CN109628645A - For detecting the DNA tetrahedron probe and kit of virus - Google Patents
For detecting the DNA tetrahedron probe and kit of virus Download PDFInfo
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- CN109628645A CN109628645A CN201910058979.0A CN201910058979A CN109628645A CN 109628645 A CN109628645 A CN 109628645A CN 201910058979 A CN201910058979 A CN 201910058979A CN 109628645 A CN109628645 A CN 109628645A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The present invention provides a kind of for detecting the DNA tetrahedron probe and kit of virus.The probe generally DNA tetrahedral structure, extend DNA identification sequence and stem sequence on a tetrahedral vertex, the stem sequence can identify that sequence is combined with DNA and form loop-stem structure, donor fluorescent molecule and acceptor fluorescence molecule are modified on the DNA tetrahedron probe, wherein the donor fluorescent molecule is located in DNA identification sequence, and the distance when being not added with test object between the donor fluorescent molecule and acceptor fluorescence molecule is not more than 7nm.Rapidly and efficiently virus can be detected using above-mentioned probe.
Description
Technical field
The present invention relates to a kind of for detecting the DNA tetrahedron probe and kit of virus.
Background technique
In recent years, with the development of DNA chemical synthesising technology, DNA becomes a kind of biomaterial simple and easy to get, and DNA is glimmering
Light probe also has become to analyze the powerful of sensing and optical imagery.DNA tetrahedron have good bio-compatibility and
It is easy the property of coding.It is had existed in the prior art using DNA tetrahedron as skeleton, in conjunction with fluorescence resonance energy transfer
(FRET), constructing can be to DNA tetrahedron " nano-tweezers " (DNAtetrahedron that mRNA in living cells is imaged
Nanotweezer, DTNT).
Hepatitis type B virus (HBV), abbreviation hepatitis B are a kind of DNA virus, belong to Hepadnaviridae, can lead to
The generation of cirrhosis and liver cancer brings serious Disease Spectrum to the whole world.Normal host cell how is infected simultaneously about HBV at present
The process replicated in host cell has been achieved with common recognition.When HBV infects normal host cell, outer membrane, HBV are sloughed first
DNA double chain formed it is loose there is " notch " cyclic structure (rcDNA), by the effect of enzyme, space knot is converted into after polishing
Structure is the covalently closed circular DNA (covalently closed circular DNA, cccDNA) of superhelix.cccDNA
It is the primary template of hepatitis B pregenome RNA (pg RNA) duplication, pg RNA is then used as the template of reverse transcription.Although existing
Antiviral drugs can control the duplication of HBV virus, but cannot it is fully erased it.Since cccDNA " hiding " is in nucleus
In, once stopping treatment, the HBV in patient's liver can be reactivated.Pg RNA is transcribed by the cccDNA in infected liver cell core
It generates.
With the development of nucleic acid extraction purification technique and real-time fluorescence quantitative polymerase chain reaction (Real Time
Polymerase Chain Reaction, qPCR) and RT-polymerase chain reaction (Reverse Transcription
Polymerase Chain Reaction, RT-PCR) technology extensive use, have been realized in and HBV pg RNA quantified
Analysis, and have relevant detection kit.RNA is easy degradation, and method of the initial in vitro for HBV pg RNA detection operates
Next complex, time-consuming, and sensitivity is not high enough.
The detection and imaging of intracellular HBV pg RNA are realized in living cells level, will be able to achieve and HBV virion is entered
The dynamic process of host cell is invaded, and enters the transhipment after cell, the whereabouts of viral nucleic acid and albumen and positioning progress in real time
Dynamic monitoring, facilitate clear virus and membrane molecule adherency, in conjunction with, enter the dynamic mechanism of born of the same parents.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of for detecting the DNA of virus
Tetrahedron probe and kit, for solving low to viral diagnosis specificity in the prior art, the low problem of sensitivity, especially
HBV。
As shown in Figure 1, being constructed based on DNA tetrahedron nanostructure and FRET effect a kind of for detecting the DNA of virus
Tetrahedron namo fluorescence probe.By taking HBV as an example, which is expected to realize multiple sites in the HBV pgRNA in living cells
It carries out while detecting and being imaged.The advantage of this DNA tetrahedron nano-probe combination DNA tetrahedron nanostructure, bio-compatible
Property is good, is easy to encode, can be stabilized by cellular uptake and in the cell in complex biological system.We will form
The DNA terminal modified CY3 of tetrahedral A chain one, the interior modification CY5 of chain form molecular beacon probe.Can have between CY3 and CY5
The FRET phenomenon of effect, when being excited with the laser of CY3, the energy of CY3 can partially be transferred to CY5, so that CY5 is issued
Fluorescence.After target molecules are added, target molecule can be opened the loop-stem structure of probe, formed rigid duplex structure, led
The distance of CY3 and CY5 is caused to become remote, FRET phenomenon, which weakens, even to disappear.Pass through fluorescent molecule signal caused by FRET efficiency change
Variation, realize the conveniently detection of pgRNA；By the FRET-TDN-Probe that identifies a variety of pgRNA with transcrypted
The liver cancer cells of Hepatitis B virus-DNA are incubated for, and realize intracellular detection and imaging to intracellular multidigit point pgRNA.
