CN105218536A - A kind ofly exempt from mark fluorescent probe and detecting the application in amphiploid G-tetra-stranded structure - Google Patents
A kind ofly exempt from mark fluorescent probe and detecting the application in amphiploid G-tetra-stranded structure Download PDFInfo
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Abstract
The invention discloses and a kind ofly exempt from mark fluorescent probe and detecting the application in amphiploid G-tetra-stranded structure, this probe is the symmetrical Berberine dimer derivate that ether chain connects.Of the present invention pair of berberinc derivate can be used under ultraviolet lamp and exempts from mark fluorescent detecting amphiploid G-tetra-serobila DNA structure, there is Selective recognition, highly sensitive and do not affect the characteristic of its conformation and thermostability, and can cell be entered, G-tetra-stranded structure in cell can be detected, this type of probe is had broad application prospects.
Description
Technical field
The present invention relates to a class Novel free mark fluorescent probe, and its application in intraor extracellular fluoroscopic examination amphiploid G-tetra-serobila nucleic acid secondary structure.
Background technology
G-tetra-serobila (G-quadruplex) is a kind of special DNA secondary structure.Much be rich in guanine (G) region in human genome and there is the ability forming this structure, comprise telomerase guanine tumor-necrosis factor glycoproteins, and the promoter region of several genes, as c-kit, c-myc, c-myb, bcl-2, PDGF, kRAS, VEGF, Rb and insulin gene etc.Current research shows, the RNA sequence of a lot of non-coding region also can form G-tetra-stranded structure, G-tetra-stranded structure of RNA is more stable than DNA, and this structure can reach the effect of Tumor suppression growth by stoping the process of the translation of gene or the identification of participation protein binding.All regulating and controlling effect is there is in the formation of G-tetra-stranded structure for a series of physiological processs in body.Research proves, G-tetra-stranded structure of some promoter region understands transcribing and translation skill of remarkably influenced gene, therefore G-tetra-stranded structure is considered to the function playing molecular switch, and it is formed and breaks physiological process important in a series of bodies such as may relating to intracellular signaling, apoptosis and cell proliferation.So, in vivo or in vitro tests, can detect existence or the formation of G-tetra-stranded structure specifically, the aspects such as the cancer therapy drug that related biological function and exploitation for research G-tetra-stranded structure are target spot with G-tetra-stranded structure all have very important effect.
At present, all have made some progress with the research of vitro detection G-tetra-stranded structure in vivo.Due to the existence of duplex DNA greatly excessive in body, and the intracellular environment of complexity, make proper interior detection need to solve a more difficult problem relative to vitro detection, more existing fluorescence molecules can realize the detection of G-tetra-stranded structure in body at present; G-tetra-stranded structure is detected mainly by the means of instrument in experiment in vitro, the methods such as such as circular dichroism detector, nucleus magnetic resonance, surface plasma resonance, FRET (fluorescence resonance energy transfer), the requirement of these methods to instrument and technological operation is all higher, and price costly, substantially use can not be popularized.And, current great majority research interest concentrates on selectivity to monoploid G-tetra-stranded structure of a certain conformation (as parallel, antiparallel or Combination) and sensitivity technique, simultaneously an excellent fluorescent probe is while detection target G-tetra-serobila, can not affect conformation and the thermostability of targeted molecular, but such fluorescence molecule report seldom.
People's telomeric dna is the duplex district that end of chromosome is made up of repetitive dna sequence TTAGGG, its end is that the strand being rich in G base containing 100-200nt Nucleotide is overhang, this telomeric dna is overhang for the substrate of Telomerase, and the activation of Telomerase and the prolongation of telomere will lead oncogenic generation.And the strand of these rich G is overhang, the complicated amphiploid connected by TTA base-pair sequence or polyploid G-tetra-stranded structure can be formed under certain conditions, realize the detection to amphiploid or polyploid G-tetra-stranded structure by some fluorescence molecules, to aspects such as the cancer therapy drugs that related biological function and the exploitation of research G-tetra-stranded structure are target spot with G-tetra-stranded structure, all there is important effect.And being at present still in the fluoroscopic examination of amphiploid or polyploid G-tetra-serobila the starting stage, the fluorescence molecule of report is few.
At present, the epiberberine only having bibliographical information single fluoroscopic examination can be present in K
+mixed type monoploid in solution and polyploid G-tetra-serobila, and this fluorescence molecule cannot distinguish monoploid and polyploid G-tetra-serobila.
Summary of the invention
For the blank of amphiploid G-tetra-serobila context of detection, the invention provides the fluorescent probe that a class is exempted to mark, monoploid and polyploid G-tetra-serobila can be distinguished in the solution; And G-tetra-serobila in cell can be detected.Probe of the present invention not only has highly sensitive and Selective recognition, and does not affect G-tetra-stranded structure conformation and thermostability.
The symmetrical Berberine dimer derivate that the object of the invention is to provide ether chain to connect is as the application of G-tetra-stranded structure detection probes.
The technical solution used in the present invention is:
The symmetrical Berberine dimer derivate that ether chain connects, its molecular structure is such as formula shown in I:
(I), wherein n is 2 or 3.
The symmetrical Berberine dimer derivate that above-mentioned ether chain connects is as the application of G-tetra-stranded structure detection probes.
Further, above-mentioned G-tetra-serobila DNA structure is amphiploid G-tetra-stranded structure.
