CN107573289B - The compound and labeling method of a kind of specific chemical label 5- aldehyde radical uracil and application - Google Patents
The compound and labeling method of a kind of specific chemical label 5- aldehyde radical uracil and application Download PDFInfo
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Abstract
The invention discloses a kind of compound of specific chemical label 5- aldehyde radical uracil and labeling method and applications.It is reacted by 1 property of can choose of compound with azido group and 5- aldehyde radical uracil aldehyde radical, adds the biotin DBCO-S-S-PEG with alkynyl3- biotin and nitrine do not need the click reaction of catalyst.Using 5- aldehyde radical uracil specific chemical labeling method of the invention, it can be achieved that the nucleic acid samples of the specific enrichment uracil of aldehyde radical containing 5-, the sequence distributed intelligence of 5- aldehyde radical uracil in analyzing nucleic acid molecules.The present invention provides effective research method for epigenetics and nucleic acid chemistry biological study field.
Description
Technical field
The present invention relates to the chemical labeling of epigenetic modification base and detection method more particularly to a kind of compound 1 and
Using the method for the compound specificity chemical labeling 5- aldehyde radical uracil, and its label, detection, in terms of answer
With.
Background technique
In nature other than the presence of four kinds of natural bases, there is also a large amount of modified bases.Wherein DNA oxidative damage
Important products --- 5- aldehyde radical uracil (hereinafter referred to as 5fU) is modification alkali naturally occurring in genome and less content
Base.And since it is in the reproduction process of DNA, mispairing can be generated, such as 5fU and guanine match, and then cause gene prominent
Become, at the same be also induce a variety of diseases an important factor for one of.During the active demethylation of organism nucleic acid, inherently
It is repaired along with the oxidative damage to modified base, excision.Univ Munich Germany Thomas professor Carell in 2014 is for the first time
It reports TET enzyme and can aoxidize thymidine and become 5-hydroxylmethyluracil.The oxidation that this result discloses thymidine produces
There may be certain epigenetics meanings for object (5-hydroxylmethyluracil, 5- aldehyde radical uracil etc.).Cambridge is big within 2015
It learns Balasubramanian Shanker and has screened a series of biotinylated small organic molecule for 5- aldehyde radical uracil
Enrichment, and in 2017, it is used in for Eukaryotic helminth Leishmania (eukaryote parasite
Leishmania the genome sequencing of 5hmU).5fU can be generated via free-radical oxidation, or by Fenton effect, gamma
Ray and oxydasis generate.5fU can induce DNA that base mispairing occurs, and DNA structure is caused to change, and occur with albumen
Interaction.It is widely present in vivo and content is seldom.
Comparision contents just because of 5fU are few, and there is presently no the technologies that 5fU in full-length genome is sequenced.Therefore,
Need to develop a kind of new highly selective, efficient 5fU tagging and testing method, this is to being pushed further into epigenetic demethyl
Change research has the function of positive.
Summary of the invention
In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a kind of compound 1 and utilizationizations
Close 1 specific chemical of object label 5- aldehyde radical uracil method, it can be achieved that the specific enrichment uracil of aldehyde radical containing 5- nucleic acid sample
Product, the sequence distributed intelligence of 5- aldehyde radical uracil in analyzing nucleic acid molecules.
