WO2021004379A1 - 一种荧光标记核酸及其合成方法 - Google Patents

一种荧光标记核酸及其合成方法 Download PDF

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WO2021004379A1
WO2021004379A1 PCT/CN2020/100030 CN2020100030W WO2021004379A1 WO 2021004379 A1 WO2021004379 A1 WO 2021004379A1 CN 2020100030 W CN2020100030 W CN 2020100030W WO 2021004379 A1 WO2021004379 A1 WO 2021004379A1
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dye
nucleic acid
base
fluorescently labeled
formula
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French (fr)
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陈锦森
李竑
吴政宪
王�锋
杨涛
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南京金斯瑞生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the invention relates to the field of nucleic acid fluorescent labeling, in particular to fluorescently labeled primers and probes, in particular to a broad-band DNA fluorescent probe with a visible red-purple region synthesized by the principle of fluorescence resonance energy transfer and a method.
  • a primer (oligonucleotide) probe refers to a short-stranded single-stranded DNA or RNA used to detect a complementary nucleic acid sequence (target sequence) by hybridization.
  • the realization of probe signal reading is usually achieved by using radioisotopes, epitopes, biotin or fluorophores to label primer probes so that they can be detected.
  • the fluorescently labeled primer probes are used to identify biological organisms through a similar converter function. (Hybridization, ligand binding, etc.) is converted into a fluorescent signal.
  • Fluorescent labeling has several advantages, such as high sensitivity and multiple transduction methods (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer and excimer-monomer light conversion). These multiple signal options, combined with the design flexibility of identification elements (DNA, RNA, PNA, LNA) and various labeling strategies, help to develop a variety of selective and sensitive biological tests, such as individual identification and profiling in the field of forensic medicine. Quantitative short tandem repeat (STR) analysis method.
  • the fluorescent signal of the fluorescent label of the primer probe is usually excited by the fluorescent dye by the excitation light wave and then emits a light wave longer than the excitation wavelength, and the wavelength difference is called the Stokes shift.
  • the fluorescent dyes corresponding to the emission spectrum in the visible red-violet region (590-750 nm) in the market are very limited, and the above-mentioned dyes also have some disadvantages, such as (1) very expensive; (2) only through the purchase of kits , The current price is $176/8 times, that is, $22/time, and the structural form and modified functional groups of the dye products are single, which cannot meet the needs of customized and diversified R&D; (3) There is no long-wavelength region in the red-violet region in China Sales of raw materials for dyestuffs that generate emission spectra. These limiting factors have greatly restricted the development and utilization of fluorescent primer probe analysis technology.
  • the present invention provides a fluorescently labeled nucleic acid comprising a first dye and a second dye, characterized in that the first dye generates an emission spectrum after being excited under an excitation light source with a wavelength of 488-515 nm And is absorbed by the second dye, and the second dye further emits a spectrum with a wavelength of 590-750 nanometers after absorbing the emission spectrum of the first dye.
  • the first dye generates an emission spectrum after being excited under an excitation light source with a wavelength of 488 nm, 505 nm or 515 nm and is absorbed by the second dye, and the second dye absorbs After the first dye emission spectrum, it further emits a spectrum with a wavelength of 590-750 nanometers.
  • the fluorescently labeled nucleic acid comprises the structure represented by formula (I):
  • a and D are both dyes, when A is the first dye, D is the second dye, and when A is the second dye, D is the first dye;
  • X 1 is a nucleic acid sequence containing any base from 0-35 nt;
  • X 2 is a nucleic acid sequence containing any base from 0-35 nt;
  • L 1 is a linker, and the linker is selected from linear alkane, linear PEG, branched alkane, glycosyl rigid linker or a combination thereof;
  • n 0 or 1
  • X 3 is a nucleic acid sequence containing at least 1 nt base
  • the distance between A and D is 10-100 angstroms
  • the separation distance between the first dye and the second dye of the fluorescent-labeled nucleic acid is any value in the range of 10-100 angstroms, preferably 10-30 angstroms, more preferably 15-25 angstroms, and most preferably 15 or 20 angstroms.
  • Other optional separation distances are 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 angstroms.
  • X 1 in the formula (I) is a nucleic acid sequence comprising any base of 0-35nt, preferably 1-20nt, more preferably 1-15nt, preferably 1-10nt, most preferably 1- 8nt; In some embodiments, X 1 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16 , 17 , 18 , 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 nt nucleic acid sequence of any number of bases.
  • X 2 in the formula (I) is a nucleic acid sequence containing any base of 0-35nt, preferably 1-20nt, more preferably 1-15nt, preferably 1-10nt, most preferably 1- 8nt;
  • X 1 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16 , 17 , 18 , 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 nt nucleic acid sequence of any number of bases.
  • the fluorescently labeled nucleic acid comprises a structure represented by formula (II):
  • Both A'and D'are dyes when A'is the first dye, D'is the second dye, and when A'is the second dye, D'is the first dye;
  • X 4 is a nucleic acid sequence containing any base from 0-35 nt;
  • X 5 is a nucleic acid sequence containing any base from 0-35 nt;
  • L 2 is a linker, and the linker is selected from linear alkanes, linear PEGs, branched alkanes, glycosyl rigid linkers or combinations thereof;
  • X 6 is a nucleic acid sequence containing at least 1 nt base
  • the separation distance between the first dye and the second dye of the fluorescent-labeled nucleic acid is any value in the range of 10-100 angstroms, preferably 10-30 angstroms, more preferably 15-25 angstroms, and most preferably 15 or 20 angstroms.
  • Other optional separation distances are 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 angstroms.
  • X 4 in the formula (II) is a nucleic acid sequence comprising any base of 0-35 nt, preferably 1-20 nt, more preferably 1-15 nt, preferably 1-10 nt, most preferably 1 -8nt;
  • X 1 is comprised of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16 , 17 , 18 , 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35nt nucleic acid sequence of any number of bases.
  • X 5 in the formula (II) is a nucleic acid sequence comprising any base of 0-35 nt, preferably 1-20 nt, more preferably 1-15 nt, preferably 1-10 nt, most preferably 1 -8nt;
  • X 1 is comprised of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16 , 17 , 18 , 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35nt nucleic acid sequence of any number of bases.
  • the first dye and the second dye are selected from Cy5, Cy3, Texas Red, Alexa Fluor 610, BODIPY TR, AP593, FAM, Alexa Fluor 488, Alexa Fluor 514, BODIPY FL, BODIPY 500 /510, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, BODIPY 630/650, BODIPY 650/665.
  • the first dye is selected from Alexa Fluor 488, Alexa Fluor 514, BODIPY FL, BODIPY 500/510, Cy3, or FAM
  • the second dye is selected from Cy5, Texas Red, Alexa Fluor 610, BODIPY TR, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, BODIPY 630/650, BODIPY 650/665 or AP593.
  • the first dye and the second dye are Cy3 and Texas Red, Cy3 and Cy5, Cy3 and Alexa Fluor 610, Cy3 and BODIPY TR, Cy3 and AP593, FAM and BODIPY TR, respectively , Or FAM and AP593.
  • the linker when n is 1, the linker includes:
  • X S or O.
  • A is the first dye Cy3
  • D is the second dye Texas Red, Cy5, Alexa Fluor 610 or BODIPY TR
  • L 1 is n is 1
  • X 1 is a nucleic acid sequence containing Ont base
  • X 2 is a nucleic acid sequence containing 5 nt any base
  • A is the first dye FAM or FAM dT
  • D is the second dye BODIPY TR
  • L 1 is n is 1
  • X 1 is a nucleic acid sequence containing Ont base
  • X 2 is a nucleic acid sequence containing 5 nt any base
  • X 3 is a nucleic acid sequence containing at least 1 nt base
  • A is the first dye FAM or FAM dT
  • D is the second dye BODIPY TR
  • L 1 is n is 1
  • X 1 is an Ont base
  • X 2 is an Ont base
  • X 3 is a nucleic acid sequence that contains at least 1 nt base
  • A is the first dye Cy3
  • D is the second dye BODIPY TR or Texas Red
  • L 1 contains 5 With 1 Linked structure
  • n is 1
  • X 1 contains Ont base
  • X 2 contains Ont base
  • A is the first dye FAM or FAM dT
  • D is the second dye AP593
  • L 1 is n is 1
  • X 1 is an Ont base
  • X 2 is an Ont base
  • X 3 is a nucleic acid sequence that contains at least 1 nt base
  • A is a first dye Cy3
  • D is a second dye AP593
  • n is 0,
  • X 2 contains a 5nt any base or X 1 and X plus 5nt bases containing 2
  • X 3 a base comprising at least 1nt
  • the structure of formula (I) is 5'to 3'or 3'to 5'from left to right.