Further, above-mentioned amphiploid G-tetra-stranded structure is 5 '-A (GGGTTA)
7g
3-3 ' or 5 '-A (GGGTTA)
4(TTAGGG)
4-3 ' amphiploid G-tetra-stranded structure of Sequence composition.
The symmetrical Berberine dimer derivate that above-mentioned ether chain connects detects the method for amphiploid G-tetra-stranded structure, its the method is the symmetrical Berberine dimer derivate solution by adding the connection of ether chain in DNA solution to be measured, mixing, detect the fluorescence intensity of mixed solution, if the fluorescence intensity of DNA solution to be measured enhances at least 160 times, illustrate in DNA to be measured containing amphiploid G-tetra-stranded structure.
Further, the solvent of above-mentioned DNA solution to be measured is for containing Na
+the Tris-HCl damping fluid of pH6.0 ~ 8.0.
Further, the solvent of the symmetrical Berberine dimer derivate solution of above-mentioned ether chain connection is for containing Na
+the Tris-HCl damping fluid of pH6.0 ~ 8.0.
Further, the mol ratio of symmetrical Berberine dimer derivate that above-mentioned DNA to be measured is connected with ether chain is (1 ~ 10): 1.
The symmetrical Berberine dimer derivate that above-mentioned ether chain connects detects the method for amphiploid G-tetra-stranded structure, comprises the following steps:
1) concentration of DNA in DNA solution to be measured is measured;
2) in DNA solution to be measured, add the symmetrical Berberine dimer derivate that ether chain connects, mixing, detect the fluorescence intensity of mixed solution;
3) the standard fluorescence spectrum that the fluorescence intensity upper pacing obtained and described derivative detect amphiploid G-tetra-stranded structure is compared, can judge whether containing amphiploid G-tetra-stranded structure in DNA to be measured, and determine specifically containing which kind of amphiploid G-tetra-stranded structure.
The symmetrical Berberine dimer derivate that above-mentioned ether chain connects detects the method for G-tetra-stranded structure in cell, the method is after cell and described derivative are incubated 2.5 ~ 3.5h altogether, under fluorescent microscope, fluoroscopic examination is carried out to cell, if cell produces obvious fluorescence, illustrate in cell containing G-tetra-stranded structure.
The invention has the beneficial effects as follows:
1) probe steady of the present invention, is easy to get, and Stability Analysis of Structures, be convenient to store.
2) probe provided by the invention specifically in conjunction with amphiploid G-tetra-stranded structure, can realize the differentiation to amphiploid G-tetra-stranded structure and other nucleic acid constructs; The present invention only just need can identify DNA secondary structure G-tetra-stranded structure with simple ultraviolet lamp or fluorescence spectrum, fast, simple to operate, with low cost.
3) probe of the present invention is exempted from mark fluorescent and is detected amphiploid G-tetra-serobila, does not affect its conformation and thermostability.
4) probe of the present invention
1be 0.65nM to the detection sensitivity of amphiploid G-tetra-serobila G2T1, probe
21.6nM can be reached to the detection sensitivity of amphiploid G-tetra-serobila G2T2.
5) probe of the present invention can enter cell, can detect, this type of probe is had broad application prospects to G-tetra-stranded structure in cell.
Accompanying drawing explanation
Fig. 1 is probe
1to the luminous situation of different configuration DNA under ultraviolet lamp and fluorescence intensity histogram;
Fig. 2 is probe 1 fluorescence intensity change figure at 530nm when adding the different configuration DNA of different concns;
Fig. 3 is monoploid G-tetra-serobila G1 titration probe
1fluorescence spectrum figure; (a) monoploid G1 titration probe
1the fluorescence spectrum of (0.5 μM); (b) probe
1the fluorescence intensity of (0.5 μM) and the linear graph of monoploid G1 concentration.Experiment condition: 10mMTris-HClbuffer, 100mMNaCl, pH7.2. λ
ex/ λ
em=355/530nm;
Fig. 4 is amphiploid G-tetra-serobila G2T1 titration probe
1fluorescence spectrum; (a) probe
1the fluorescence spectrum of titration amphiploid G2T1; (b) probe
1the fluorescence intensity of (0.5 μM) and the linear graph of amphiploid G2T1 concentration.Experiment condition: 10mMTris-HClbuffer, 100mMNaCl, pH7.2. λ
ex/ λ
em=355/530nm;
Fig. 5 is amphiploid G-tetra-serobila G2T2 titration probe
1fluorescence spectrum; (a) probe
1the fluorescence spectrum of titration amphiploid G2T2; (b) probe
1the fluorescence intensity of (0.5 μM) and the linear graph of amphiploid G2T2 concentration.Experiment condition: 10mMTris-HClbuffer, 100mMNaCl, pH7.2. λ
ex/ λ
em=355/530nm;
Fig. 6 is probe
1(0.5 μM) G1 (a) to different concns (0 ~ 0.5 μM) and the sensitivity technique figure of G2T1 (b);
Fig. 7 is that monoploid G-tetra-serobila G1 and amphiploid G-tetra-serobila G2T1 exist probe
1time CD spectrum;
Fig. 8 is that Hela cancer cells is at λ
ex=488nm place is not containing (a, d and g) with containing (b, e and h) probe
1confocal fluorescent cell imaging figure during (5 μMs) reaction 3h, and stacking diagram (c, f and i).Figure a ~ c, d ~ f and g ~ i represent blank cancer cells (Control) respectively, added the cancer cells (DNase) of DNA digestive ferment and have added the cancer cells (RNase) of RNA digestive ferment;