In order to achieve the goal above, the invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of compound 1, and structural formula is shown in formula I:
The second aspect of the present invention provides a kind of method based on 1 specific chemical of compound label 5- aldehyde radical uracil,
Include the following steps:
(1) aldehyde radical of compound 1 and 5- aldehyde radical uracil with azido group is subjected to dehydration condensation, generationization
Object B is closed, is shown below:
(2) compound B is reacted with the generation of compound 2 click, and the compound 2 is the biotin DBCO-S- with alkynyl
S-PEG3-biotin。
The third aspect of the present invention provides a kind of based on 1 specific chemical of compound label 5- aldehyde radical uracil progress 5-
The method of aldehyde radical uracil genome sequencing, includes the following steps:
(1) genomic DNA is extracted, interrupts the segment that instrument is broken into 250-400bp with ultrasound, a part is used as gene
The label of group DNA, another part give over to control group and directly build library；
(2) genomic DNA that mark group interrupts is added compound 1 and is handled, and 5fU is carried out on compound 1 and genome
Reaction；
(3) click occurs with compound 2 again by genomic DNA that compound 1 marks react, the compound 2 for
The biotin DBCO-S-S-PEG of alkynyl3-biotin；
(4) it after being incubated for step (3) treated DNA chain with the magnetic bead for having modified Streptavidin, collects containing biology
The chain of element has the DNA chain of 5fU；
(5) DNA chain for the mark group that control group and step (4) obtain is subjected to library construction respectively, carries out the sequencing of two generations
(NGS), data are analyzed；Using control group data as background, data enrichment times are greater than 4 times for effectively enrichment, with ginseng in mark group
It examines genome GRCm38.p5.genome.fa to be compared, obtains distribution of the segment containing fU in mouse genome.
The mode for interrupting genomic DNA in above-mentioned steps (1) are as follows: with Qubit Fluorometer by the gene of extraction
Group DNA is quantified, and is configured to the solution of 20 μ g/mL, is interrupted instrument with Covaris ultrasound and interrupt genomic DNA, and parameter is arranged
As follows: power 175W ultrasonic time 420 seconds, controls temperature between 4 DEG C -16 DEG C；The DNA interrupted is solidifying with 1% agarose
The verifying of gel electrophoresis progress fragment length.
The mode that compound 1 is handled is added in the genomic DNA interrupted in step (2) are as follows: the gene interrupted to 20 μ g
Sodium acetate (NaOAc) buffer (1M, pH 5.0) of 5 μ L is added in group DNA, adding 5 μ L compounds 1, (250mM uses DMSO
Dissolution), adding water to reaction system volume is 50 μ L；System in oscillator 37 DEG C reaction 12 hours after with gel column remove it is extra
Small molecule compound, such as compound 1.
With with alkynyl-modified biotin (biotin) side that click reacts occurs for the 5fU being labeled in step (3) again
Formula are as follows: the DBCO-S-S-PEG of 5 μ L is added into the genomic DNA purified in step (2)3- biotin (20mM), system are being shaken
Extra small molecule compound such as DBCO-S-S-PEG is removed with gel column after swinging in device 37 DEG C of reactions 2 hours3-biotin。
The processing mode of the genomic DNA with biotin is transferred in step (4) with magnetic bead are as follows: by after purification DNA with
After magnetic bead with Streptavidin is incubated for 15 minutes, in standing 5 minutes on magnetic frame, clear liquid, magnetic bead binding& are discarded
Washing buffer is washed 3 times, discards clear liquid, and the dithiothreitol (DTT) (DTT) of 100 μ L (50mM) is added；37 DEG C in oscillator
After reaction 2 hours, magnetic bead is fixed on magnetic frame 3 minutes, collects liquid, and product removes DTT with Purification Kit；It obtains
DNA quantified with Qubit Fluorometer.
Genomic DNA chain carries out the processing mode of library construction in step (5) are as follows: the genome for taking 20ng magnetic bead to transfer
DNA is used for library construction, and control group is that the genomic DNA 20ng directly interrupted is used for library construction；Other operation according to
The specification that the Thruplex DNA-Seq kit of Rubicon Genomics builds library kit carries out.
The principle of the present invention as shown in Figure 1, the present invention be first by ultrasound interrupt after genomic DNA reacted with compound 1,
Click reaction occurs with the azido group of biotin and compound 1 with alkynyl again.Finally with the magnetic with Streptavidin
Pearl transfers the segment containing 5fU.
The fourth aspect of the present invention provides the kit for detecting 5- aldehyde radical uracil base, includes compound 1.
Another object of the present invention is to provide the application of compound 1 in the following aspects:
(1) in sequencing analysis genome 5- aldehyde radical uracil sequence distributed intelligence；
(2) DNA molecular of the uracil base of aldehyde radical containing 5- is enriched with；
(3) preparation detects the kit of the distributed intelligence of 5- aldehyde radical uracil base in genome DNA sample.