In order to achieve the above objects and other related objects, the present invention provides a kind of DNA tetrahedron spy for detecting virus
Needle, the DNA probe generally tetrahedral structure extend stem sequence, the stem sequence on a tetrahedral vertex
DNA identification sequence is extended at 5 ' ends, and the stem sequence can identify that sequence is combined with DNA and form loop-stem structure,
It is modified with donor fluorescent molecule and acceptor fluorescence molecule on the DNA tetrahedron probe, wherein the donor fluorescent
Molecule is located in DNA identification sequence, when being not added with test object the donor fluorescent molecule and acceptor fluorescence molecule it
Between distance be not more than 7nm.
The excitation of the donor fluorescent molecule can induce acceptor molecule and issue fluorescence.
Preferably, the donor fluorescent molecule and acceptor fluorescence molecule are respectively selected from CY3 and CY5.
CY3 (cyanine-3) and CY5 (cyanine-5) is fluorescent molecule pair commonly used in the prior art.
Further, the DNA tetrahedron is assembled by tetra- DNA of A, B, C and D are single-stranded, and the stem sequence is from A chain
5 ' ends extend.Further, the donor fluorescent molecule is located at 5 ' ends of DNA identification sequence.
Further, the acceptor fluorescence molecule is located at 1/2 site of B chain between 1/4 site, at preferably 1/3 site.
1/2 site, 1/3 site, 1/4 site refer to that the length at 5 ' ends of site distance A chain is the 1/2,1/3,1/4 of B chain length.
In the preferred scheme, the acceptor fluorescence molecule is located at 5 ' end positions of A chain.
A, B, the C and D DNA single-stranded is the prior art.Wherein A chain nucleotide sequence such as SEQ ID NO.33:ACAT
TCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA.
B chain such as SEQ ID NO.34:TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCC
AATAC。
C chain such as SEQ ID NO.35:CAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCT
CTTC。
D chain such as SEQ ID NO.36:TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCT
CGCAT。
For example, the DNA identification sequence is selected from SEQ ID NO.3, SEQ ID NO.8, SEQ when needing to detect HBV
Any one in ID NO.13, SEQ ID NO.17 or SEQ ID NO.19.The DNA tetrahedron probe A chain after extension
Respectively as shown in NO.23~27 SEQ ID.
Further, the containing for DNA identification sequence is not less than 20 bases, then may cause probe lower than this length
It cannot be effectively combined with object to be detected.
Further, the stem sequence contain 6~8 bases.The base sequence of length in this way holds that loop-stem structure very
Easily it is opened.
For example, the acceptor fluorescence molecule is located at 5 ' end positions of A chain, the donor fluorescent when needing to detect HBV
Molecule is located at 5 ' ends of DNA identification sequence, and the DNA identifies that sequence extends from 5 ' ends of stem sequence, the A chain nucleotide after extension
Any one of sequence in SEQ ID NO.23-27.
Another aspect of the present invention provides a kind of pharmaceutical composition, and the combination is comprising above-mentioned for detecting virus
DNA tetrahedron probe and auxiliary material.
Another aspect of the present invention provides a kind of virus detection kit, and the kit includes above-mentioned for examining
Survey the DNA tetrahedron probe or pharmaceutical composition of virus.
Another aspect of the present invention provides above-mentioned DNA tetrahedron probe in preparing above-mentioned virus detection kit
Purposes.
Another aspect of the present invention provides aforementioned pharmaceutical compositions and is preparing the use in above-mentioned virus detection kit
On the way.
Another aspect of the present invention provides use of the above-mentioned DNA tetrahedron probe in the drug of preparation detection virus
On the way.
Another aspect of the present invention provides purposes of the aforementioned pharmaceutical compositions in the drug of preparation detection virus.
As described above, the DNA tetrahedron probe and kit for being used to detect virus of the invention, has below beneficial to effect
Fruit:
Can specific recognition virus, sensitivity with higher, by taking HBV as an example, some detections is limited up to 1nM, and special
The opposite sex, while detection time is rapid, can be realized within 30min.
Detailed description of the invention
Fig. 1 is shown as DNA tetrahedron probe of the present invention and plays a role schematic illustration.
Fig. 2 a is shown as in the embodiment of the present invention two the potential secondary structure analysis using software to FRET-TDN-Probe1
Figure.
Fig. 2 b is shown as in the embodiment of the present invention two the potential secondary structure analysis using software to FRET-TDN-Probe2
Figure.