Fig. 9 is probe
2to the luminous situation of different configuration DNA under ultraviolet lamp and fluorescence intensity histogram;
Figure 10 is monoploid G-tetra-serobila G1 titration probe
2fluorescence spectrum figure; (a) probe
2the fluorescence spectrum of titration monoploid G1; (b) probe
2the fluorescence intensity of (0.5 μM) and the linear graph of monoploid G1 concentration.Experiment condition: 10mMTris-HCl buffered soln, 100mMNaCl, pH7.2. λ
ex/ λ
em=355/530nm;
Figure 11 is amphiploid G-tetra-serobila G2T1 titration probe
2fluorescence spectrum figure; (a) probe
2the fluorescence spectrum of titration amphiploid G2T1; (b) probe
2the fluorescence intensity of (0.5 μM) and the linear graph of amphiploid G2T1 concentration.Experiment condition: 10mMTris-HCl buffered soln, 100mMNaCl, pH7.2. λ
ex/ λ
em=355/530nm;
Figure 12 is amphiploid G-tetra-serobila G2T2 titration probe
2fluorescence spectrum figure; (a) probe
2the fluorescence spectrum of titration amphiploid G2T2; (b) probe
2the fluorescence intensity of (0.5 μM) and the linear graph of amphiploid G2T2 concentration.Experiment condition: 10mMTris-HCl buffered soln, 100mMNaCl, pH7.2. λ
ex/ λ
em=355/530nm;
Figure 13 is probe
2(0.5 μM) G1 (a), G2T1 (b) to different concns (0 ~ 0.5 μM) and the sensitivity technique figure of G2T2 (c);
Figure 14 is that monoploid G-tetra-serobila G1 and amphiploid G-tetra-serobila G2T2 exist probe
2time CD spectrum.
Embodiment
The symmetrical Berberine dimer derivate that ether chain connects, its molecular structure is such as formula shown in I:
(I), wherein n is 2 or 3.
The symmetrical Berberine dimer derivate that above-mentioned ether chain connects is as the application of G-tetra-stranded structure detection probes.
Preferably, above-mentioned G-tetra-serobila DNA structure is amphiploid G-tetra-stranded structure.
Preferably, above-mentioned amphiploid G-tetra-stranded structure is 5 '-A (GGGTTA)
7g
3-3 ' or 5 '-A (GGGTTA)
4(TTAGGG)
4-3 ' amphiploid G-tetra-stranded structure of Sequence composition.
The symmetrical Berberine dimer derivate that above-mentioned ether chain connects detects the method for amphiploid G-tetra-stranded structure, its the method is the symmetrical Berberine dimer derivate solution by adding the connection of ether chain in DNA solution to be measured, mixing, detect the fluorescence intensity of mixed solution, if the fluorescence intensity of DNA solution to be measured enhances at least 160 times, illustrate in DNA to be measured containing amphiploid G-tetra-stranded structure.
Preferably, excitation wavelength during above-mentioned fluorescence intensity is 355nm, and emission wavelength is 530nm.
Preferably, the solvent of above-mentioned DNA solution to be measured is for containing Na
+the Tris-HCl damping fluid of pH6.0 ~ 8.0.
Preferably, the solvent of the symmetrical Berberine dimer derivate solution of above-mentioned ether chain connection is for containing Na
+the Tris-HCl damping fluid of pH6.0 ~ 8.0.
Preferably, above-mentioned Tris-HCl damping fluid is containing 95 ~ 105mMNa
+.
Preferably, the mol ratio of symmetrical Berberine dimer derivate that above-mentioned DNA to be measured is connected with ether chain is (1 ~ 10): 1.
The symmetrical Berberine dimer derivate that above-mentioned ether chain connects detects the method for amphiploid G-tetra-stranded structure, comprises the following steps:
1) concentration of DNA in DNA solution to be measured is measured;
2) in DNA solution to be measured, add the symmetrical Berberine dimer derivate that ether chain connects, mixing, detect the fluorescence intensity of mixed solution;
3) the standard fluorescence spectrum that the fluorescence intensity upper pacing obtained and described derivative detect amphiploid G-tetra-stranded structure is compared, can judge whether containing amphiploid G-tetra-stranded structure in DNA to be measured, and determine specifically containing which kind of amphiploid G-tetra-stranded structure.
The symmetrical Berberine dimer derivate that above-mentioned ether chain connects detects the method for G-tetra-stranded structure in cell, the method is after cell and described derivative are incubated 2.5 ~ 3.5h altogether, under fluorescent microscope, fluoroscopic examination is carried out to cell, if cell produces obvious fluorescence, illustrate in cell containing G-tetra-stranded structure.
Below in conjunction with specific embodiment, the invention will be further described, but be not limited thereto.
embodiment 1: the synthesis of probe 2
The fluorescent probe that the present invention detects amphiploid G-tetra-serobila is the symmetrical Berberine dimer derivate that ether chain connects, and its molecular structure is such as formula shown in I:
(I), wherein n is 2 or 3; As n=2, it is fluorescent probe
1(hereinafter referred to as probe
1), as n=3, be fluorescent probe
2(hereinafter referred to as probe
2).
The symmetrical Berberine dimer derivate 2(probe that ether chain of the present invention connects
2), according to document (
med.Chem.Commun.,
2013,
4, 1400-1404) and method synthesis, its synthetic route is as shown in (II):
(II), wherein
2be probe
2.
embodiment 2: the synthesis of probe 1
Probe
1use similar probe
2method synthesis.