The present invention is occurred by 1 property of can choose of compound with azido group and 5- aldehyde radical uracil aldehyde radical
Reaction, and other bases discord compound 1 reacts；It adds biotin and nitrine with alkynyl and does not need catalyst
Click reaction.It is transferred by the magnetic bead with Streptavidin modification and is connected with the segment of biotin, it can be by the segment of fU from complete
It is taken out in genome.After building library kit building library, the sequencing of two generations is carried out, is realized for the first time from hippocampus of mice body
The distributed intelligence of the segment containing 5fU is obtained in full-length genome.
Detailed description of the invention
Fig. 1 is the flow diagram of fU in marker gene group of the present invention.
Fig. 2 is high performance liquid chromatography (HPLC) figure of compound 1 and the DNA chain reaction containing 5fU, in order to be confirmed
Compound 1 and DNA chain are reacted.
Fig. 3 is the gel electrophoresis figure after the DNA chain with different modifying base is labeled, in order to confirm compound
1 and the DNA chain with fU are reacted.
Fig. 4 is the distribution map for passing through fU segment in the hippocampus of mice body genome that compound 1 is transferred in the present invention.
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
The raw material of compound used in the specific embodiment of the invention is purchased from Beijing Yi Nuokai Science and Technology Ltd..
The synthesis process and characterization of [embodiment 1] compound 1
Take 3,4- diaminobenzoic acid (3g, 19.7mmol) and ethyl cyanoacetate (7mL, 65.8mmol) in the circle of 100mL
In the flask of bottom, 1h is stirred to react at 180 DEG C.After reaction, reaction system is poured into 100mL ether and is precipitated solid, subtract
Pressure filters, and solid uses silica gel column chromatography, methylene chloride: methanol: acetic acid=100:1:0.1 elution obtains compound as white solid A
(1g, 25%).1H NMR(400MHz,DMSO-d6) δ 12.92 (s, 1H), 8.15 (s, 1H), 7.83 (d, J=8.4Hz, 1H),
7.61 (d, J=7.7Hz, 1H), 4.46 (s, 2H) .HRMS (ESI+) C10H8N3O2 +[M+H]+calculated 202.06110,
found 202.06082。
Specific reaction route is as follows:
Take compound A (290mg, 1.4mmol), 3- azidopropylamine (720mg, 7.2mmol) and HATU (1.1g,
2.9mmol) in 25mL round-bottomed flask, 10mL n,N-Dimethylformamide and 5 drop triethylamines is added, is stirred to react at 25 DEG C
4h.After reaction, reaction system decompression rotation is evaporated, solid uses silica gel column chromatography, methylene chloride: methanol=100:1
It is eluted to 40:1, obtains compound as white solid 1 (330mg, 80%).1H NMR(400MHz,DMSO-d6)δ12.86(s,1H),
8.53 (t, J=5.5Hz, 1H), 8.08 (s, 1H), 7.74 (dd, J=8.4,1.3Hz, 1H), 7.59 (d, J=8.2Hz, 1H),
4.44 (s, 2H), 3.43 (t, J=6.8Hz, 4H), 1.80 (p, J=6.8Hz, 2H)13C NMR(100MHz,DMSO-d6)δ
167.12,147.33,129.10,122.12,116.96,49.05,37.19,28.96,18.95.HRMS(ESI+)C13H14N7O+
[M+H]+calculated 284.12543,found 284.12497。
Specific reaction route is as follows:
[embodiment 2] compound 1 and 5fU monomer reaction route and characterization
Take 5- aldehyde radical uracil (30mg, 0.12mmol) and compound 1 (33mg, 0.12mmol) in 25mL round-bottomed flask
In, 10mL methanol and 5 drop acetic acid is added, is stirred to react 15h at 50 DEG C.After reaction, reaction system decompression rotation is evaporated,
Solid use silica gel column chromatography, methylene chloride: methanol=50:1 to 15:1 elution, obtain yellow solid compound B (36mg,