  • the fluorescently labeled nucleic acid comprises the structure represented by formula (II):
  • A' is the first dye Cy3
  • D' is the second dye AP593
  • X 4 contains Ont bases
  • X 5 contains Ont bases
  • the nucleic acid in the fluorescently labeled nucleic acid is deoxyribonucleic acid or ribonucleic acid.
  • the nucleic acid in the fluorescently labeled nucleic acid is a probe or a primer.
  • the present invention also provides an application of the above-mentioned fluorescently labeled nucleic acid in STR detection.
  • the present invention also provides a method for preparing the above fluorescently labeled nucleic acid.
  • solid-phase synthesis of nucleic acids by the phosphoramidite triester method, specifically by an automated DNA synthesizer, after uploading the designed nucleic acid sequence, the synthesis proceeds in the 3'to 5'direction , Including the step of removing trityl, the coupling step of phosphoramidite monomer, the capping step of connecting unreacted 5'-OH, the oxidation step of converting phosphite triester into stable phosphate triester , And the cleavage step of cleaving the oligonucleotide from the solid-phase carrier after the complete sequence is synthesized, and also includes a deprotection step of removing the primary amino protecting groups outside the adenine, cytosine and guanine ring.
  • Different linkers (Linker) and nucleic acid connection methods all adopt solid-phase synthesis by phosphoramidite triester method.
  • connection of the dye to the nucleic acid through a linker includes four connection methods:
  • the dye donor the first dye (such as Cy3 or FAM-dT) is a phosphoramidite monomer
  • the dye acceptor which is the second dye (such as Cy5, Texas Red, Alexa Fluor 610 or BODIPY TR) is N-hydroxysuccinate Imide (NHS) activated ester form.
  • the synthesis proceeds in the 3'to 5'direction, including the de-tritylization step, the coupling step of the phosphoramidite monomer, and the The 5'-OH connection of the reaction is a closed capping step, an oxidation step that converts the phosphite triester into a stable phosphate triester.
  • the nucleic acid is cleaved from the solid-phase carrier and includes a deprotection step of removing the primary amino protecting groups outside the adenine, cytosine and guanine ring.
  • the nucleic acid containing the dye donor and linker After obtaining the nucleic acid containing the dye donor and linker, it needs to be separated and purified by HPLC. After the acceptor dye in the form of NHS activated ester is fully dissolved in DMSO, it is oscillated to react with the nucleic acid at a temperature of 37°C. After the NHS condensation reaction, the product is purified and recovered by HPLC again, and the concentration is measured for spectral analysis with a spectrum analyzer.
  • the dye donor (such as Cy3) is in the form of NHS activated ester
  • the acceptor (such as The following abbreviated as AP593) is a phosphoramidite monomer.
  • Both the dye donor (such as Cy3 or FAM-dT) and the acceptor dye (such as AP593) are in the form of phosphoramidite monomers. After editing the sequence information including the sequence position information of the dye donor, acceptor and linker, upload it to an automated DNA synthesizer. After the dye donor, acceptor, and linker are dissolved, they are placed in separate corresponding synthesis channels of the synthesizer, and the solid-phase synthesis and cleavage deprotection methods are the same as the above synthesis.
  • the separation and purification method of the product is HPLC separation and recovery, after measuring the concentration, spectral analysis is performed with a spectrum analyzer.
  • the dye donor (such as Cy3) is an azide modified form
  • the acceptor (such as AP593) is a phosphoramidite monomer.
  • After editing the sequence information including the sequence position information of the dye receptor and the linker upload it to the automated DNA synthesizer.
  • the dye acceptor and linker are dissolved and placed in a separate corresponding synthesis channel of the synthesizer, the solid-phase synthesis and cleavage and deprotection methods are the same as the above synthesis.
  • the azide form donor After the azide form donor is fully dissolved in DMSO, it reacts with nucleic acid at 37°C under the catalyzed condition of cuprous bromide and sodium ascorbate. After the click reaction, it is purified and recovered by HPLC again. After measuring the concentration, use a spectrum analyzer for spectrum analysis.
  • Fluorescent groups, fluorescent compounds, or fluorophores include, but are not limited to, discrete compounds, molecules, proteins, and macromolecular complexes. Fluorophores also include compounds that exhibit long-lived fluorescence decay, such as lanthanide ions and complexes of lanthanide and organic ligand sensitizers.
  • the fluorophore is a chemical compound that can be excited by light of a specific wavelength and emit light of different wavelengths.
  • Fluorophore is a molecule capable of providing a detectable signal using one or more detection techniques (eg, spectrometry, spectroscopy, or visual inspection), and suitable examples of detectable signals may include optical signals.
  • the fluorophore can be described in terms of its emission profile or "color”.
  • Green fluorophores such as Cy3, FITC, and Oregon Green
  • Red fluorophores such as Texas Red, Cy5, and tetramethylrhodamine
  • fluorophores include, but are not limited to, 4-acetylamino-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine, acridine, and acridine isothiocyanate derivatives
  • EDANS 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid
  • Luciifer Yellow VS N-(4-anilino-1-naphthyl) maleimide
  • anthranilamide Brilliant Yellow
  • coumarin coumarin derivatives
  • Substances 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-trifluoromethyl coumarin (Coumaran 151), cyanosine dye (cyanosine); 4',6-diam
  • rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxy Rhodamine (R6G), lissamide rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine isothiocyanate X, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride Sulfonyl chloride derivative of Gyrhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine, tetramethyl isothiocyanate Rhodamine (TRITC); riboflavin; rosolic acid and lanthanide chelate derivatives, cyanine, pyrelium
  • the fluorophore may be cyanine, rhodamine, BODIPY, BODIPY TR (product of Thermo Fisher), AP593 (product of Glen research), Alexa Fluor 488, Alexa Fluor 514, BODIPY FL, BODIPY 500/510 , Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, BODIPY 630/650, BODIPY 650/665 or 1,3-Dichloro-7-hydroxy-9,9-dimethyl- 2(9H)-Acridone (DDAO) dye.
  • DDAO 1,3-Dichloro-7-hydroxy-9,9-dimethyl- 2(9H)-Acridone
  • fluorescently labeled nucleic acid refers to a nucleic acid coupled to a fluorophore.
  • Fluorescence refers to molecules that can absorb light of a specific frequency and emit light of different frequencies. Fluorescence is well known to those of ordinary skill in the art.
  • nucleic acid refers to a polymer of nucleotides (such as ribonucleotides, deoxyribonucleotides, nucleotide analogs, etc.), and includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA-RNA Hybrids, oligonucleotides, polynucleotides, aptamers, peptide nucleic acids (PNA), PNA-DNA conjugates, PNA-RNA conjugates, locked nucleic acids (LNA), etc., which include linear or branched co- Nucleotides linked together.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA-RNA Hybrids DNA-RNA Hybrids
  • oligonucleotides polynucleotides
  • PNA peptide nucleic acids
  • PNA PNA-DNA conjugates
  • PNA-RNA conjugates locked nucleic acids
  • Nucleic acids are usually single-stranded or double-stranded, and generally contain phosphodiester bonds, although in some cases, nucleic acid analogs that can have alternative backbones are included, including, for example, phosphoramidites (Beaucage et al. (1993) Tetrahedron 49 (10 ): 1925); phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Patent No. 5,644,048), phosphorothioate (Briu et al. (1989) J. Am. Chem.
  • nucleic acid backbone and bonds see Egholm (1992) J. Am. Chem. Soc. 114:1895).
  • Other analog nucleic acids include those with a positively charged backbone (Denpcy et al. (1995) Proc. ) And those with non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506. Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al. (1995) Chem.
  • FRET fluorescence resonance energy transfer.
