSO-d6): 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 C17H16N5O [M+H]+: 306.1355. Found: 306.1356.
Wherein, compound 5-(N-methyl-N-(2-hydroxyethyl)amino) pyrimidine-2-carbaldehyde
With reference to the synthetic method of compound III-4Y (0.42 g, 72%). 1H NMR (400 MHz, DMSO-d6): 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 C8H12N3O2 [M+H]+: 182.1. Found: 182.1.
With reference to the synthetic method of compound III-1, (0.5g, 89%). 1H NMR (400 MHz, DMSO-d6): 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 C17H16N5O [M+H]+: 306.1. Found: 306.1.
Compound III-12 was synthesised with reference to the synthetic method of compound III-4, (0.36 g, 96%). 1H NMR (400 MHz. DMSO d6): 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 C19H21N6 [M+H]+: 333.1828. Found: 333.1829.
Wherein, 5-cyano-2- acetonitrile-pyridine:
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 Na2SO4 and evaporation under reduced pressure, the residue was purified by column chromatography on silica gel to afford target compound.(0.19 g, 56%). 1H NMR (400 MHz, DMSO-d6): δ=8.78 (s, 1H), 7.95 (m, 1H), 7.56 (m, 1H), 4.01 (s, 2H). HRMS (ESI-TOF): Calcd. For C8H6 N3 [M+H]+: 144.1, Found: 144.1.
Compound III-13 was synthesised with reference to the synthetic method of compound III-1, (0.45 g, 95%). 1H NMR (400 MHz, DMSO-d6): 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 C18H 17 N4O [M+H]+: 305.1402. Found: 305.1403.
Wherein, 5-cyano-2-acetonitrile-pyrazine:
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 Na2SO4 and evaporation under reduced pressure the residue was purified by column chromatography on silica gel to afford target compound (0.20 g, 69%). 1H NMR (400 MHz, DIVISO-d6): δ=8.60 (s, 1H), 8.48 (s, 1H), 3.92 (s, 2H). HRMS (ESI-TOF): Calcd, For C7H5N4 [M+H]+: 145.1. Found: 145.1.
Compound III-14 was synthesised with reference to the synthetic method of compound III-1, (0.25 g, 91%). 1H NMR (400 MHz, DMSO-d6): 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 C17H16N5O [M+H]+: 306.1355. Found: 306.1354.
With reference to the synthetic method of compound III-1, (0.25 g, 91%). 1H NMR (400 MHz, DMSO-d6): 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 C1pH18N3O2 [M+H]+: 320.1399. Found: 320.1397.
With reference to the synthetic method of compound III-1, (0.29 g, 94%). 1H NMR (400 MHz, DMSO-d6): 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 C19H18N3OS [M+H]+: 336.1171. Found: 336.1170.
Wherein, 6-(methylamino)benzo[b]thiophene-2-carbaldehyde
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), K3PO4.H2O (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 Na2SO4 and evaporation under reduced pressure, the residue was purified by column chromatography on silica. gel to afford target compound (0.23 g, 68%). 1H NMR (400 MHz, DMSO-d6): 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 C10H10NOS [M+H]+: 192.0, Found: 192.0.
Compound III -17 was synthesised with reference to the synthetic method of compound III-1, (0.29 g, 94%). 1H NMR (400 MHz, DMSO-d6): 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 C19H14N3S [M+H]+: 360.1171. Found: 360.1173.
Wherein, 64(((2-hydroxyethyl)(methyl)arnino)benzo[b]thlophene-2-carbaldehyde:
With reference to the synthetic method of compound 6-(methylamino)benzo[b]thiophene-2-carbaldehyde, (0.54 g 79%3). 1NMR (400 MHz, DMSO-d6): 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 C12H14NO2S, [M+H]+: 235.1. Found 236.1.
Compound III-18 was synthesised with reference to the synthetic method of compound III-1, (0.21 g, 95%). 1H NMR (400 MHz, DMSO-d6): 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 C21H19N3OS [M+H]+: 360.1171. Found: 360.1173.
Wherein, 5-(N, N-dimethylamino)-thieno[3,2-b]thiophene-2-carbaldehyde:
With reference to the synthetic method of compound 6-((2-hydroxyethyl)(methyl)amino)benzo[b]thiophene-2-carbaldehyde, (0.54 g, 79%). 1H NMR (400 MHz, DMSO-d6): 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 C9H12NOS2 [M+H]+: 214.0. found 214.0.
Compound III-19 was Synthesised with reference to the synthetic method of compound III-1(0.31 g,90%). 1H NMR (400 MHz, DMSO-d6): 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 C18H14N3S2 [M+H]30 : 336.0629. Found: 336.0630.
Wherein, 5-(N,N-diethylamino)-thieno[3,2-b]thiophene-2-carbaldehyde:
With reference to the synthetic method of compound 5-(N,N-dimethylamino)- thieno[3,2- b]thiophene-2-carbaldehyde, (0.44 g, 75%). 1H NMR (400 MHz, DMSO-d6): 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 C9H12NOS2 [M+H]+: 214.0; found 214.0.
Compound III-20 was synthesised with reference to the synthetic method of compound III-1, 0.31 g, 90%), 1H NMR (400 MHz, DMSO-d6): 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 C20H18N3S2 [M+H]+: 364.0942. Found: 364.0943.
Wherein,:
5-(2-hydroxyethyl)(methyl)amino)-thieno[3,2-b]thiophene-2-carbaldehyde:
With reference to the synthetic method of compound 6-((2-hydroxyethyl)(methylamino)benzo[b]thiophene-2-carbaldehyde (0.44 g, 75%). 1H NMR (400 MHz, DMSO-d6): 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 C10H12NO2S2 [M+H]+: 241.0; found 242.0.
Compound III-21 was synthesised with reference to the synthetic method of compound III-1, (0.31 g, 90%). 1H NMR (400 MHz. DMSO-d6): 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 C19H16N3OS2 [M+H]+: 366.0735. Found: 366.0736.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910348701.7 | Apr 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/087415 | 4/28/2020 | WO |