60%).1H NMR(400MHz,DMSO-d6)δ13.39(s,1H),11.98(s,1H),8.95(s,1H),8.57(s,1H),
8.36-7.90 (m, 2H), 7.67 (dd, J=78.5,12.8Hz, 2H), 6.20 (t, J=6.6Hz, 1H), 5.35 (s, 1H),
4.99 (s, 1H), 4.35-4.25 (m, 1H), 3.90 (dd, J=6.8,4.1Hz, 1H), 3.63 (d, J=4.0Hz, 2H), 3.52-
3.40(m,4H),2.37–2.11(m,2H),1.90–1.72(m,2H).13C NMR(100MHz,DMSO-d6)δ167.04,
162.12,149.76,143.38,142.24,138.00,135.09,129.33,123.61,122.04,118.71,116.68,
111.55,107.86,100.39,88.68,86.20,71.21,61.86,49.06,40.78,37.22,28.94.HRMS(ESI
+)C23H24N9O6 +[M+H]+calculated 522.18441,found 522.18419。
Specific reaction route is as follows:
The label of [embodiment 3] mouse gene group DNA
The genomic DNA (gDNA) of extraction is dissolved in ultrapure water after being interrupted with Ultrasound Instrument, and with Qubit Fluorometer
Quantitative instrument quantifies gDNA.The label of genomic DNA mainly divides 2 steps, as shown in Figure 1, step 1: to 1.5mL's
The gDNA (1 μ g/ μ L) of 20 μ L is added in EP pipe, 5 μ L sodium acetate buffer (pH 5.0), (250mM is dissolved in DMSO to 5 μ L compounds 1
In), the ultrapure water of 20 μ L is put into oscillating reactions device after oscillation centrifugation, and 37 DEG C are reacted 12 hours.After having reacted, gel column is used
Remove extra compound 1.Step 2: the DBCO-S-S-PEG of 5 μ L is added in the product purified to the first step3- biotin, together
Sample, oscillation are put into oscillator 37 DEG C after reaction 2 hours and remove extra small molecule compound with gel column after mixing.Finally
The mixture of purifying adds ultrapure water polishing to the volume of 100 μ L.
The enrichment of the segment containing 5fU is transferred in [embodiment 4] mouse gene group DNA
The genomic DNA for the label after purification that embodiment 3 obtains is transferred for magnetic bead, as shown in Figure 1, detailed process is such as
Under: (1) after the magnetic bead with Streptavidin shakes up manually, takes the magnetic bead of 50 μ L in the EP pipe of 1.5mL, rest on magnetic frame
Liquid was siphoned away with pipettor upper 3 minute, magnetic bead washes 3 times (2) 50 μ of addition with 500 μ 1 × binding&washing of L buffer
2 × binding&washing of L buffer makes magnetic bead dispersion in the solution, is added in 50 μ L embodiments 1 into magnetic bead dispersion liquid
Product after final purification, room temperature rotation is incubated for 15 minutes after being mixed with liquid-transfering gun piping and druming.(3) reaction tube is fixed on magnetic frame
On, standing siphons away liquid after five minutes.It is washed 3 times with 500 μ 1 × binding&washing of L buffer, in order to wash off
Segment without containing biotin.(4) DTT (50mM) of the 100 fresh configurations of μ L is added into magnetic bead, 37 DEG C of reactions in oscillator
2 hours.(5) reaction tube is fixed on magnetic frame 3 minutes, clear liquid is collected, with DNA Purification Kit.Obtained product is used
The building of sequencing library is carried out after Qubit Fluorometer is quantitative.
The building of [embodiment 5] control group and mark group library
The template that the genomic DNA for taking 20ng ultrasound to interrupt is used as control group carries out library construction, takes 20ng label purifying
The template that genomic DNA afterwards is used as mark group carries out library construction.The detailed process of library preparation is according to Thruplex DNA-
Seq kit (Rubicon Genomics) operating guidance carries out.