  • FRET refers to an energy transfer process between at least two fluorescent compounds, between a fluorescent compound and a non-fluorescent component, or between a fluorescent component and a non-fluorescent component.
  • separation distance refers to the physical length distance between two dyes.
  • STR refers to short tandem repeats (STRs), which is a type of polymorphism, or microsatellite (Microsatellite), or simple sequence repeats (SSRs), which refers to two Or a form in which multiple nucleotides are repeatedly arranged, and different repetitive sequences are adjacent, about 2 to 10 base pairs in length, and are commonly found in non-coding introns. Due to the different repeat units and the number of repeats, the distribution among different races and different populations is very different, which constitutes STR genetic polymorphism. The number of repetitions at a homologous STR site varies between individuals. It is possible to create a personal genetic profile by identifying specific sequence duplications at specific locations in the genome. At present, more than 10,000 STR loci have been published. The STR analysis method has become an important analysis method for individual identification and paternity identification in the field of forensic medicine, and can be applied to judicial investigations, that is, genetic fingerprint analysis.
  • acceptor In this context, “acceptor”, “acceptor dye”, “dye acceptor” or “second dye” have the same meaning.
  • the present invention provides a fluorescently labeled nucleic acid, which uses at least two dyes to label the nucleic acid.
  • the emission spectrum of the dye donor is absorbed by the dye acceptor through fluorescence resonance energy transfer and then emits fluorescence in the red-violet region.
  • it can increase nucleic acid, such as probes or primers, by about 11 times the fluorescence intensity, so that fluorescent primers or probes can detect sensitivity and accuracy.
  • connection mode of the fluorescently labeled nucleic acid and the dye of the present invention can be adjusted according to needs, and the modified functional groups used are diversified, so as to meet the needs of customized and diversified research and development.
  • the raw material cost of the technical solution of the present invention is relatively low, with an average of RMB 10 per time, which is far lower than the price of a single fluorescent dye emitting in the red-violet region.
  • the technical solution of the present invention solves the problems of low detection sensitivity of the existing fluorescent dyes emitting high-wavelength spectra, single applicable modification functional group, and high price, which restrict the development and application of fluorescent probe technology.
  • Figure 1 Fluorescence scanning spectra of samples 1, 2 and 3 under 488 nm excitation.
  • Figure 2 Fluorescence scanning spectra of samples 1, 4, 5, 6, 7, 8, and 9 under excitation at 488 nm.
  • Figure 3 Fluorescence scanning spectrum of sample 11 under excitation conditions of 488 and 515 nm.
  • Figure 4 Fluorescence scanning spectrum of sample 12 under excitation conditions of 488 and 515 nm.
  • Figure 5 Fluorescence scanning spectra of samples 13, 14, and 15 under excitation at 488 nm.
  • Figure 6 Fluorescence scanning spectrum of sample 16 under excitation conditions of 488 and 515 nm.
  • Figure 7 Fluorescence scanning spectra of samples 4 and 17 under excitation at 488 nm.
  • Figure 8 Fluorescence scanning spectra of samples 6 and 18 under 488 nm excitation.
  • Figure 9 Fluorescence scanning spectra of samples 7 and 18 under excitation at 488 nm.
  • Figure 10 Fluorescence scanning spectra of samples 12 and 19 under excitation at 488 nm.
  • the difficulty of large fluorescent probes is to attach suitable fluorescent dyes in the visible red-violet region to the primers.
  • the dye has the following requirements: (1) It can be excited by the light source on the current analyzer, namely 488 nanometer, 505 nanometer or 515 nanometer light source; (2) Its emission wavelength should fall between 590-750 nanometers; (3) Various connection methods are suitable for the modification of a variety of functional dyes.
  • the present invention uses the principle of fluorescence resonance energy transfer, and uses the overlap of the emission spectrum of one fluorescent group (donor) and the absorption spectrum of another group (acceptor) to make the two fluorescent groups Under the condition of a suitable distance (10-100 angstroms), the fluorescence energy is transferred from the donor to the acceptor, that is, the energy of the donor group is transferred from the donor to the acceptor by a pair of dipoles in the excited state.
  • the fluorescently labeled nucleic acid of the present invention uses multiple combinations of fluorescent donors and acceptors that meet the above conditions to label the nucleic acid, and uses different linkers to connect it to the nucleic acid through a variety of coupling chemical schemes.
  • Sample 1 contains the dye donor Cy3, linker C6 and dye acceptor Texas Red primers, which constitute fluorescence resonance energy transfer; samples 2 and 3 only contain the dye donor Cy3 and dye acceptor Texas Red primer samples, respectively.
  • the fluorescence scanning spectra of the three samples under the excitation conditions of 488 nm after the normalized concentration are shown in Figure 1. It can be seen that: (1) The emission of the dye donor Cy3 at 530-570 nm is absorbed due to fluorescence resonance energy transfer. Quenching; (2) The emission of the dye acceptor Texas Red at 590-650 nm is significantly increased due to the effect of fluorescence resonance energy transfer. For example, the fluorescence intensity at 610-660 nm is increased by 11 times.
  • the fluorescence intensity is reduced by about 600-650 nanometers compared with that of sample 6. half.
  • the present invention chooses STR analysis.
  • the sample 10 with the same scheme as sample 1 but with a different sequence is tested by the Applied Biosystems 3730xl DNA analyzer commonly used in STR analysis. It is equivalent to the FAM, HEX, TAMRA, and ROX used in the instrument.
  • the fluorescent primer probe sample 10 in the purple band excited by 488 nm gives a signal with the same fluorescence intensity as FAM, HEX, TAMRA, and ROX.
  • the dye donor and acceptor are connected by branched linkers, as shown in Figure 3.
  • the fluorescence emission of the donor can also be seen to be absorbed.
  • the fluorescence emission spectrum of the body is higher under the excitation of 515 nm than the fluorescence intensity obtained under the excitation of 488 nm.
  • the method of solid-phase synthesis and cleavage deprotection is the same as that of conventional primer solid-phase synthesis.
  • the dye donor FAM-dT coupling time is 10 minutes ⁇ 2 times; the dye acceptor AP593 coupling time is 3 minutes ⁇ 2 times; the linker C3, C9, C18 monomer coupling time is 2.5 minutes ⁇ 2 times.
  • sample 12 The characteristic of sample 12 is that the primer containing the dye donor Cy3 and the acceptor AP593 can be directly obtained by solid-phase synthesis, which can avoid the modification after the completion of the primer synthesis and the loss caused by repeated chromatographic purification to improve the synthesis yield, as shown in Figure 4, at 488 Or under the condition of excitation at 515 nm, the fluorescence emission of the donor can also be seen to be absorbed.
  • the fluorescence emission of the combination of the schemes is shown as the fluorescence emission pattern of the acceptor, which is higher under excitation at 515 nm than under excitation at 488 nm. . It can be seen in Fig. 5 that the fluorescence resonance energy transfer effect of samples 13, 14 and 15 under the excitation condition of 488 nanometers is also seen. It is worth noting that as the length of the linear linker increases, the fluorescence intensity increases.
  • Dye acceptor AP593 and linker dT-yne monomer are dissolved in acetonitrile at concentrations of 0.067 M- and 0.10 M, respectively, and placed in the corresponding synthesis channel of the synthesizer.
  • the method of solid-phase synthesis and cleavage and deprotection is the same as that of conventional primers.
  • the coupling time of the dye acceptor AP593 is 3 minutes ⁇ 2 times
  • the coupling time of the linker dT-yne monomer is 2.5 minutes ⁇ 2 times.
  • sample 16 Compared with sample 11 and sample 12, although sample 16 uses the same dye donor (Cy3) and acceptor (AP593), and the connection method uses click chemistry, it can still achieve fluorescence resonance energy transfer. As shown in Figure 6, sample 16 has a higher fluorescence intensity under excitation at 515 nm than under excitation at 488 nm.
  • AF610 is the abbreviation of AFAlexa Fluor 610.