Fig. 2 c is shown as in the embodiment of the present invention two the potential secondary structure analysis using software to FRET-TDN-Probe3
Figure.
Fig. 2 d is shown as in the embodiment of the present invention two the potential secondary structure analysis using software to FRET-TDN-Probe4
Figure.
Fig. 2 e is shown as in the embodiment of the present invention two the potential secondary structure analysis using software to FRET-TDN-Probe5
Figure.
Fig. 3 is shown as the gel electrophoresis figure of the embodiment of the present invention three.
Fig. 4 a is shown as the schematic diagram of FRET efficiency of the FRET to modification in one DNA chain of tetrahedron in the present invention.
Fig. 4 b is shown as FRET of the present invention to the assembling schematic diagram in tetrahedron top position.
Fig. 4 c is shown as the schematic diagram of FRET efficiency of the FRET of the present invention to modification in one DNA chain of tetrahedron.
Fig. 5 a is shown as the concentration of FRET-TDN-Probe1DNA target molecule in the embodiment of the present invention four, gradually increases from 0
Fluorescence signal figure when to 200nM.
Fig. 5 b is shown as the concentration of FRET-TDN-Probe2DNA target molecule in the embodiment of the present invention four, gradually increases from 0
Fluorescence signal figure when to 200nM.
Fig. 5 c is shown as the concentration of FRET-TDN-Probe3DNA target molecule in the embodiment of the present invention four, gradually increases from 0
Fluorescence signal figure when to 200nM.
Fig. 5 d is shown as the concentration of FRET-TDN-Probe4DNA target molecule in the embodiment of the present invention four, gradually increases from 0
Fluorescence signal figure when to 200nM.
Fig. 5 e is shown as the concentration of FRET-TDN-Probe5DNA target molecule in the embodiment of the present invention four, gradually increases from 0
Fluorescence signal figure when to 200nM.
Fig. 6 a is shown as FRET-TDN-Probe1 I562 when target DNA concentration gradually increases in the embodiment of the present invention four
(nm) numerical curve of/I664 (nm).
Fig. 6 b is shown as FRET-TDN-Probe2 I562 when target DNA concentration gradually increases in the embodiment of the present invention four
(nm) numerical curve of/I664 (nm).
Fig. 6 c is shown as FRET-TDN-Probe3 I562 when target DNA concentration gradually increases in the embodiment of the present invention four
(nm) numerical curve of/I664 (nm).
Fig. 6 d is shown as FRET-TDN-Probe4 I562 when target DNA concentration gradually increases in the embodiment of the present invention four
(nm) numerical curve of/I664 (nm).
Fig. 6 e is shown as FRET-TDN-Probe5 I562 when target DNA concentration gradually increases in the embodiment of the present invention four
(nm) numerical curve of/I664 (nm).
Fig. 7 a is shown as FRET-TDN-Probe1 550-750nm when various target molecules are added in the embodiment of the present invention five
Fluorescence intensity curves figure at wavelength.
Fig. 7 b is shown as FRET-TDN-Probe2 550-750nm when various target molecules are added in the embodiment of the present invention five
Fluorescence intensity curves figure at wavelength.
Fig. 7 c is shown as FRET-TDN-Probe3 550-750nm when various target molecules are added in the embodiment of the present invention five
Fluorescence intensity curves figure at wavelength.
Fig. 7 d is shown as FRET-TDN-Probe4 550-750nm when various target molecules are added in the embodiment of the present invention five
Fluorescence intensity curves figure at wavelength.
Fig. 7 e is shown as FRET-TDN-Probe5 550-750nm when various target molecules are added in the embodiment of the present invention five
Fluorescence intensity curves figure at wavelength.
Fig. 8 a is shown as the data statistics figure to 7a.
Fig. 8 b is shown as the data statistics figure to 8b.
Fig. 8 c is shown as the data statistics figure to 8c.
Fig. 8 d is shown as the data statistics figure to 8d.
Fig. 8 e is shown as the data statistics figure to 8e.
Fig. 9 a is shown as FRET-TDN-Probe1 in the embodiment of the present invention six when the target molecule exactly matched therewith is added
The fluorescence intensity curves figure of (0,10,20,30,40,50,60min) at 550-750nm wavelength.
Fig. 9 b is shown as FRET-TDN-Probe2 in the embodiment of the present invention six when the target molecule exactly matched therewith is added
The fluorescence intensity curves figure of (0,10,20,30,40,50,60min) at 550-750nm wavelength.
Fig. 9 c is shown as FRET-TDN-Probe3 in the embodiment of the present invention six when the target molecule exactly matched therewith is added
The fluorescence intensity curves figure of (0,10,20,30,40,50,60min) at 550-750nm wavelength.