The analysis of [embodiment 6] two generation sequencing result
DNA library prepared after by Suzhou Jin Weizhi company carry out the sequencing of two generations, microarray dataset be Illumina Hiseq
150PE, mode are pair end sequencing.It is GRCm38.p5.genome.fa (GENCODE number with reference to genome file
Downloaded according to library), data are analyzed using HOMER (v4.9) software.Using control group as background, wherein obtained in mark group
4 times bigger than control group of segment reads number or more of the effective rich segment of conduct.By reference to genome
GRCm38.p5.genome.fa is compared, and obtains distribution of the segment containing fU in mouse genome.From Fig. 2 result
It sees, fU is mainly distributed in intergenic region and introne, and wherein intergenic region accounts for 62.12%, introne 3 6.02%, other areas
Domain such as exon, promoter, translational termination site, the regions such as 5 '-UTR, 3 '-UTR account for 1.86% altogether.
The analysis of [embodiment 7] high performance liquid chromatography (RP-HPLC)
In the EP pipe of 1.5mL, the DNA of 2 μ L (100 μM) containing 5fU base, 5 μ L sodium acetate buffer (pH is added
5.0), 2 μ L compound 1 (100mM is dissolved in DMSO), the ultrapure water of 38 μ L are put into oscillating reactions device, 37 DEG C after oscillation centrifugation
Reaction 6 hours.After having reacted, extra compound 1 is removed with gel column.The RP-HPLC tracer of the product of reaction is collected
The signal of 260nm, by retention time it can be seen that compound 1 on the DNA fragmentation pass flag containing fU, as shown in Figure 3.
The analysis of [embodiment 8] reaction selectivity
(ODN-T, ODN-5hmU, ODN-5fU, ODN-5hmC, ODN-5fC, ODN-AP) DNA with different modifying
(ODN-T, ODN-5fU are synthesized by Wuhan Jim Carrey company, other DNA are synthesized by Dalian treasured biotech firm, the DNA of different modifying
Sequence be shown in Table 1) according to the reaction step in embodiment 3 respectively and after 1 reaction purification of compound, every kind of DNA and compound 1 are anti-
Product after purification is answered to analyze by 20% denaturing gel electrophoresis, since the segment of fU is marked by compound 1, molecular weight increase is led
Electrophoretic velocity is caused to slow down.It can be seen that a new band on gel electrophoresis figure as shown in Figure 4.
The sequence of DNA of the table 1 with different modifying
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (6)
1. a kind of compound 1, which is characterized in that its structural formula is shown in formula I:
2. a kind of method based on 1 specific chemical of compound described in claim 1 label 5- aldehyde radical uracil, feature exist
In including the following steps:
(1) compound 1 with azido group is reacted with the aldehyde radical of 5- aldehyde radical uracil, generates compound B, it is specific anti-
Answer route as follows:
(2) compound B is reacted with the generation of compound 2 click, and the compound 2 is the biotin DBCO-S-S- with alkynyl
PEG3-biotin。
3. one kind is phonetic based on 1 specific chemical of compound described in claim 1 label 5- aldehyde radical uracil progress 5- aldehyde radical urine
The method of pyridine genome sequencing, which comprises the steps of:
(1) genomic DNA is extracted, interrupts the segment that instrument is broken into 250-400bp with ultrasound, a part is used as genome
The label of DNA, another part give over to control group and directly build library；
(2) genomic DNA that mark group interrupts is added compound 1 and is handled, and compound 1 is reacted with 5fU on genome；
(3) click occurs with compound 2 again by the genomic DNA that compound 1 marks to react, the compound 2 is with alkynyl
Biotin DBCO-S-S-PEG3-biotin；
(4) after being incubated for step (3) treated DNA chain with the magnetic bead for having modified Streptavidin, collection contains biotin
Chain has the DNA chain of 5fU；
(5) DNA chain for the mark group that control group and step (4) obtain is subjected to library construction respectively, carries out the sequencing of two generations
(NGS), data are analyzed；Using control group data as background, data enrichment times are greater than 4 times for effectively enrichment, with ginseng in mark group
It examines genome GRCm38.p5.genome.fa to be compared, obtains distribution of the segment containing fU in mouse genome.