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Abstract

本发明提供一种荧光标记核酸及其合成方法，涉及核酸荧光标记领域，为解决现有技术中红紫区高波长范围信号检测难的问题，本发明提供一种包含第一染料和第二染料的荧光标记核酸，其特征在于，所述第一染料在波长488-515纳米的激发光源下被激发后产生发射光谱并被第二染料吸收，且所述第二染料在吸收第一染料发射光谱后进一步发射波长为590-750纳米的光谱。本发明的荧光标记核酸可应用于可见光红紫区域的荧光检测中。

Description

一种荧光标记核酸及其合成方法 技术领域
本发明涉及核酸荧光标记领域，具体涉及荧光标记的引物及探针，尤其是采用荧光共振能量转移原理合成的具有可见光红紫区域宽波段DNA荧光探针及方法。
背景技术
引物(寡核苷酸)探针是指用于通过杂交检测互补核酸序列(靶序列)的短链单链DNA或RNA。探针信号读取的实现通常是用放射性同位素、表位、生物素或荧光团等手段来标记引物探针以使其能够被检测，其中荧光标记的引物探针通过类似转换器作用将生物识别(杂交，配体结合等)转化为荧光信号。荧光标记具有若干优点，例如，高灵敏度和多种转导方法(荧光猝灭或增强，荧光各向异性，荧光寿命，荧光共振能量转移和准分子-单体光转换)。这些多种信号选择与识别元件(DNA，RNA，PNA，LNA)的设计灵活性和各种标记策略相结合，有助于开发多种选择性和敏感的生物检测，例如，法医学领域个体识别和亲权鉴定的短串联重复序列(STR)分析方法。而引物探针的荧光标记的荧光信号通常由荧光染料被激发光波激发后发射长于激发波长的光波，波长差值称为斯托克斯(stokes)位移。目前荧光引物探针开发需要在有限的可见光区域内增加检测通道，所以对于可以被短波长激发而在红紫区域的长波长发射即斯托克斯位移大(约100纳米)的染料需求巨大，而市场中对应于在可见光红紫区域(590-750纳米)发射光谱的荧光染料非常有限，并且上述染料也存在着一些缺陷，如(1)价格十分昂贵；(2)只能通过购买试剂盒，目前售价是$176/8次，即$22/次，且染料产品的结构形式及修饰官能团单一，无法满足定制化、多样化的研发需求；(3)国内没有在红紫区域的长波长区域产生发射光谱的染料原料原料销售。这些限制因素极大限制了对荧光引物探针分析技术的开发与利用。
发明内容
针对现有技术的不足，本发明提供一种包含第一染料和第二染料的荧光标记核酸，其特征在于，所述第一染料在波长488-515纳米的激发光源下被激发后产生发射光谱并被第二染料吸收，且所述第二染料在吸收第一染料发射光谱后进一步发射波长为590-750纳米的光谱。
在本发明的一些优选实施方案中，所述第一染料在波长488纳米、505纳米或515纳米的激发光源下被激发后产生发射光谱并被第二染料吸收，且所述第二染料在吸收第一染料发射光谱后进一步发射波长为590-750纳米的光谱。
本发明的一些实施方案中，所述的荧光标记核酸包含式(I)所示的结构：
-AX 1(L 1) nX 2DX 3-
(I)
其中，A和D均为染料，当A为第一染料时D为第二染料，当A为第二染料时D为第一染料；
X 1为包含0-35nt任意碱基的核酸序列；
X 2为包含0-35nt任意碱基的核酸序列；
L 1为连接子，所述连接子选自直链烷烃、直链PEG、支链烷烃、糖基刚性连接子或其组合；
n为0或1；
X 3为包含至少1nt碱基的核酸序列；
A和D的间隔距离为10-100埃；
且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向。
本发明的一些实施方案中，所述的荧光标记核酸第一染料与第二染料的间隔距离为10-100埃范围内的任意值，优选10-30埃，更优选15-25埃，最优选15或20埃。另一些可选的间隔距离为10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100埃。
本发明的一些实施方案中，所述式(I)中X 1为包含0-35nt任意碱基的核酸序列，优选1-20nt，更优选1-15nt，在优选1-10nt，最优选1-8nt；在一些实施方案中，X 1为包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35nt任意碱基数的核酸序列。
本发明的一些实施方案中，所述式(I)中X 2为包含0-35nt任意碱基的核酸序列，优选1-20nt，更优选1-15nt，在优选1-10nt，最优选1-8nt；在一些实施方案中，X 1为包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35nt任意碱基数的核酸序列。
本发明的另一些实施方案中，所述的荧光标记核酸包含式(II)所示的结构：
Figure PCTCN2020100030-appb-000001
其中，
A＇和D＇均为染料，当A＇为第一染料时D＇为第二染料，当A＇为第二染料时D＇为第一染料；
X 4为包含0-35nt任意碱基的核酸序列；
X 5为包含0-35nt任意碱基的核酸序列；
L 2为连接子，所述连接子选自直链烷烃、直链PEG、支链烷烃、糖基刚性连接子或其组合；
X 6为包含至少1nt碱基的核酸序列；
A＇和D＇的间隔距离为10-100埃；
且式(II)所示的结构从左向右是5’至3’方向或是3’至5’方向。
本发明的一些实施方案中，所述的荧光标记核酸第一染料与第二染料的间隔距离为10-100埃范围内的任意值，优选10-30埃，更优选15-25埃，最优选15或20埃。另一些可选的间隔距离为10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100埃。
本发明的又一些实施方案中，所述式(II)中X 4为包含0-35nt任意碱基的核酸序列，优选1-20nt，更优选1-15nt，在优选1-10nt，最优选1-8nt；在一些实施方案中，X 1为包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35nt任意碱基数的核酸序列。
本发明的再一些实施方案中，所述式(II)中X 5为包含0-35nt任意碱基的核酸序列，优选1-20nt，更优选1-15nt，在优选1-10nt，最优选1-8nt；在一些实施方案中，X 1为包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35nt任意碱基数的核酸序列。
本发明的一些实施方案中，所述第一染料和第二染料选自Cy5、Cy3、Texas Red、Alexa Fluor 610、BODIPY TR、AP593、FAM、Alexa Fluor 488、Alexa Fluor 514、BODIPY FL、BODIPY 500/510、Alexa Fluor 594、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、BODIPY 630/650、BODIPY 650/665中的任意两种。
本发明的一些实施方案中，所述的第一染料选自Alexa Fluor 488、Alexa Fluor 514、BODIPY FL、BODIPY 500/510、Cy3或FAM，所述的第二染料选自Cy5、Texas Red、Alexa Fluor 610、BODIPY TR、Alexa Fluor 594、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、BODIPY 630/650、BODIPY 650/665或AP593。
本发明的一些优选的实施方案中，所述的第一染料和第二染料分别为Cy3和Texas Red、Cy3和Cy5、Cy3和Alexa Fluor 610、Cy3和BODIPY TR、Cy3和AP593、FAM和BODIPY TR、或FAM和AP593。
本发明的又一些优选的实施方案中，当n为1时，所述连接子包含：
Figure PCTCN2020100030-appb-000002
Figure PCTCN2020100030-appb-000003
和/或
Figure PCTCN2020100030-appb-000004
其中，X＝S或O。
本发明的一些优选的实施方案中，所述式(I)中：
-AX 1(L 1) nX 2DX 3-
(I)
A为第一染料Cy3，D为第二染料Texas Red、Cy5、Alexa Fluor 610或BODIPY TR，L 1是
Figure PCTCN2020100030-appb-000005
n为1，X 1为包含0nt碱基，X 2为包含5nt任意碱基的核酸序列，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
本发明的另一些优选的实施方案中，所述式(I)中：
-AX 1(L 1) nX 2DX 3-
(I)
A为第一染料FAM或FAM dT，D为第二染料BODIPY TR，L 1是
Figure PCTCN2020100030-appb-000006