1) synthesis of triethylene glycol two p-methyl benzenesulfonic acid ester: by triethylene glycol (2.25g, 15mmol) with p-methyl benzene sulfonic chloride (5.72g, 30mmol) dissolve with 15ml anhydrous methylene chloride, be cooled to 0 DEG C, gradation under agitation condition, add KOH (6.72g, 120mmol) on a small quantity, holding temperature 0 DEG C.Under 0 DEG C of condition after stirring reaction 5h, add 15ml methylene dichloride and 30ml frozen water termination reaction.Be separated organic layer, water layer uses 15ml dichloromethane extraction twice respectively, merge organic layer, and with 10ml water washing once, with underpressure distillation after anhydrous sodium sulfate drying remove desolventizing obtain white object compound solid 6.6g, yield 96%.
1HNMR(400MHz,CDCl
3)
δ7.81(d,
J=8.4Hz,4H),7.36(d,
J=8.0Hz,4H),4.16(t,
J=4.8Hz,4H),3.67(t,
J=4.8Hz,4H),3.55(s,4H),2.46(s,6H);
13CNMR(100MHz,CDCl
3)δ144.8,132.9,129.8,127.9,70.6,69.1,68.7,21.6；ESI-MS:
m/z481.8([M+Na]
+)。
2) probe
1synthesis: berberrubine (310mg, 0.96mmol) and triethylene glycol two p-methyl benzenesulfonic acid ester (200mg, 0.44mmol) acetonitrile (20mL) is dissolved, stirring and refluxing reaction 84h under 90 DEG C of conditions.Underpressure distillation removes desolventizing, after gained crude product Chioro-anion exchange resin is carried out ion-exchange, through reversed-phase silica gel column chromatography purifying (eluent: H
2o-CH
3oH=100/0 ~ 90/10, v/v), obtain glassy yellow target-probe compound triethylene glycol-9-two Berberine, i.e. probe
1290mg, productive rate 87%.
This probe
1be soluble in organic solvent and the water such as methyl alcohol, ethanol, DMSO, m.p.163.4 ~ 164.8 DEG C;
1hNMR (400MHz, DMSO-
d 6)
δ9.71 (s, 2H), 8.89 (s, 2H), 8.11 (d,
j=9.2Hz, 2H), 7.93 (d,
j=9.2Hz, 2H), 7.68 (s, 2H), 7.05 (s, 2H), 6.14 (s, 4H), 4.94 (t,
j=5.6Hz, 4H), 4.32 (t,
j=4.0Hz, 4H), 3.95 (s, 6H), 3.81 (t,
j=4.4Hz, 4H), 3.67 (s, 4H), 3.20 (t,
j=6.2Hz, 4H);
13cNMR (100MHz, DMSO) δ 150.7,150.1,148.0,145.7,142.8,137.7,133.2,130.9,126.7,123.9,122.0,120.7,120.5,108.7,105.7,102.5,73.6,70.1,69.8,57.3,55.8,26.7; ESI-MS:
m/z379.6 ([M-2Cl]
2+) and HRMSforC
44h
42n
2o
10([M-2Cl]
2+) Calcd.:379.1420, found:379.1414.
embodiment 3: probe 1 is for the detection of extracellular DNA sample
1, sample is prepared
DNA sample: DNA sample is purchased from upper marine life Sheng Gong Technology Co., Ltd., appropriate DNA is dissolved in the buffered soln (10mMTris of Tris-HCl, 100mMNaCl, pH7.2), slowly room temperature is down to after 95 DEG C of heating 5min, 4 DEG C of overnight incubation, finally measure the absorbancy at 260nm place on SMA1000, calculate the concentration of DNA according to langbobier law.
The DNA sample sequence of test comprises:
Ss-ATDNA:5 '-CGCGATATCGCG-3 ', single stranded DNA;
Ds-ATDNA:ds (5 '-CGCGATATCGCG-3 '), double-stranded DNA;
Ss-CGDNA:5 '-CGCGCGCGCGCG-3 ', single stranded DNA;
Ds-CGDNA:ds (5 '-CGCGCGCGCGCG-3 '), double-stranded DNA;
CtDNA: double-stranded linear DNA;
C-kit:5 '-(CG
3)
2(CG)
2(AG
3)
2t-3 ', intramolecular parallel monoploid G-tetra-serobila DNA;
G1 (K
+): 5 '-A (G
3tTA)
3g
3-3 ', intramolecular parallel and antiparallel mixing monoploid G-tetra-serobila DNA;
G1:5 '-A (G
3tTA)
3g
3-3 ', antiparallel monoploid G-tetra-serobila DNA in molecule;
G2T1:5 '-A (GGGTTA)
7g
3-3 ', amphiploid G-tetra-serobila DNA;
G2T2:5 '-A (GGGTTA)
4(TTAGGG)
4-3 ', amphiploid G-tetra-serobila DNA;
Detect amphiploid G-tetra-stranded structure:
Probe
1solution: take a certain amount of probe
1solid, becomes the storage liquid of 50 μMs with Tris-HCl buffer solution, then is diluted to 0.5 μM of solution for test with damping fluid.
Buffered soln: 10mMTris-HCl buffered soln, containing 100mMNaCl, pH7.2.