Fig. 9 d is shown as FRET-TDN-Probe4 in the embodiment of the present invention six when the target molecule exactly matched therewith is added
The fluorescence intensity curves figure of (0,10,20,30,40,50,60min) at 550-750nm wavelength.
Fig. 9 e is shown as FRET-TDN-Probe5 in the embodiment of the present invention six when the target molecule exactly matched therewith is added
The fluorescence intensity curves figure of (0,10,20,30,40,50,60min) at 550-750nm wavelength.
Figure 10 a is shown as the target molecule that FRET-TDN-Probe1 is exactly matched therewith in addition in the embodiment of the present invention six
When I562 (nm)/I664 (nm) numerical curve figure within 1 hour.
Figure 10 b is shown as the target molecule that FRET-TDN-Probe2 is exactly matched therewith in addition in the embodiment of the present invention six
When I562 (nm)/I664 (nm) numerical curve figure within 1 hour.
Figure 10 c is shown as the target molecule that FRET-TDN-Probe3 is exactly matched therewith in addition in the embodiment of the present invention six
When I562 (nm)/I664 (nm) numerical curve figure within 1 hour.
Figure 10 d is shown as the target molecule that FRET-TDN-Probe4 is exactly matched therewith in addition in the embodiment of the present invention six
When I562 (nm)/I664 (nm) numerical curve figure within 1 hour.
Figure 10 e is shown as the target molecule that FRET-TDN-Probe5 is exactly matched therewith in addition in the embodiment of the present invention six
When I562 (nm)/I664 (nm) numerical curve figure within 1 hour.
Figure 11 a is shown as FRET-TDN-Probe1 in the embodiment of the present invention six and DNase I is being added and DNase is not added
I562 (nm)/I664 (nm) numerical curve figure in the case of I；Illustration is two groups of subsequent 550- after being separately added into target molecules
Fluorescence intensity curves figure at 750nm wavelength.
Figure 11 b is shown as FRET-TDN-Probe1 in the embodiment of the present invention six and DNase I is being added and DNase is not added
I562 (nm)/I664 (nm) numerical curve figure in the case of I；Illustration is two groups of subsequent 550- after being separately added into target molecules
Fluorescence intensity curves figure at 750nm wavelength.
Figure 11 c is shown as FRET-TDN-Probe1 in the embodiment of the present invention six and DNase I is being added and DNase is not added
I562 (nm)/I664 (nm) numerical curve figure in the case of I；Illustration is two groups of subsequent 550- after being separately added into target molecules
Fluorescence intensity curves figure at 750nm wavelength.
Figure 11 d is shown as FRET-TDN-Probe1 in the embodiment of the present invention six and DNase I is being added and DNase is not added
I562 (nm)/I664 (nm) numerical curve figure in the case of I；Illustration is two groups of subsequent 550- after being separately added into target molecules
Fluorescence intensity curves figure at 750nm wavelength.
Figure 11 e is shown as FRET-TDN-Probe1 in the embodiment of the present invention six and DNase I is being added and DNase is not added
I562 (nm)/I664 (nm) numerical curve figure in the case of I；Illustration is two groups of subsequent 550- after being separately added into target molecules
Fluorescence intensity curves figure at 750nm wavelength.
Figure 12 is shown as the gel electrophoresis picture in the embodiment of the present invention seven.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text
In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Following embodiment will illustrate the content of present invention by taking HBV as an example.
Material and facility:
Laboratory customary salt MgCl2(analyzing pure grade) is bought from Chinese medicines group chemical reagents corporation with reagents such as KCl；
Testing water used is Millipore Q, 18.3M Ω .cm.
The sequence of hepatitis B pgRNA is provided by Shanghai Public Health Center；
Deoxyribonuclease I is bought in precious bioengineering (Dalian) Co., Ltd；
Ultraviolet specrophotometer (UV3010, Hitachi, Japan)；
Sepectrophotofluorometer (F900, Edinburgh, Britain instrument company)；
PCR instrument (96well, Applied Biosystems company, the U.S.)；
High-speed refrigerated centrifuge (Hitachi)；
Nucleic acid gel imager (G:BOX Chenmi XL)；
Electrophoresis apparatus (Bio-Rad Laboratories)；
Oligonucleotides is purchased from Sangon Biotech (Shanghai) Co., Ltd. and uses high-efficient liquid phase chromatogram purification.