n为1，X 1为包含0nt碱基，X 2为包含5nt任意碱基的核酸序列， X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
本发明的又一些优选的实施方案中，所述式(I)中：
-AX 1(L 1) nX 2DX 3-
(I)
A为第一染料FAM或FAM dT，D为第二染料BODIPY TR，L 1是
Figure PCTCN2020100030-appb-000007
n为1，X 1为包含0nt碱基，X 2为包含0nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
本发明的再一些优选的实施方案中，所述式(I)中：
-AX 1(L 1) nX 2DX 3-
(I)
A为第一染料Cy3，D为第二染料BODIPY TR或Texas Red，L 1是包含5个
Figure PCTCN2020100030-appb-000008
与1个
Figure PCTCN2020100030-appb-000009
连接的结构，n为1，X 1为包含0nt碱基，X 2为包含0nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
本发明的更一些优选的实施方案中，所述式(I)中：
-AX 1(L 1) nX 2DX 3-
(I)
A为第一染料FAM或FAM dT，D为第二染料AP593，L 1是
Figure PCTCN2020100030-appb-000010
Figure PCTCN2020100030-appb-000011
n为1，X 1为包含0nt碱基，X 2为包含0nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
本发明的又一些优选的实施方案中，所述式(I)中：
-AX 1(L 1) nX 2DX 3-
(I)
A为第一染料Cy3，D为第二染料AP593，n为0，X 1、X 2任意一个包含5nt碱基或X 1与X 2的加和包含5nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向。
本发明的一些优选的实施方案中，所述的荧光标记核酸包含式(II)所示的结构：
Figure PCTCN2020100030-appb-000012
其中，A＇为第一染料Cy3，D＇为第二染料AP593，X 4包含0nt碱基，X 5包含0nt碱基，L 2为包含
Figure PCTCN2020100030-appb-000013
和/或
Figure PCTCN2020100030-appb-000014
的结构，X 6为包含至少1nt碱基的核酸序列，且式(II)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
本发明的一些实施方案中，所述的荧光标记核酸中的核酸是脱氧核糖核酸或核糖核酸。
本发明的另一些实施方案中，所述的荧光标记核酸中的核酸是探针或引物。
本发明还提供一种上述荧光标记核酸在STR检测中的应用。
本发明又提供一种制备上述荧光标记核酸的方法。
本发明的一些实施方案中，通过亚磷酰胺三酯法固相合成核酸(如引物)，具体为通过自动化DNA合成仪，上载设计好的上述核酸序列后，合成以3'至5'方向进行，包括脱三苯甲基化步骤、亚磷酰胺单体的偶联步骤、将未反应的5'-OH连接的封闭的盖帽步骤、将亚磷酸三酯转化为稳定的磷酸三酯的氧化步骤，以及在合成全序列之后将寡核苷酸从固相载体中切割下来的切割步骤，还包含将腺嘌呤、胞嘧啶和鸟嘌呤环外伯氨基保护基团去除的脱保护步骤。不同的连接子(Linker)与核酸的连接方法均采用通过亚磷酰胺三酯法固相合成。
本发明的又一些实施方案中，染料通过连接子(Linker)与核酸的连接包含四种连接方法：
a)染料供体即第一染料(如Cy3或FAM-dT)为亚磷酰胺单体，染料受体即第二染料(如Cy5、Texas Red、Alexa Fluor 610或BODIPY TR)为N-羟基琥珀酰亚胺(NHS)活化酯形式。将包含染料供体及连接子的序列位置信息的序列信息编辑完毕后，上载于自动化DNA合成仪。染料供体及连接子溶解后置于合成仪单独的对应合成通道后，合成以3'至5'方向进行，包括脱三苯甲基化步骤、亚磷酰胺单体的偶联步骤、将未反应的5'-OH连接的封闭的盖帽步骤、将亚磷酸三酯转化为稳定的磷酸三酯的氧化步骤。合成全序列之后将核酸从固相载体中切割下来，并包含将腺嘌呤、胞嘧啶和鸟嘌呤环外伯氨基保护基团去除的脱保护步骤。在得到包含染料供体及连接子的核酸后，需要经过HPLC分离纯化。NHS活化酯形式的受体染料充分溶解于DMSO中后，与核酸在37℃温度条件下振荡反应，NHS缩合反应结束后产物再次经HPLC纯化回收，并测量浓度后用光谱分析仪进行光谱分析。
b)染料供体(如Cy3)为NHS活化酯形式，受体(如
Figure PCTCN2020100030-appb-000015
以下缩写为AP593)为亚磷酰胺单体。将包含染料受体及连接子的序列位置信息的序列信息编辑完毕后，上载于自动化DNA合成仪。染料受体及连接子溶解后置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同上述合成。在得到包含染料受体及连接子的核酸后，需要经过HPLC分离纯化。NHS活化酯形式的供体充分溶解于DMSO中后，与核酸在37℃温度条件下振荡反应，NHS缩合反应结束后再次经HPLC纯化回收，并测量浓度后用光谱分析仪进行光谱分析。
c)染料供体(如Cy3或FAM-dT)与受体染料(如AP593)都为亚磷酰胺单体形式。将包含染料供体与受体及连接子的序列位置信息的序列信息编辑完毕后，上载于自动化DNA合成仪。染料供体与受体及连接子溶解后分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同上述合成。产物的分离纯化方法为HPLC分离回收，测量浓度后，用光谱分析仪进行光谱分析。
d)染料供体(如Cy3)为叠氮修饰形式，受体(如AP593)为亚磷酰胺单体。将包含染料受体及连接子的序列位置信息的序列信息编辑完毕后，上载于自动化DNA合成仪。染料受体及连接子溶解后置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同上述合成。在得到包含染料受体及连接子的核酸后，需要经过HPLC分离纯化。叠氮形式的供体充分溶解于DMSO中后，与核酸在37℃温度，在溴化亚铜及抗坏血酸钠催化条件下振荡反应，点击化学反应(click reaction)结束后再次经HPLC纯化回收，并测量浓度后用光谱分析仪进行光谱分析。
术语
“染料”、“荧光基团(fluorescent group)”、或“荧光团(fluorophore)”互换使用，是指为荧光性的分子、基团或基。术语“荧光性的”当应用至化合物的分子时，用来指化合物吸收能量(如UV、可见光或IR辐射)并将所述能量的至少一部分随时间再次发射为光的特性。荧光基团、荧光化合物或荧光团包括但不限于离散的化合物、分子、蛋白质和大分子复合物。荧光团还包括显示出长期荧光衰减(long-lived fluorescence decay)的化合物，如镧系元素离子和镧系元素与有机配体敏化剂(sensitizer)的络合物。该荧光团是能够被特定波长的光激发，并发射不同的波长的光的化学化合物。荧光团能够使用一种或多种检测技术(例如，光谱测定法、光谱法或肉眼检查)提供可检测的信号的分子，可检测的信号的合适实例可包括光信号。荧光团可根据其发射概况(profile)或“色彩”进行描述。绿色荧光团(例如Cy3、FITC和俄勒冈绿(Oregon Green))的特征可在于其以通常在515-540纳米范围内的波长发射。红色荧光团(例如得克萨斯红(Texas Red)、Cy5和四甲基罗丹明)的特征可在于其以通常在590-690纳米范围内的波长发射。荧光团的实例包括，但不限于，4-乙酰氨基-4'-异硫氰酸芪(isothiocyanatostilbene)-2,2'二磺酸、吖啶、吖啶和吖啶异硫氰酸酯的衍生物、5-(2'-氨基乙基)氨基萘-1-磺酸(EDANS)、4-氨基-N-[3-乙烯基磺酰基)苯基]萘二甲酰亚胺-3,5二磺酸盐(Lucifer Yellow VS)、N-(4-苯胺基-1-萘基)马来酰亚胺、邻氨基苯甲酰胺、亮黄(Brilliant Yellow)、香豆素、香豆素衍生物、7-氨基-4-甲基香豆素(AMC、Coumarin 120)、7-氨基-三氟甲基香豆素(Coumaran 151)、焰红染料(cyanosine)；4',6-二脒基(diaminidino)-2-苯基吲哚(DAPI)、5',5”-二溴邻苯三酚(dibromopyrogallol)-磺酞(溴邻苯三酚红)、7-二乙基氨基-3-(4'-异硫氰酸基苯基)-4-甲基香豆素、-4,4'-二异硫氰酸基二氢-芪-2,2'-二磺酸、4,4'-二异硫氰酸芪-2,2'-二磺酸、5-[二甲基氨基]萘-1-磺酰氯(DNS、丹磺酰氯)、荧光素和衍生物例如5-羧基荧光素(FAM)、5-(4,6-二氯三嗪-2-基)氨基荧光素(DTAF)、2'7'-二甲氧基-4'5'-二氯-6-羧基荧光素(JOE)、荧光素、异硫氰酸荧光素(FITC)、QFITC(XRITC)；荧光胺衍生物(荧光与胺反应时)；IR144；IR1446；异硫氰酸孔雀绿；4-甲基伞形酮；邻甲酚酞；硝基酪氨酸；副品红；酚红、B-藻红蛋白；邻苯二甲醛衍生物(荧光与胺反应时)；芘和衍生物例如芘、丁酸芘和1-芘丁酸琥珀酰亚胺基酯；活性红4(Cibacron.RTM.