2. detect
2.1. visual inspection under ultraviolet lamp
By probe
1solution mixes with testing sample, probe after mixing
1concentration be 0.5 μM, the concentration of sample DNA is 5 μMs, under mixed solution is placed on ultraviolet lamp, visual inspection.If fluorescence does not obviously strengthen, then can be judged as non-G-tetra-stranded structure; If the fluorescence of system obviously strengthens, then can judge that testing sample is G-tetra-serobila DNA structure; For the DNA sample that fluorescence obviously strengthens, what fluorescence was the strongest can judgement sample be amphiploid G-tetra-serobila G2T1 structure.
As illustrated in fig. 1 and 2, Fig. 1 is 530nm place to detected result, the probe of 0.5 μM
1add fluorescence color changing conditions and fluorescence intensity in 5 μMs of different configuration DNA sample, therefrom can find out, add probe
1different configuration DNA sample fluorescence intensity under ultraviolet light different, ss-ATDNA, ds-ATDNA, ss-CGDNA, ds-CGDNA, CTDNA wherein without G-tetra-stranded structure are the same with blank group (Blank), almost do not have fluorescence; And c-kit, the G1(K containing monoploid G-tetra-stranded structure
+) and G1 group have more weak fluorescence, not obvious; And containing having obvious fluorescence in G2T1 and the G2T2 group of amphiploid G-tetra-stranded structure, Fluorescence Increasing at least 200 times, especially the Fluorescence Increasing amplitude of G2T1 group is the most obvious.
The above results illustrates fluorescent probe of the present invention
1can specific detection amphiploid G-tetra-stranded structure, if testing sample adds probe
1after, its fluorescence increase at least 200 times, can judge in this DNA containing amphiploid G-tetra-stranded structure.
Fig. 2 is 0.5 μM of probe
1when adding in the DNA of different concns (0 ~ 5 μM) different configuration, at the fluorescence intensity change figure at 530nm place, Fig. 2 further demonstrate that the result of Fig. 1: along with the continuous increase of DNA concentration, ss-ATDNA, ds-ATDNA, ss-CGDNA, ds-CGDNA and CTDNA group does not still almost have fluorescence, containing c-kit, G1(K of monoploid G-tetra-stranded structure
+) and the fluorescence of G1 group slightly to strengthen but not obvious, and the fluorescence in amphiploid G-tetra-stranded structure G2T1 and G2T2 significantly strengthens, and especially the Fluorescence Increasing amplitude of G2T1 is the most obvious.
The above results illustrates, fluorescent probe of the present invention
1there is obvious dose relationship in the concentration of the fluorescence intensity sent and amphiploid G-tetra-serobila, further illustrates fluorescent probe
1can specific detection amphiploid G-tetra-stranded structure.
2.2. fluorescence spectrometry
Probe
1concentration be 0.5 μM, toward probe
1drip different testing sample solutions in solution, after piping and druming evenly, by the fluorescent emission of fluorescence spectrometry system, sosbestic point 355nm is set as excitation wavelength.
Fig. 3 ~ 5 are respectively stationary probe
1concentration is 0.5 μM
,continuous increase monoploid G1(Fig. 3), amphiploid G2T1(Fig. 4), amphiploid G2T2(Fig. 5) concentration time probe
1fluorescence spectrum variation diagram, DNA concentration increases to till fluorescence spectrum no longer changes.As can be seen from Figure 3, along with the continuous increase of DNA concentration, the fluorescence of monoploid G1 group slightly strengthens, and maximum fluorescence intensity is only about 120; As can be seen from Fig. 4 ~ 5, along with the continuous increase of DNA concentration, the fluorescence intensity of amphiploid G-tetra-serobila G2T1 and G2T2 group strengthens fast, in G2T1 group when DNA concentration is only 0.25 μM, its fluorescence intensity just reaches about 120, along with the increase of DNA concentration, fluorescence intensity can reach more than 240 (Fig. 4); Equally, the fluorescence intensity of G2T2 group also can reach more than 220 (Fig. 5) along with the increase of DNA concentration.
The above results illustrates, according to probe
1detect the standard fluorescence spectrum of monoploid G1, amphiploid G2T1, amphiploid G2T2, add probe by the testing sample DNA of concentration known
1, the fluorescence intensity recorded and standard fluorescence spectrum are compared, can judge whether containing amphiploid G-tetra-stranded structure in DNA to be measured, and determine specifically containing which kind of amphiploid G-tetra-stranded structure.
On the basis of the change in fluorescence spectrum of Fig. 3 ~ 5, according to following formula:
To add probe after DNA
1fluorescence intensity
ito DNA concentration [DNA]
0carry out curve fitting, as Fig. 3 ~ 5, obtain probe
1with the binding constant of G-tetra-stranded structure
k a.I in formula
0the fluorescence intensity of probe at 530nm place during for not adding DNA, [D]
0for probe
1concentration, Δ
i' be constant.Probe is obtained by Fig. 3 ~ 5
1be respectively with the binding constant of G1, G2T1 and G2T2: (2.8 ± 0.2) × 10
5m
-1, (2.4 ± 0.7) × 10
7m
-1, (4.4 ± 0.5) × 10
6m
-1.Probe is further illustrated by binding constant
1monoploid G-tetra-serobila is significantly higher than to amphiploid G-tetra-serobila binding ability, wherein the strongest to the selective binding ability of amphiploid G2T1.