The structure of one FRET-TDN-Probe of embodiment designs
(1) the structure design of FRET-TDN-Probe
Firstly, we select one section of pgRNA sequence of AJ344117.1 on Gene bank, this part is hepatitis B
There is more repetitive sequence in pgRNA (this part is provided by Shanghai Public Health Center).SEQ ID NO.1:
ggagcttctgtggagttactctcttttttgccttctgacttctttccttctattcgagatctcctcga
caccgcctctgctctgtatcgggaggccttagagtctccggaacattgttcacctcaccatacggcactcaggcaa gctattctgtgttggggtgagttgatgaatctagccacctgggtgggaagtaatttggaagatccatcatccaggg
aattagtagtcagctatgtcaacgttaatatgggcataaaaatcagacaactattgtggtttcacatttcctgtct
cacttttgggagagaaactgttcttgaatatttggtgtcttttggagtgtggattcgcactcctcctgcatataga
ccaccaaatgcccctatcttatcaacacttccggaaactactgttgttagacgaagaggcaggtcccctagaagaa
gaactccctcgcctcgcagacgaaggtctcaatcgccgcgtcgcagaagatctcaatctcgggaatctcaatgtta
gtattccttggacacataaggtgggaaactttacggggctttattcttctactgtaccttgctttaatcctaaagg gcaaactccttcttttcctgatattcatttgcaggaggacattgttgatagatgtaagcaatttgtggggcccctt
acagtaaatgaaaacaggagactaaaattaattatgcctgctagattttatcccaatgttactaaatatttgccct
tagataaagggatcaaaccgtattatccagagtatgtagttgatcattacttccagacgcgacattatttacacac
tctttggaaggcggggattttatataaaagagagtccacacgtagcgcctcattttgcgggtcaccatattcttgg
gaacaagatctacagcatgggaggttggtcttccaaacctcgaaaaggcatggggacaaatctttct
In websiteHttps: it on //www.biosearchtech.com/stellaris-designer, utilizes Stellaris Probe Designer 4.2 software realization of version designs the complementary series of pgRNA sequence, and source is set It is set to human, masking level is 5, and design maximum number of probes is 21, and the length of nucleotide is selected as 22nt, the smallest Train interval is 10nt, obtains a plurality of alternative probe (as shown in table 1)；Table 1
Having chosen five sequences is pgRNA probe, has separately designed five kinds of probes (in SEQ ID NO.1 according to its sequence
Underscore part), the sequence of probe such as table 2.
2 five kinds of probes of table and its target spot
The synthesis of two FRET DNA tetrahedron namo fluorescence probe (FRET-TDN-Probe) of embodiment
FRET-TDN-Probe by four DNA of equimolar single-stranded (A, B, C, D) TM buffer (10mM Tris-HCl,
5mM MgCl2, pH 8.0) in a step annealing self assembly complete.95 DEG C of heating 10min, are rapidly cooled to 4 DEG C, stop at 4 DEG C
20min completes assembling.
There is 6-9 base pair complementarity in the stem of the molecular beacon probe of design, on the one hand guarantees the formation of structure, another
Aspect does not influence the combination of object again.The potential secondary structure of FRET-TDN-Probe can be enterprising in website NUPAKC software
The stability of row stem ring is analyzed, such as Fig. 2 a-2e, the results showed that five kinds of FRET-TDN-Probe can form stable stem ring knot
Structure and the synthesis that will not influence DNA tetrahedron nanostructure.We are set by our probes of the obtained structural confirmation of simulation
The reasonable validity of meter.
The electrophoresis of three FRET-TDN-Probe of embodiment characterizes
The DNA tetrahedron of the 1uM of synthesis is subjected to structural characterization with polyacrylamide gel electrophoresis (PAGE).Configuration 8%
PAGE glue after, by the FRET-TDN-Probe of 5uL be added to 1uL 6 × DNA loading buffer solution carry out loading.
1 × TBE electrophoresis liquid in electrophoresis tank containing 12.5mM magnesium ion.Under the conditions of ice-water bath, voltage 100V, electrophoresis time are set
100min.After electrophoresis, dyeing 30min is carried out with 1 × Gel Red solution, is finally observed in nucleic acid gel imaging system
It takes pictures.
As a result it such as Fig. 3, while being demonstrated by PAGE gel electrophoresis and can be effectively synthesized DNA tetrahedral structure.
The hybrid experiment of example IV FRET-TDN-Probe
Certain density nano-probe solution (final concentration of 100nM) is chosen, the target point exactly matched therewith is separately added into
Sub (synthetic DNA target) is incubated for, and with the concentration of target molecule gradually increases (0,5,10,20,40,60,80,100,
200nM).After being incubated for 1 hour at 37 DEG C, with sepectrophotofluorometer, exciting light: 488nm is designed, emission light gathering range:
550-750nm.Such as Fig. 4 a-4c.The result shows that: by the modification of a pair of of fluorescent molecule in two DNA chain, respectively in one end of A chain
At 1/3 position of B chain, FRET efficiency can reach 91.66% (such as Fig. 4 a).By fluorescent molecule to modification a chain also
It is the both ends of A, FRET efficiency can reach 92.18% (such as Fig. 4 b).