亮红3B-A)、罗丹明和衍生物例如6-羧基-X-罗丹明(ROX)、6-羧基罗丹明(R6G)、丽丝胺罗丹明B磺酰氯、罗丹明(Rhod)、罗丹明B、罗丹明123、异硫氰酸罗丹明X、磺基罗丹明B、磺基罗丹明101和磺基罗丹明101的磺酰氯衍生物(得克萨斯红)；N,N,N',N'-四甲基-6-羧基罗丹明(TAMRA)；四甲基罗丹明、异硫氰酸四甲基罗丹明(TRITC)；核黄素；玫红酸和镧系元素螯合物衍生物、花青、pyrelium染料、方酸箐(squaraines)、1,3-二氯-7-羟基-9,9-二甲基-2(9H)-吖啶酮(DDAO)和二甲基吖啶酮(DAO)。在一些实施方案中，荧光团可以是花青、罗丹明、BODIPY、BODIPY TR(Thermo Fisher公司产 品)、AP593(Glen research公司产品)、Alexa Fluor 488、Alexa Fluor 514、BODIPY FL、BODIPY 500/510、Alexa Fluor 594、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、BODIPY 630/650、BODIPY 650/665或1,3-二氯-7-羟基-9,9-二甲基-2(9H)-吖啶酮(DDAO)染料。
术语“荧光标记核酸”指偶联于荧光团的核酸。
术语“荧光”指能够吸收特定频率的光并发射不同频率的光的分子。荧光是本领域普通技术人员熟知的。
术语“核酸”指核苷酸(例如核糖核苷酸、脱氧核糖核苷酸、核苷酸类似物等)的聚合物，并且包含脱氧核糖核酸(DNA)、核糖核酸(RNA)、DNA-RNA杂交体、寡核苷酸、多核苷酸、适体、肽核酸(PNA)、PNA-DNA缀合物、PNA-RNA缀合物、锁核酸(LNA)等，其包含以线性或分支方式共价连接在一起的核苷酸。核酸通常是单链或双链的，并且一般含有磷酸二酯键，尽管在一些情况下，包括可以具有替代主链的核酸类似物，包括例如磷酰胺(Beaucage等人(1993)Tetrahedron 49(10):1925)；硫代磷酸酯(Mag等人(1991)Nucleic Acids Res.19:1437；和美国专利号5,644,048)，二硫代磷酸酯(Briu等人(1989)J.Am.Chem.Soc.111:2321)，O-甲基亚磷酰胺键(参见Eckstein，Oligonucleotides and Analogues:APractical Approach，Oxford University Press(1992))，以及肽核酸主链和键(参见Egholm(1992)J.Am.Chem.Soc.114:1895)。其他类似物核酸包括具有带正电的主链(Denpcy等人(1995)Proc.Natl.Acad.Sci.USA 92:6097)；非离子主链(美国专利号5,386,023，5,637,684，5,602,240，5,216,141和4,469,863)和非核糖主链的那些，包括在美国专利号5,235,033和5,034,506中描述的那些。含有一种或多种碳环糖的核酸也包括在核酸的定义内(参见Jenkins等人(1995)Chem.Soc.Rev.第169-176页)，并且类似物也在例如Rawls，C&E News Jun.2，1997第35页中描述。可以完成磷酸核糖主链的这些修饰，以促进另外部分例如标记物的添加，或改变此类分子在生理环境中的稳定性和半衰期。
术语“FRET”是指荧光共振能量转移。在本发明中，FRET是指至少两种荧光化合物之间、荧光化合物和非荧光组分之间，或荧光组分和非荧光组分之间发生的能量转移过程。
术语“埃”，指长度计量单位，符号为
Figure PCTCN2020100030-appb-000016
与国际制单位换算，即
Figure PCTCN2020100030-appb-000017
米＝0.1纳米。
术语“间隔距离”，指两个染料之间的物理长度距离。
术语“STR”，指短串联重复序列(short tandem repeats，STRs)是多型性的一种类型，或称微卫星(Microsatellite)，或称简单重复序列(Simple Sequence Repeats，SSRs)，指两个或多个核苷酸重复排列，且不同的重复序列相邻的形式，长度约2到10个碱基对，常见 于非编码的内含子中。由于重复单位及重复次数不同，使其在不同种族，不同人群之间的分布具有很大差异性，构成了STR遗传多态性。不同个体之间在一个同源STR位点的重复次数不同。通过识别基因组在特定位点的特定序列重复，可能创建一个个人基因档案。目前已经有超过10000个STR位点被公开。STR分析法已经成为法医学领域个体识别和亲权鉴定的重要分析方法，可应用于司法案件调查，也就是遗传指纹分析。
在本文中，“供体”、“供体染料”、“染料供体”或“第一染料”表达相同的含义。
在本文中，“受体”、“受体染料”、“染料受体”或“第二染料”表达相同的含义。
有益效果
本发明提供了一种的荧光标记核酸，其采用至少两个染料对核酸进行标记，通过荧光共振能量转移使染料供体发射光谱被染料受体吸收后在红紫区发射荧光，其可依据需求采用不同的染料组合在不同可见光红紫区域波长范围内发射光谱，与单染料荧光标记相比可以提高核酸，如探针或引物荧光强度约11倍，从而使荧光引物或探针检测灵敏度和准确性更高；同时本发明的荧光标记核酸与染料的连接方式可根据需要进行调整，使用的修饰官能团多种化，从而满足定制化、多样化的研发需求。另外，本发明的技术方案原料成本相对较低，平均为每次10元人民币，远低于单个在红紫区发射荧光染料的价格。本发明的技术方案解决了现有发射高波长光谱的荧光染料检测灵敏度低、适用修饰官能团单一、售价昂贵从而制约荧光探针技术开发及应用的问题。
附图说明
图1样品1、2和3在488纳米激发条件下的荧光扫描图谱。
图2样品1、4、5、6、7、8和9在488纳米激发条件下的荧光扫描图谱。
图3样品11在488与515纳米激发条件下的荧光扫描图谱。
图4样品12在488与515纳米激发条件下的荧光扫描图谱。
图5样品13、14、15在488纳米激发条件下的荧光扫描图谱。
图6样品16在488与515纳米激发条件下的荧光扫描图谱。
图7样品4和17在488纳米激发条件下的荧光扫描图谱。
图8样品6和18在488纳米激发条件下的荧光扫描图谱。
图9样品7和18在488纳米激发条件下的荧光扫描图谱。
图10样品12和19在488纳米激发条件下的荧光扫描图谱。
具体实施方式
如果要实现在有限的可见光区域内增加检测通道，需要能够被短波长激发而在红紫区域的长波长发射即斯托克斯位移大(约100纳米)的染料，而具有斯托克斯位移大的荧光探针的难点在于，将合适的可见光红紫区域荧光染料连接于引物中。对该染料有以下要求：(1)可以被现在的分析仪上的光源即488纳米、505纳米或515纳米光源激发；(2)其发射在波长应该落于590-750纳米间；(3)连接方法多样，适用于多种官能染料修饰。基于以上要求，本发明利用荧光共振能量转移原理，利用一个荧光基团(供体Donor)的发射光谱与另一个基团(受体Acceptor)的吸收光谱的重叠，使这两个荧光基团间在距离合适(10-100埃)的条件下，荧光能量由供体向受体转移，即供体基团在激发状态下由一对偶极子介导的能量从供体向受体转移。该过程具体为供体分子被激发后，当受体分子与供体分子相距一定距离，且供体和受体的基态及第一电子激发态两者的振动能级间的能量差相互适应时，处于激发态的供体将把一部分或全部能量转移给受体，使荧光受体被激发，呈现出受体的荧光，并造成次级荧光光谱的红移，即增加了斯托克斯(stokes)位移。这样，实现在激发光源(488纳米、505纳米或515纳米)得到所需要的发射波长范围(590-750纳米)的发射荧光。因此，本发明的荧光标记核酸是采用满足上述条件的荧光供体及受体的多种组合来标记核酸，同时利用不同连接子通过多种偶联化学方案将其连接至核酸中。
下面通过实施例，并结合附图，对本发明的技术方案作进一步详细的说明，但本发明不限于下面的实施例。除非另有说明，本发明所用的技术和科学术语具有与本发明所属领域的普通技术员通常所理解的含义。
实施例1