The above results illustrates, if after DNA sample to be measured adds fluorescent probe of the present invention, the fluorescence intensity of system strengthens more than 200 times, then can judge in DNA to be measured containing amphiploid G-tetra-stranded structure.
embodiment 4: probe 2 is for the detection of extracellular DNA sample
By the probe of preparation
2carry out the detection of the DNA sample similar to embodiment 3, itself and embodiment 3 middle probe
1operation consistent, therefore not repeat.
As shown in Figure 9, ss-ATDNA, ds-ATDNA, ss-CGDNA, ds-CGDNA in figure, CTDNA are the same with blank group (Blank), and fluorescence does not almost strengthen, monoploid G-tetra-stranded structure c-kit, G1(K for ultraviolet lamp detected result
+) and G1 group fluorescence slightly to strengthen but not obvious, and the fluorescence in amphiploid G-tetra-stranded structure G2T1, G2T2 group significantly strengthens, and especially the Fluorescence Increasing amplitude of G2T2 group is the most obvious.
The above results illustrates fluorescent probe of the present invention
2can specific detection amphiploid G-tetra-stranded structure, if testing sample adds probe
2after, its fluorescence increase at least 160 times, can judge in this DNA containing amphiploid G-tetra-stranded structure.
Figure 10 ~ 12 are respectively stationary probe
2concentration is 0.5 μM
,the probe during concentration of continuous increase monoploid G1, amphiploid G2T1, amphiploid G2T2
2fluorescence spectrum variation diagram, DNA concentration increases to till fluorescence spectrum no longer changes.When detecting containing monoploid G-tetra-stranded structure G1 group, when DNA concentration reaches 4 μMs, its fluorescence intensity is not also more than 80(Figure 10); When detecting containing amphiploid G-tetra-stranded structure G2T1, when DNA concentration reaches 2.5 μMs, maximum fluorescence intensity can be reached and be about 260(Figure 11); When detecting containing amphiploid G-tetra-stranded structure G2T2, when DNA concentration is only 0.5 μM, its fluorescence intensity just can increase more than 200 times (Figure 12); Thus, probe can be judged
2under lower concentration, when detecting G-tetra-stranded structure, that fluorescence is the strongest is amphiploid G-tetra-stranded structure G2T2.
The above results illustrates, according to probe
2detect the standard fluorescence spectrum of monoploid G1, amphiploid G2T1, amphiploid G2T2, add probe by the testing sample DNA of concentration known
2, the fluorescence intensity recorded and standard fluorescence spectrum are compared, can judge whether containing amphiploid G-tetra-stranded structure in DNA to be measured, and determine specifically containing which kind of amphiploid G-tetra-stranded structure.
The basis of Figure 10 ~ 12 carries out curve fitting, obtains probe
2with the binding constant of G1, G2T1 and G2T2: (3.9 ± 0.5) × 10
5m
-1, (2.6 ± 0.2) × 10
6m
-1, (2.0 ± 0.5) × 10
7m
-1.Probe is further illustrated by binding constant
2monoploid G-tetra-serobila is significantly higher than to the binding ability of amphiploid G-tetra-serobila, wherein the strongest to the selective binding ability of amphiploid G2T2.
The above results illustrates, if after DNA sample to be measured adds fluorescent probe of the present invention, the fluorescence intensity of system strengthens more than 160 times, then can judge in DNA to be measured containing amphiploid G-tetra-stranded structure.
enforcement case 5: the sensitivity technique of probe 1 pair of G-tetra-serobila DNA structure
Detectability experiment is tested on luminoscope.
Get 0.5 μM of probe
1solution, measures the fluorescence spectrum of solution using 355nm as excitation wavelength, and excite and launch that seam is wide is 3nm, sweep limit is 365-700nm, and the absorbance of continuously measured 5 solution, then calculates standard deviation δ.According to the data of fluorometric titration, choose the last period data and carry out fitting of a straight line, detectability is 3 δ/slope (slope is the slope of straight line).
As shown in Figure 6, calculation result shows probe to experimental result
1be 6.0nM to monoploid G1 detection sensitivity, be 0.65nM to antiparallel amphiploid G2T1 detection sensitivity, probe is described
1to amphiploid G2T1, there is higher detection sensitivity.
enforcement case 6: the sensitivity technique of probe 2 couples of G-tetra-serobila DNA structures G1, G2T1 and G2T2
With the probe of preparation
2for example, carry out the detection of the DNA sample similar to embodiment 6, compound in its result and embodiment 6
1unanimously.
As shown in figure 13, calculation result shows probe to experimental result
2be 36.9nM to monoploid G1 detection sensitivity, be 7.0nM to amphiploid G2T1 detection sensitivity, be 1.6nM to amphiploid G2T2 detection sensitivity, probe is described
2to amphiploid G-tetra-stranded structure, there is good detection sensitivity.
embodiment 7: probe 1 couple of G-tetra-serobila DNA structure G1 and G2T1 conformation and heat stability test
CD spectrum is containing Na
+test in damping fluid (10mMTris-HCl, pH7.2 containing 100mMNaCl).The wide 4mm of cuvette, reaction solution is 700 μ L, and sweep limit is 220-340nm.In titration experiments, to containing in 5 μMs of G1 or 5 μM of G2T1 solution, drip probe gradually
1, test CD spectrum.Add probe at every turn
1after, measure after stirring, measuring terminal is that CD signal no longer changes.Attention: do not have bubble in cuvette wall.Use Origin6.0 software data processing.