It can be seen that from Fig. 5 a-5e when the concentration for changing DNA target molecule, 200nM progressively increased to from 0, for each
The fluorescence intensity that FRET-TDN-Probe, CY3 are generated gradually increases, and the fluorescence intensity that CY5 is generated gradually weakens,
What this showed really has occurred hybridization reaction because of nano-probe and DNA target molecule, weakens FRET effect, generates fluorescence
The variation of signal.
Such as Fig. 6 a-6e, we measure synthesis target using the fluorescent emission of receptor and the ratio of the Fluorescence emission values of donor
The concentration of DNA.With I562 (nm)/I664 (nm) for the longitudinal axis, to synthesize target level as horizontal axis, by five kinds of FRET-TDN-Probe's
Detection data is simulated.When there is no target DNA to occur, the FRET efficiency highest of fluorescent molecule pair, I562 (nm)/I664 (nm)
Value it is minimum.Concentration with synthesis target DNA increases, and molecular beacon structure is opened, fluorescent molecule to the distance between increase,
FRET effect weakens, and I562 (nm)/I664 (nm) value gradually increases.
When the concentration for synthesizing target DNA increases to 100nM, curve reaches balance, illustrates the FRET-TDN- that we are added
Probe is already close to complete reaction.When the concentration of target DNA is 100nM, I562 (nm)/I664 (nm) value be can reach
3.1.The result shows that FRET-TDN-Probe can effectively identify target DNA and change its secondary structure, so that FRET effect subtracts
Weak, this result and our design schemes are expected identical.
It can be seen that (Tmix represents the mixed of five kinds of target DNAs for the target DNA concentration and the relationship of fluorescence signal ratio of various concentration
It closes).There are two types of its detection limits can achieve 1nM (FRET-TDN-Probe2, FRET-TDN-Probe3) in five kinds of probes, there is three
Kind of its its detection is limited to 5nM (background can still be distinguished plus 3 times of SD), and fitting result is good, R2 value 0.9 with
On.
The specificity and selectivity experiment of five FRET-TDN-Probe of embodiment
The DNA target molecule of the corresponding complete complementary pairing of each FRET-TDN-Probe and other targets of incomplementarity pairing
Molecule (concentration of target molecule is 50nM), is surveyed after carrying out hybridization incubation with concentration respectively for the buffer solution of 100nM nano-probe
Obtain its fluorescence data.All experiments are at least in triplicate.
From Fig. 7 a-7e can be seen that (mixing that Tmix represents five kinds of target DNAs) every kind of FRET-TDN-Probe can with it is right
The DNA target molecular specific for the exact matching answered combines, and the fluorescence signal intensity ratio of generation is 5-7 times of other DNA target molecules
(Fig. 8 a-8e)；In contrast, in the presence of other target molecules, even if in the case where concentration is much higher than 100 times of object,
FRET-TDN-Probe probe has specificity accordingly just for target molecule, for other target DNAs without obviously accordingly, this illustrates us
Detection architecture have very high specificity to object.In addition to this, in the mixed system of target DNA and non-target DNA, FRET-
TDN-Probe still is able to detect target DNA, and little with the signal value difference of independent detection target DNA.Illustrate ours
FRET-TDN-Probe has preferable selectivity.
The dynamics research of six FRET-TDN-Probe of embodiment is tested
Certain density nano-probe solution (final concentration of 100nM) is chosen, the target point exactly matched therewith is separately added into
Sub (concentration is 100nM) carries out hybridization incubation, surveys the change of its fluorescence intensity (0,10,20,30,40,50,60min) at any time
Change.
As Fig. 9 a-10e can be quick in 1 hour when showing that nano-probe encounters the DNA target molecule of exact matching
Response, and very strong fluorescence signal variation is generated, we screen in obtained probe, there are three types of (Probe2, Prob3,
Probe4 completion) can be reacted in 30min.Quick kinetic reaction proves our potential use of FRET-TDN-Probe
To detect pgRNA and be imaged into the cell.
The nuclease stability of seven FRET-TDN-Probe of embodiment is tested
Two groups of nano-probe solution (final concentration of 100nM), are placed in water-bath at 37 DEG C.To 10 minutes sample solutions
After stable equilibrium, DNase I (final concentration 0.5U/mL) is added in one group of probe solution, another group is added without DNase I, in 1h
The continuously fluorescence intensity of two groups of nano-probe solution of detection.Two groups of probes of 1h will be reacted, the stability of glue verifying structure is run.