将包含染料供体及连接子的序列位置信息(表1样品1、4-10)的序列信息编辑完毕后，上载于自动化DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；染料供体Cy3或者FAM-dT溶解于乙腈，浓度分别为0.058M与0.039M；连接子C6单体与连接子dSpacer单体溶解于乙腈，浓度分别为0.10M，0.10M，分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成参见(Beaucage et al.,J.Tetrahedron Letters.22.20:1859-1862(1981))，其中，染料供体Cy3偶联时间为3分钟×2次，染料供体FAM-dT偶联时间为10分钟×2次；对于样品1、4-8，连接子C6单体偶联时间为2.5分钟×2次；对于样品9-10，连接子dSpacer单体及C6单体偶联时间均为2.5分钟×2次。在得到包含染料供体及连接子的引物后，经过HPLC分离纯化回收。
纯化后的引物溶解于去离子水中，最终浓度为25ug/uL；250ug活化酯形式的受体(Cy5、Texas Red、AF610、BODIPY TR)均溶解在14uL DMSO中后，取14uL的活化酯溶液至一 个2mL的氨解管中，并依次加入以下试剂：7.0uL去离子水，75uL硼酸盐缓冲液(pH＝8.5)，4.0uL的引物溶液(25ug/uL)；引物混合物在25℃温度条件下振荡反应过夜，NHS缩合反应结束后再次经HPLC纯化回收，并测量浓度后用光谱分析仪进行光谱分析，图谱横坐标为波长(纳米)，纵坐标为荧光强度(无单位，为相对荧光强度)。
样品1包含染料供体Cy3、连接子C6及染料受体Texas Red的引物，构成了荧光共振能量转移；样品2与3分别只包含染料供体Cy3、染料受体Texas Red的引物样品。三个样品在归一化浓度后488纳米的激发条件下的荧光扫描图谱如图1，可以看出：(1)由于荧光共振能量转移作用，染料供体Cy3在530-570纳米的发射被吸收淬灭；(2)染料受体Texas Red在590-650纳米处的发射由于荧光共振能量转移作用得到明显增加，如在610-660纳米处荧光强度均增加到原来的11倍。
在保持染料供体Cy3情况下将染料受体换为AF610后，如图2中的样品5与样品1相比，荧光强度在600-650纳米间有少许降低，将染料受体换为BODIPY TR后，图2中的样品6，荧光强度在620-650纳米间有增加；而将染料受体换为Cy5后，尽管其激发与Cy3的发射重合度低，但其荧光与样品1对比，在较大波长在650-720纳米范围内发射仍然有约30％的荧光发射强度。在保持染料受体BODIPY TR条件下，将染料供体换成FAM-dT的条件下，如图2中的样品7与8，对其荧光强度影响不大。在保持染料供体Cy3受体BODIPY TR条件下，改变连接子单体，如图2中的样品9，在488纳米的激发下荧光在600-650纳米间与样品6相比，强度下降了约一半。为了验证样品的应用，本发明选择了STR分析，与样品1方案相同但不同序列的样品10经过STR分析常用的Applied Biosystems 3730xl DNA分析仪测试，在与仪器使用的FAM、HEX、TAMRA、ROX同等浓度下，经488纳米激发在紫色波段荧光引物探针样品10给出了与FAM、HEX、TAMRA、ROX同等荧光强度的信号。
实施例2
将包含染料受体及连接子的序列位置信息(表1样品11)的序列信息编辑完毕后，上载于自动化DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；染料受体AP593及支链连接子C6-br单体溶解溶解于乙腈，浓度分别为0.067M与0.10M，分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成，其中，染料受体AP593偶联时间为3分钟×2次；连接子C6-br单体偶联时间为2.5分钟×2次。在得到包含染料受体及连接子的引物后，经过HPLC分离纯化回收。
纯化后的引物溶解于去离子水中，最终浓度为25ug/uL；250ug供体Cy3活化酯溶解在14uL DMSO中后，取14uL的活化酯溶液至一个2mL的氨解管中，并依次加入以下试剂：7.0uL去离子水，75uL硼酸盐缓冲液(pH＝8.5)，4.0uL的引物溶液(25ug/uL)；引物混合物 在25℃温度条件下振荡反应过夜，NHS缩合反应结束后再次经HPLC纯化回收，并测量浓度后用光谱分析仪进行光谱分析。染料供体、受体通过支链连接子相连这种方案，如图3，在488纳米或者515纳米激发的条件下，同样看到供体的荧光发射被吸收，方案组合的荧光发射表现为受体的荧光发射图谱，在515纳米的激发下比在488纳米激发下得到的荧光强度高。
实施例3
将包含染料供体Cy3、受体AP593的序列位置信息(表1样品12)的序列信息编辑完毕后，上载于自动化DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；染料供体Cy3溶解于乙腈，浓度分别为0.058M；染料受体AP593溶解乙腈，浓度为0.067M；分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成；其中，染料供体Cy3偶联时间为3分钟×2次；染料受体AP593偶联时间为3分钟×2次。在得到包含染料供体、受体的引物样品后，经过HPLC分离纯化回收，样品12经过测量浓度后用光谱分析仪进行光谱分析。
将包含染料供体FAM-dT、受体AP593及连接子C3、C9、C18的序列位置信息(表1样品13-15)的序列信息编辑完毕后，上载于自动化DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；染料供体FAM-dT溶解于乙腈，浓度分别为0.039M；染料受体AP593溶解乙腈，浓度为0.067M；连接子C3、C9、C18单体溶解于乙腈，浓度均为0.10M，分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成，其中，染料供体FAM-dT偶联时间为10分钟×2次；染料受体AP593偶联时间为3分钟×2次；连接子C3、C9、C18单体偶联时间为2.5分钟×2次。在得到包含染料供体、受体及连接子的引物样品13、14与15，经过HPLC分离纯化回收，测量浓度后用光谱分析仪进行光谱分析。
样品12的特点在于直接通过固相合成得到含有染料供体Cy3和受体AP593的引物，可以避免引物合成完成后的修饰及重复色谱纯化导致的损失从而提高合成产率，如图4，在488或者515纳米激发的条件下，同样看到供体的荧光发射被吸收，方案组合的荧光发射表现为受体的荧光发射图谱，在515纳米的激发下比在488纳米激发下得到的荧光强度高。可以在图5中看到样品13、14与15在488纳米激发条件下，同样看到荧光共振能量转移作用，值得指出的是，随着直链连接子长度的增加，荧光强度增加。
实施例4
将包含染料受体及连接子的序列位置信息的序列信息编辑完毕后(表1样品16)，上载于自动化DNA合成仪。染料受体AP593及连接子dT-yne单体溶解乙腈，浓度分别为0.067 M-和0.10M，后置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成，其中，染料受体AP593偶联时间为3分钟×2次，连接子dT-yne单体偶联时间为2.5分钟×2次。在得到包含染料受体及连接子的引物后，经过HPLC分离纯化回收测定浓度。
将8.8mg L-抗坏血酸溶于1.0mL灭菌水配制50mM的L-抗坏血酸溶液，将7.2mg溴化亚铜溶于1.0mL DMSO配制50mM的溴化亚铜溶液，8.2mg叠氮形式的供体Cy3充分溶解于1.0mL DMSO。
从回收得到的引物水溶液中取已知摩尔数对应体积量的引物于2mL的反应管中，计算所需要的叠氮Cy3、L-抗坏血酸、溴化亚铜的摩尔数以及所对应已经配制好溶液的需要的体积数(物料摩尔比为：引物：叠氮：L-抗坏血酸：溴化亚铜＝1:0.83:15:15)。往反应管中依次加入100uL的pH＝7.0的2M TEAA水溶液、对应体积的叠氮染料、50mM的L-抗坏血酸溶液、50mM的溴化亚铜溶液及100uL的DMSO。将反应管震荡摇匀，并用氩气保护，在55℃密封避光恒温机械振荡2小时，待点击化学反应(click reaction)结束后反应物再次经HPLC纯化回收，得到的产物测量浓度后用光谱分析仪进行光谱分析。
与样品11和样品12相比，样品16虽然采用了相同的染料供体(Cy3)与受体(AP593)，连接方法采用了点击化学，仍然可以实现荧光共振能量转移作用。如图6，样品16在515纳米的激发下比在488纳米激发下得到的荧光强度高。
表1中2、3、17、18、19号样品采用实施例1-4相对应的方法制备，并用光谱分析仪进行光谱分析。三个样品在488纳米的激发条件下的荧光扫描图谱如图7-10，从图中可以看出，与单独采用受体染料标记相对，采用供体染料与受体染料组合标记的引物探针其在590-750纳米范围处的发射由于荧光共振能量转移作用得到明显增加。
表1利用荧光共振能量转移制备荧光引物探针
Figure PCTCN2020100030-appb-000018
Figure PCTCN2020100030-appb-000019
注:AF610为AFAlexa Fluor 610的简写。