Experimental result as shown in Figure 7, at Na
+in damping fluid, people's telomere G-tetra-serobila (G1 and G2T1) has large negative peak and posivtive spike respectively at 265nm and 290nm, and now System forming antiparallel G-tetra-stranded structure is described; Probe is added along with in DNA solution
1, find all not change in the absorption peak strength at 265nm and 290nm place, show probe
1do not stablize the antiparallel conformation of people's telomere G-tetra-serobila DNA, i.e. probe
1the conformation of G-tetra-serobila is not affected.
In CD-Melting experiment, containing 4 μMs of G1 or 2 μM G2T1 in damping fluid, gather the absorption value (temperature interval is 5 DEG C) at 295nm place in 20 DEG C to 95 DEG C temperature ranges.Origin6.0 software data processing, obtains T
dNA.Then, containing 4 μMs of G1 or 2 μM G2T1 in damping fluid, then 8 μMs of probes are added wherein respectively
1, gather the absorption value (temperature interval is 5 DEG C) at 295nm place in 20 DEG C to 95 DEG C temperature ranges.Origin6.0 software data processing, obtains T
m.Wherein T
m=T
m-T
dNA, T
mrefer to and add probe
1the fusing point of rear DNA, T
dNArefer to the fusing point of DNA itself.
Experimental result shows, probe
1to the T of G1
m=-0.7 ± 0.1 DEG C, to the T of G2T1
m=-0.5 ± 0.1 DEG C, this result illustrates probe
1the thermostability of G-tetra-serobila is not also affected.
enforcement case 8: probe 2 couples of G-tetra-serobila DNA structure G1 and G2T2 conformation and heat stability test
With the probe of preparation
2for example, carry out the detection of the DNA sample similar to embodiment 7, its result and embodiment 7 middle probe
1similar.
Experimental result as shown in figure 14, at Na
+in damping fluid, people's telomere G-tetra-serobila (G1 and G2T2) has large negative peak and posivtive spike respectively at 265nm and 290nm, and now System forming antiparallel G-tetra-stranded structure is described; Probe is added along with in DNA solution
2, find all not change in the absorption peak strength at 265nm and 290nm place, show probe
2do not stablize the antiparallel conformation of people's telomere G-tetra-serobila DNA; I.e. probe
2the conformation of G-tetra-serobila is not affected.
In CD-Melting experiment, probe
2to the T of G1
m=-8.9 ± 0.3 DEG C, to the T of G2T2
m=-1.2 ± 0.2 DEG C, this result illustrates probe
2the thermostability of antiparallel amphiploid G-tetra-serobila G2T1 is not also affected.
enforcement case 9: the detection of G-tetra-serobila DNA in probe 1 pair of cell
What this experiment adopted is Hela cervical cancer cell, and nurturing an environment is the foetal calf serum containing 10%, the PRMI-1640 substratum of dual anti-(penicillin and the Streptomycin sulphate) of 1%, in 37 DEG C, and 5%CO
2cell culture incubator in cultivate.Get 3.0 × 10
5individual cell in good condition to be inoculated in the glass dish of 3cm and overnight incubation, adds sterilized probe
1mother liquor, making it to mix rear concentration is 7 μMs, at 37 DEG C, 5%CO
23h is educated altogether with cell under environment.After the time, discard nutrient solution, and carefully rinse twice by the PBS solution of 0.1M, discard washing fluid, add 2mLPBS, finally use the radium-shine confocal fluorescent microscopic examination of FV1000,60 times of eyepieces, excitation wavelength is 488nm, experiment repetition twice.
As shown in Figure 8, wherein Fig. 8 a ~ c is respectively Hela cervical cancer cell and does not add probe experimental result
1, add probe
1with the superposition confocal fluorescent image of the first two collection of illustrative plates, this common Coloration experiment illustrates probe
1have the reaction of significant fluorescence developing to nucleus, but the reaction of this fluorescence developing does not occur in whole nucleus, may be tenuigenin or part cell benevolence, possible cause be probe
1molecular structure is larger, and whole molecular structure enters in nucleus more difficult.Because the cell benevolence in nucleus is the region that rDNA is transcribed into rRNA, in other words, be the G-tetra-serobila domain of the existence in rDNA, in order to confirm probe further
1whether the target spot of fluorescence developing is cell benevolence, and we have done again following thymus nucleic acid and Yeast Nucleic Acid digestion experiment.
Thymus nucleic acid and Yeast Nucleic Acid digestion experiment: Hela cervical cancer cell first fixes 15 minutes with 4% paraformaldehyde, to be fixed completely after rinse 2 times with 0.1MPBS, then the Triton permeation cell film 30 minutes of 0.3% is used, after the time, carefully rinse ware twice at the bottom of glass with 0.1MPBS, residual Triton is cleaned up; With the deoxyribonuclease (RNase-freeDNaseI) adding 100 units/mL successively in three cells processed, the rnase (DNaseandProtease-freeRNaseA) of 100 units/mL or add PBS in contrast, carry out mark, at the bottom of three glass, ware is all at 37 DEG C, 5%CO
2react 2h under environment to digest, then add probe
1its final concentration is made to be 7 μMs, at 37 DEG C, 5%CO
23h is cultivated under environment; Reacted rear 0.1MPBS and carefully washed twice, discarded washing fluid, add 2mLPBS, taken pictures with the radium-shine confocal fluorescent microscopic examination of FV1000,60 times of eyepieces, excitation wavelength is 488nm, experiment repetition twice.