4. according to the method described in claim 3, it is characterized in that,
The mode for interrupting genomic DNA in step (1) are as follows: carried out the genomic DNA of extraction with Qubit Fluorometer
It is quantitative, it is configured to the solution of 20 μ g/mL, instrument is interrupted with Covaris ultrasound and interrupts genomic DNA, setting parameter is as follows: power
For 175W, ultrasonic time 420 seconds, temperature is controlled between 4 DEG C -16 DEG C；The DNA interrupted with 1% agarose gel electrophoresis into
The verifying of row fragment length；
The mode that compound 1 is handled is added in the genomic DNA interrupted in step (2) are as follows: the base interrupted to 20 μ g mark groups
The sodium-acetate buffer that pH5.0 concentration because 5 μ L are added in group DNA is 1M, adding 5 μ L with the concentration that DMSO dissolves is
The compound 1 of 250mM, adding water to reaction system volume is 50 μ L；System in oscillator 37 DEG C reaction 12 hours after use gel
Column removes extra small molecule compound, wherein including extra compound 1；
With with alkynyl-modified biotin (biotin) mode that click reacts occurs for the 5fU being labeled in step (3) again
Are as follows: the DBCO-S-S-PEG that 5 μ L concentration are 20mM is added into the genomic DNA purified in step (2)3- biotin, system exist
Extra small molecule compound is removed with gel column after reaction 2 hours for 37 DEG C in oscillator, wherein including extra DBCO-S-S-
PEG3-biotin；
The processing mode of the genomic DNA with biotin is transferred in step (4) with magnetic bead are as follows: by after purification DNA with have
After the magnetic bead of Streptavidin is incubated for 15 minutes, in standing 5 minutes on magnetic frame, clear liquid, magnetic bead binding& are discarded
Washing buffer is washed 3 times, discards clear liquid, and the dithiothreitol (DTT) (DTT) that 100 μ L concentration are 50mM is added；In oscillator
After 37 DEG C are reacted 2 hours, magnetic bead is fixed on magnetic frame 3 minutes, collects liquid, and product removes DTT with Purification Kit；
Obtained DNA is quantified with Qubit Fluorometer；
Genomic DNA chain carries out the processing mode of library construction in step (5) are as follows: the genomic DNA for taking 20ng magnetic bead to transfer is used
In library construction, control group is that the genomic DNA 20ng directly interrupted is used for library construction；Other operations are according to Rubicon
The specification that the Thruplex DNA-Seq kit of Genomics builds library kit carries out.
5. a kind of kit for detecting 5- aldehyde radical uracil base, which is characterized in that include compound 1 described in claim 1.
6. the application of compound 1 described in claim 1 in the following aspects:
(1) in sequencing analysis genome 5- aldehyde radical uracil sequence distributed intelligence；
(2) DNA molecular of the uracil base of aldehyde radical containing 5- is enriched with；
(3) preparation detects the kit of the distributed intelligence of 5- aldehyde radical uracil base in genome DNA sample.
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| CN111041023A (en) * | 2018-10-11 | 2020-04-21 | 中国科学院天津工业生物技术研究所 | Specific nucleic acid binding proteins and methods for enriching for specific nucleic acids |
| CN109678802B (en) * | 2019-01-28 | 2020-12-29 | 四川大学 | Method for deriving aldehyde pyrimidine, method for detecting 5-aldehyde cytosine and application of aldehyde pyrimidine derivative |
| CN111088252B (en) * | 2019-12-30 | 2021-07-13 | 西安交通大学 | Specific labeling method of 5-hydroxymethyl uracil on DNA |
| CN114106012B (en) * | 2021-12-13 | 2023-05-23 | 宜昌博仁凯润药业有限公司 | Preparation method and application of compound Biotin-PEG3-SS-DBCO |
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