以下是合成表1中对应的荧光引物探针所用到的连接子单体结构式：
Figure PCTCN2020100030-appb-000020
表2实施例1-4中所用染料的来源
Figure PCTCN2020100030-appb-000021

Claims (18)

  1. 一种包含第一染料和第二染料的荧光标记核酸，其特征在于，所述第一染料在波长488-515纳米的激发光源下被激发后产生发射光谱并被第二染料吸收，且所述第二染料在吸收第一染料发射光谱后进一步发射波长为590-750纳米的光谱。
  2. 权利要求1所述的荧光标记核酸，其包含式(I)所示的结构：
    -AX 1(L 1) nX 2DX 3-
    (I)
    其中，
    A和D均为染料，当A为第一染料时D为第二染料，当A为第二染料时D为第一染料；
    X 1为包含0-35nt任意碱基的核酸序列；
    X 2为包含0-35nt任意碱基的核酸序列；
    L 1为连接子，所述连接子选自直链烷烃、直链PEG、支链烷烃、糖基刚性连接子或其组合；
    n为0或1；
    X 3为包含至少1nt碱基的核酸序列；
    A和D的间隔距离为10-100埃；
    且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向。
  3. 权利要求1所述的荧光标记核酸，其包含式(II)所示的结构：
    Figure PCTCN2020100030-appb-100001
    其中，
    A＇和D＇均为染料，当A＇为第一染料时D＇为第二染料，当A＇为第二染料时D＇为第一染料；
    X 4为包含0-35nt任意碱基的核酸序列；
    X 5为包含0-35nt任意碱基的核酸序列；
    L 2为连接子，所述连接子选自直链烷烃、直链PEG、支链烷烃、糖基刚性连接子或其组合；
    X 6为包含至少1nt碱基的核酸序列；
    A＇和D＇的间隔距离为10-100埃；
    且式(II)所示的结构从左向右是5’至3’方向或是3’至5’方向。
  4. 权利要求1-3中任一项所述的荧光标记核酸，所述第一染料和第二染料选自Cy5、Cy3、Texas Red、Alexa Fluor 610、BODIPY TR、AP593、FAM、Alexa Fluor 488、Alexa Fluor 514、BODIPY FL、BODIPY 500/510、Alexa Fluor 594、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、BODIPY 630/650、BODIPY 650/665中的任意两种。
  5. 权利要求4所述的荧光标记核酸，所述的第一染料选自Alexa Fluor 488、Alexa Fluor 514、BODIPY FL、BODIPY 500/510、Cy3或FAM，所述的第二染料选自Cy5、Texas Red、Alexa Fluor 610、BODIPY TR、AP593、Alexa Fluor 594、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、BODIPY 630/650或BODIPY 650/665。
  6. 权利要求4所述的荧光标记核酸，所述的第一染料和第二染料分别为Cy3和Texas Red、Cy3和Cy5、Cy3和Alexa Fluor 610、Cy3和BODIPY TR、Cy3和AP593、FAM和BODIPY TR、或FAM和AP593。
  7. 权利要求6所述的荧光标记核酸，当n为1时，所述连接子包含：
    Figure PCTCN2020100030-appb-100002
    Figure PCTCN2020100030-appb-100003
    和/或
    Figure PCTCN2020100030-appb-100004
    其中，X＝O或者S。
  8. 权利要求2所述的荧光标记核酸，所述式(I)中：
    -AX 1(L 1) nX 2DX 3-
    (I)
    A为第一染料Cy3，D为第二染料Texas Red、Cy5、Alexa Fluor 610或BODIPY TR，L 1是
    Figure PCTCN2020100030-appb-100005
    n为1，X 1为包含0nt碱基，X 2为包含5nt任意碱基的核酸序列，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
  9. 权利要求2所述的荧光标记核酸，所述式(I)中：
    -AX 1(L 1) nX 2DX 3-
    (I)
    A为第一染料FAM或FAM dT，D为第二染料BODIPY TR，L 1是
    Figure PCTCN2020100030-appb-100006
    n为1，X 1为包含0nt碱基，X 2为包含5nt任意碱基的核酸序列，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
  10. 权利要求2所述的荧光标记核酸，所述式(I)中：
    -AX 1(L 1) nX 2DX 3-
    (I)
    A为第一染料FAM或FAM dT，D为第二染料BODIPY TR，L 1是
    Figure PCTCN2020100030-appb-100007
    n为1，X 1为包含0nt碱基，X 2为包含0nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
  11. 权利要求2所述的荧光标记核酸，所述式(I)中：
    -AX 1(L 1) nX 2DX 3-
    (I)
    A为第一染料Cy3，D为第二染料BODIPY TR或Texas Red，L 1是包含5个
    Figure PCTCN2020100030-appb-100008
    与1个
    Figure PCTCN2020100030-appb-100009
    连接的结构，n为1，X 1为包含0nt碱基，X 2为包含0nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
  12. 权利要求2所述的荧光标记核酸，所述式(I)中：
    -AX 1(L 1) nX 2DX 3-
    (I)
    A为第一染料FAM或FAM dT，D为第二染料AP593，L 1是
    Figure PCTCN2020100030-appb-100010
    Figure PCTCN2020100030-appb-100011
    n为1，X 1为包含0nt碱基，X 2为包含0nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
  13. 权利要求2所述的荧光标记核酸，所述式(I)中：
    -AX 1(L 1) nX 2DX 3-
    (I)
    A为第一染料FAM或FAM dT，D为第二染料AP593，L 1是
    Figure PCTCN2020100030-appb-100012
    Figure PCTCN2020100030-appb-100013
    n为1，X 1为包含0nt碱基，X 2为包含0nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
  14. 权利要求2所述的荧光标记核酸，所述式(I)中：
    -AX 1(L 1) nX 2DX 3-
    (I)
    A为第一染料Cy3，D为第二染料AP593，n为0，X 1、X 2任意一个包含5nt碱基或X 1与X 2的加和包含5nt碱基，X 3为包含至少1nt碱基的核酸序列，且式(I)所示的结构从左向右是5’至3’方向或是3’至5’方向。
  15. 权利要求3所述的荧光标记核酸，所述的荧光标记核酸包含式(II)所示的结构：
    Figure PCTCN2020100030-appb-100014
    其中，A＇为第一染料Cy3，D＇为第二染料AP593，X 4包含0nt碱基，X 5包含0nt碱基，L 2为包含
    Figure PCTCN2020100030-appb-100015
    和/或
    Figure PCTCN2020100030-appb-100016
    的结构，X 6为包含至少1nt碱基的核酸序列，且式(II)所示的结构从左向右是5’至3’方向或是3’至5’方向；其中，X＝O或者S。
  16. 权利要求1-15中任意一项所述的荧光标记核酸，所述的核酸是脱氧核糖核酸或核糖核酸，优选引物或探针。
  17. 权利要求16所述的荧光标记核酸在STR检测中的应用。
  18. 制备权利要求1-16中任意一项所述的荧光标记核酸的方法。
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