As shown in Figure 8, wherein Fig. 8 d ~ f does not add probe through the Hela cancer cells of DNase process to detected result
1, add probe
1with the superposition confocal fluorescent image of the first two collection of illustrative plates, as can be seen from Fig. 8 e and 8f: the fluorescence developing in nucleus obviously disappears; Fig. 8 g ~ i does not add probe through the Hela cancer cells of RNase process
1, add probe
1with the superposition confocal fluorescent image of the first two collection of illustrative plates, as can be seen from Fig. 8 h and 8i: the fluorescence developing in nucleus is not by the impact of RNA digestive ferment.Above-mentioned experimental result illustrates: probe
1certain part enters cell benevolence, occurs to combine and fluorescence developing occurs to react with G-tetra-serobila in the rDNA in cell benevolence.Obtain as drawn a conclusion thus: probe
1cell can be entered fluoroscopic examination is carried out to G-tetra-stranded structure.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. the symmetrical Berberine dimer derivate of ether chain connection, its molecular structure is such as formula shown in I:
(I), wherein n is 2 or 3.
2. the symmetrical Berberine dimer derivate that connects of ether chain according to claim 1 is as the application of G-tetra-stranded structure detection probes.
3. application according to claim 2, is characterized in that: described G-tetra-serobila DNA structure is amphiploid G-tetra-stranded structure.
4. application according to claim 3, is characterized in that: described amphiploid G-tetra-stranded structure is 5 '-A (GGGTTA)
7g
3-3 ' or 5 '-A (GGGTTA)
4(TTAGGG)
4-3 ' amphiploid G-tetra-stranded structure of Sequence composition.
5. the symmetrical Berberine dimer derivate that ether chain according to claim 1 connects detects the method for amphiploid G-tetra-stranded structure, it is characterized in that: the method is the symmetrical Berberine dimer derivate solution by adding the connection of ether chain in DNA solution to be measured, mixing, detect the fluorescence intensity of mixed solution, if the fluorescence intensity of DNA solution to be measured enhances at least 160 times, illustrate in DNA to be measured containing amphiploid G-tetra-stranded structure.
6. the method according to right mark 5, is characterized in that: the solvent of described DNA solution to be measured is for containing Na
+the Tris-HCl damping fluid of pH6.0 ~ 8.0.
7. the method according to right mark 5, is characterized in that: the solvent of the symmetrical Berberine dimer derivate solution that described ether chain connects is for containing Na
+the Tris-HCl damping fluid of pH6.0 ~ 8.0.
8. the method according to right mark 5, is characterized in that: the mol ratio of the symmetrical Berberine dimer derivate that described DNA to be measured is connected with ether chain is (1 ~ 10): 1.
9. the symmetrical Berberine dimer derivate that ether chain according to claim 1 connects detects the method for amphiploid G-tetra-stranded structure, it is characterized in that: comprise the following steps:
1) concentration of DNA in DNA solution to be measured is measured;
2) in DNA solution to be measured, add the symmetrical Berberine dimer derivate that ether chain connects, mixing, detect the fluorescence intensity of mixed solution;
3) the standard fluorescence spectrum that the fluorescence intensity upper pacing obtained and described derivative detect amphiploid G-tetra-stranded structure is compared, can judge whether containing amphiploid G-tetra-stranded structure in DNA to be measured, and determine specifically containing which kind of amphiploid G-tetra-stranded structure.
10. the symmetrical Berberine dimer derivate that ether chain according to claim 1 connects detects the method for G-tetra-stranded structure in cell, it is characterized in that: the method is after cell and described derivative are incubated 2.5 ~ 3.5h altogether, under fluorescent microscope, fluoroscopic examination is carried out to cell, if cell produces obvious fluorescence, illustrate in cell containing G-tetra-stranded structure.
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| CN106008560A (en) * | 2016-05-18 | 2016-10-12 | 中国医学科学院生物医学工程研究所 | Preparation method and application of diberberine derivative |
| CN110105352A (en) * | 2019-04-18 | 2019-08-09 | 南方医科大学 | A kind of jamaicin -360A conjugate and its preparation method and application |
| CN110183439A (en) * | 2019-04-18 | 2019-08-30 | 南方医科大学 | A kind of jamaicin-Pyridostatin conjugate and its preparation method and application |
| CN114295594A (en) * | 2021-12-06 | 2022-04-08 | 贵州理工学院 | Turn on type fluorescence sensor for screening triple helix DNA intercalators based on molecular beacon |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106008560A (en) * | 2016-05-18 | 2016-10-12 | 中国医学科学院生物医学工程研究所 | Preparation method and application of diberberine derivative |
| CN110105352A (en) * | 2019-04-18 | 2019-08-09 | 南方医科大学 | A kind of jamaicin -360A conjugate and its preparation method and application |
| CN110183439A (en) * | 2019-04-18 | 2019-08-30 | 南方医科大学 | A kind of jamaicin-Pyridostatin conjugate and its preparation method and application |
| CN110183439B (en) * | 2019-04-18 | 2021-10-29 | 南方医科大学 | Berberine-Pyridostatin conjugate as well as preparation method and application thereof |
| CN114295594A (en) * | 2021-12-06 | 2022-04-08 | 贵州理工学院 | Turn on type fluorescence sensor for screening triple helix DNA intercalators based on molecular beacon |
| CN114295594B (en) * | 2021-12-06 | 2023-09-19 | 贵州理工学院 | "turn on" type fluorescence sensor based on molecular beacon screening triple helix DNA intercalator |
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