It is sharp first before being realized using FRET-TDN-Probe to the loci detection simultaneously and imaging of intracellular pgRNA
The nuclease stability of nano-probe in physiological conditions has been investigated with spectrofluorimetry.In subsequent experiment, one is utilized
The common restriction endonuclease of kind, deoxyribonuclease (DNase I), to evaluate the nuclease stability of nano-probe.From (Figure 11 a-
It can be seen that compared with the FRET-TDN-Probe handled without DNase I in 11e), the FRET-TDN- handled through DNase I
Probe, there is no significant changes for fluorescence intensity；But when being separately added into corresponding DNA target molecule into two kinds of solution
After hybridization reaction occurs, the fluorescence intensity in two kinds of solution greatly enhances illustration in (11a-11e)).These were the result shows that should
FRET-TDN-Probe have extraordinary antienzyme cut property, further show that the recovery of fluorescence signal really because
Hybridization reaction has occurred between FRET-TDN-Probe and target DNA, is not to degrade and generate through nuclease.Meanwhile we
Five kinds of FRET-TDN-Probe are incubated for 2 hours respectively at nuclease in 37 DEG C jointly, guarantees that applied sample amount is consistent with concentration, passes through
Gel electrophoresis graph discovery has the incubation result of nuclease and nuclease free to there is no difference (Figure 12), this also illustrates
The nuclease stability of FRET-TDN-Probe is fine.
Above embodiment is can not to be interpreted as in order to illustrate embodiment disclosed by the invention to limit of the invention
System.In addition, in various modifications and invention listed herein method, composition variation, do not departing from the scope of the present invention
Be obvious for those skilled in the art under the premise of spirit.Although having combined of the invention a variety of specific
Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
In fact, various obviously modify as described above for those skilled in the art to obtain invention all should include
Within the scope of the invention.
Sequence table
<110>Shanghai Advanced Research Institute, Chinese Academy of Sciences
<120>for detecting the DNA tetrahedron probe and kit of virus
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 971
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ggagcttctg tggagttact ctcttttttg ccttctgact tctttccttc tattcgagat 60
ctcctcgaca ccgcctctgc tctgtatcgg gaggccttag agtctccgga acattgttca 120
cctcaccata cggcactcag gcaagctatt ctgtgttggg gtgagttgat gaatctagcc 180
acctgggtgg gaagtaattt ggaagatcca tcatccaggg aattagtagt cagctatgtc 240
aacgttaata tgggcataaa aatcagacaa ctattgtggt ttcacatttc ctgtctcact 300
tttgggagag aaactgttct tgaatatttg gtgtcttttg gagtgtggat tcgcactcct 360
cctgcatata gaccaccaaa tgcccctatc ttatcaacac ttccggaaac tactgttgtt 420
agacgaagag gcaggtcccc tagaagaaga actccctcgc ctcgcagacg aaggtctcaa 480
tcgccgcgtc gcagaagatc tcaatctcgg gaatctcaat gttagtattc cttggacaca 540
taaggtggga aactttacgg ggctttattc ttctactgta ccttgcttta atcctaaagg 600
gcaaactcct tcttttcctg atattcattt gcaggaggac attgttgata gatgtaagca 660
atttgtgggg ccccttacag taaatgaaaa caggagacta aaattaatta tgcctgctag 720
attttatccc aatgttacta aatatttgcc cttagataaa gggatcaaac cgtattatcc 780
agagtatgta gttgatcatt acttccagac gcgacattat ttacacactc tttggaaggc 840
ggggatttta tataaaagag agtccacacg tagcgcctca ttttgcgggt caccatattc 900
ttgggaacaa gatctacagc atgggaggtt ggtcttccaa acctcgaaaa ggcatgggga 960
caaatctttc t 971
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aagagagtaa ctccacagaa gc 22
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aacaatgttc cggagactct aa 22
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aacacagaat agcttgcctg ag 22
<210> 5
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ggatgatgga tcttccaaat ta 22
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
attaacgttg acatagctga ct 22
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gtgaaaccac aatagttgtc tg 22
<210> 8
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
agaacagttt ctctcccaaa ag 22
<210> 9
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gaatccacac tccaaaagac ac 22
<210> 10
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ttgataagat aggggcattt gg 22
<210> 11
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tcttcgtcta acaacagtag tt 22
<210> 12
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
aacattgaga ttcccgagat tg 22
<210> 13
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
agaataaagc cccgtaaagt tt 22
<210> 14
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
tgccctttag gattaaagca ag 22
<210> 15
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
tcctgcaaat gaatatcagg aa 22
<210> 17
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ttagtctcct gttttcattt ac 22
<210> 18
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
attgggataa aatctagcag gc 22
<210> 18
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
attgggataa aatctagcag gc 22
<210> 19
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
tacatactct ggataatacg gt 22
<210> 20
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
tgtgtaaata atgtcgcgtc tg 22
<210> 21
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
aaaatgaggc gctacgtgtg ga 22
<210> 22
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
tagatcttgt tcccaagaat at 22
<210> 23
<211> 82
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
aacacagaat agcttgcctg agtgtgtaca ttcctaagtc tgaaacatta cagcttgcta 60
cacgagaaga gccgccatag ta 82
<210> 24
<211> 84
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
attgggataa aatctagcag gcatcccaaa cattcctaag tctgaaacat tacagcttgc 60
tacacgagaa gagccgccat agta 84
<210> 25
<211> 85
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
tgtgtaaata atgtcgcgtc tgtttacaca acattcctaa gtctgaaaca ttacagcttg 60
ctacacgaga agagccgcca tagta 85
<210> 26
<211> 85
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
gaatccacac tccaaaagac acgtggattc acattcctaa gtctgaaaca ttacagcttg 60
ctacacgaga agagccgcca tagta 85
<210> 27
<211> 85
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
tgccctttag gattaaagca agaaagggca acattcctaa gtctgaaaca ttacagcttg 60
ctacacgaga agagccgcca tagta 85
<210> 28
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
ctcaggcaag ctattctgtg tt 22
<210> 29
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
gcctgctaga ttttatccca at 22
<210> 30
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
cagacgcgac attatttaca ca 22
<210> 31
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gtgtcttttg gagtgtggat tc 22
<210> 32
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
cttgctttaa tcctaaaggg ca 22
<210> 33
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
acattcctaa gtctgaaaca ttacagcttg ctacacgaga agagccgcca tagta 55
<210> 34
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tatcaccagg cagttgacag tgtagcaagc tgtaatagat gcgagggtcc aatac 55
<210> 35
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
caactgcctg gtgataaaac gacactacgt gggaatctac tatggcggct cttc 54
<210> 36
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ttcagactta ggaatgtgct tcccacgtag tgtcgtttgt attggaccct cgcat 55
Claims (13)
1. a kind of for detecting the DNA tetrahedron probe of virus, it is characterised in that:
The DNA tetrahedron probe generally tetrahedral structure extends stem sequence on a tetrahedral vertex, described
DNA identification sequence is extended at the end of stem sequence 5 ', and the stem sequence can identify that sequence is combined with DNA and form loop-stem structure,
It is modified with donor fluorescent molecule and acceptor fluorescence molecule on the DNA tetrahedron probe, wherein the donor fluorescent molecule
In DNA identification sequence, when being not added with test object between the donor fluorescent molecule and acceptor fluorescence molecule
Distance is not more than 7nm.
2. according to claim 1 for detecting the DNA tetrahedron probe of virus, it is characterised in that: the donor fluorescent
Molecule and acceptor fluorescence molecule are respectively selected from CY3 and CY5.
3. according to claim 1 for detecting the DNA tetrahedron probe of virus, it is characterised in that: the DNA tetrahedron
Assembled by tetra- DNA of A, B, C and D are single-stranded, the stem sequence extends from the 5 ' of A chain ends, and wherein A chain nucleotide sequence is such as
Shown in SEQ ID NO.33, B chain is as shown in SEQ ID NO.34, and C chain is as shown in SEQ ID NO.35, D chain such as SEQ ID
Shown in NO.36.
4. according to claim 1 for detecting the DNA tetrahedron probe of virus, it is characterised in that: the acceptor fluorescence
Molecule is located at 1/2 site of B chain to the 5 ' ends between 1/4 site or positioned at A chain, and 1/2 site refers to between 1/4 site
The length at 5 ' ends of site distance A chain is between 1/2 to the 1/4 of B chain length.
5. according to claim 1 for detecting the DNA tetrahedron probe of virus, it is characterised in that: the DNA identifies sequence
Column contain 6~8 bases containing 20 bases, the stem sequence is not less than.
6. according to claim 1 for detecting the DNA tetrahedron probe of virus, it is characterised in that: the DNA identifies sequence
Column selection is from any in SEQ ID NO.3, SEQ ID NO.8, SEQ ID NO.13, SEQ ID NO.17 or SEQ ID NO.19
It is a kind of.
7. according to claim 6 for detecting the DNA tetrahedron probe of virus, it is characterised in that: the acceptor fluorescence
Molecule is located at 5 ' ends of A chain, and the donor fluorescent molecule is located at 5 ' ends of DNA identification sequence, and the stem sequence is held from the 5 ' of A chain
Extend, any one of A chain nucleotide sequence in SEQ ID NO.23-27 after extension.
8. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes as described in claim 1~7 any one
For detecting the DNA tetrahedron probe and auxiliary material of virus.
9. a kind of virus detection kit, which is characterized in that the kit includes as described in claim 1~7 any one
For detecting viral DNA tetrahedron probe or pharmaceutical composition as claimed in claim 8.
10. DNA tetrahedron probe is preparing the purposes in virus detection kit as described in claim 1~7 any one.
11. pharmaceutical composition as claimed in claim 8 is preparing the purposes in virus detection kit.
12. purposes of the DNA tetrahedron probe in the drug of preparation detection virus as described in claim 1~7 any one.
13. purposes of the pharmaceutical composition as claimed in claim 8 in the drug of preparation detection virus.
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