CN110964073B - Probes and methods for capturing fetal-specific DNA methylation modification binding proteins - Google Patents

Abstract

Probes and methods for capturing fetal-specific DNA methylation-modified binding proteins are disclosed. The probe consists of 10-490 bases in length, and the 3 'or 5' of the probe has biotin mark and can be combined with avidin on magnetic beads. The method for capturing the binding protein comprises the following steps: preparation of probe-magnetic bead complexes: pretreatment with BSA (bovine serum albumin) and tRNAs blocked streptavidin-labeled magnetic beads; extracting and pre-precipitating cell total protein: adding non-pretreated magnetic beads into the extracted total protein sample for incubation, removing the magnetic beads and collecting the supernatant; capturing the binding protein. The DNA methylation modified probe sequence special for the fetus can effectively capture the DNA methylation modified binding protein of the fetus, and remarkably improve the enrichment of the specific binding protein.

Description

Probe and method for capturing fetal specific DNA methylation modification binding protein

【Technical field】

The invention belongs to the technical field of molecular biology, and specifically relates to a probe and a method for capturing fetal specific DNA methylation modification binding protein.

【Background technique】

Clinically, the detection of circulating cell-free fetal DNA (cff-DNA) in maternal plasma is an important method for non-invasive prenatal diagnosis. As early as 1997, Professor Lu Yuming and others reported the presence of cff-DNA in maternal plasma, showing that DNA from the fetus could be detected in maternal plasma after 2 weeks of pregnancy, and was rapidly cleared from maternal plasma within 2 hours after delivery and disappear (Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA inmaternal plasma and serum. Lancet 1997; 350:485-7). Therefore, cff-DNA in maternal plasma has become an important target for non-invasive prenatal diagnosis. However, cff-DNA only accounts for a small portion (about 10%) in maternal plasma, and the rest is derived from maternal blood cells; current noninvasive prenatal diagnosis using cff-DNA is mainly based on paternal genetic markers, such as the SYR gene ( Chiu RW, Lo YM. Non-invasive prenatal diagnosis by fetal nucleic acid analysis in maternal plasma: the coming of age. Semin Fetal Neonatal Med 2011; 16:88-93.).

Recently, researchers have attempted to develop markers of fetal DNA polymorphisms that are not related to fetal genetics. One of the important methods is to detect fetal epigenetic markers in maternal plasma, showing that there are different DNA methylation patterns between fetal tissue and maternal blood cells (Chan KC, Ding C, Gerovassili A, et al.Hypermethylated RASSF1A inmaternal plasma : a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006; 52:2211-8.). DNA methylation usually refers to the presence of methylation on the 5′ carbon of cytosine nucleotides, followed by guanine nucleotides, forming a CpG dinucleotide. At present, there are 485,577 CpG modification sites found in fetal tissue through methylation microarray, and they exist in UTR region, promoter region or gene coding region. There is evidence that circulating fetal DNA in maternal peripheral blood is primarily derived from the placenta, as it is the only channel for nutrient transport between mother and child. Thus, it is possible to develop molecular markers specific between maternal and fetal tissues based on signatures of different DNA methylation patterns.

At present, there is no report on the design of fetal-specific DNA methylation probes. Therefore, a fetal-specific DNA methylation probe and an operation method for capturing fetal-specific DNA methylation modification binding proteins have been developed. It is very important to find new drug targets for the mechanism research and clinical intervention of pregnancy-related diseases such as premature birth and repeated spontaneous abortion.

【Content of invention】

In order to solve the above problems, the present invention provides a probe and method for capturing fetal-specific DNA methylation modification binding proteins.

The purpose of the present invention is achieved in the following manner:

The present invention provides a method for designing probes for capturing fetal-specific DNA methylation modification binding proteins, which is characterized in that it comprises the following steps:

1) The base sequence of the methylation probe:

5′-biotin-TGCC(5-mC)GGACTCTGC(5-mC)GCTCTTTGG(5-mC)GAGGGCAGG(5-mC)GGCCAGGAG(5-mC)GTCCGCATT(5-mC)GCCCCGGGT(5-mC)GGCAGTGCC(5 -mC)GGACTCTGC(5-mC)GCTCTTTGG(5-mC)GAGGGCAGG(5-mC)GGCCAGGAG(5-mC)GTCCGCATT(5-mC)GCCCCGGGT(5-mC)GGCAG-3';

The base sequence of the control non-methylated probe:

5′-biotin-TGCCCGGACTCTGCCGCTCTTTGGCGAGGGCAGGCGGCCAGGAGCGTCCGCATTCGCCCCGGGTCGGCAGTGCCCGGACTCTGCCGCTCTTTGGCGAGGGCAGGCGGCCAGGAGCGTCCGCATTCGCCCCGGGTCGGCAG-3′;

2) The insertion sequence between the 5' end of the probe and the first methylation site, and between the 3' end and the second methylation site is a human genome irrelevant sequence, and the insertion sequence is not related to the probe. Complementary to other sequences in the needle.

Furthermore, the probe is composed of a base stem-loop structure, and the 3' or 5' of the probe is labeled with biotin, which can be combined with the avidin on the magnetic beads.

Further, the length of the probe is 10-490bp, preferably 140bp.

Further, the length from the 5' end of the probe to the first methylation site is 1-70 bp, preferably 4 bp.

Further, the length from the 3' end of the probe to the fourteenth methylation site is 1-70 bp, preferably 5 bp.

Further, the number of methylation modification sites of the probe is 1-49, preferably 14.

Further, the methylation probe contains 14 cytosine (C) methylation modification sites, and the cytosine methylation (5-mC) refers to the coupling of methylation at the 5' carbon position of cytosine Kylation modification.

Further, the 5'-biotin refers to the coupling of a biotin label to the 5' end of the sequence.

The present invention also provides a method for capturing fetal-specific DNA methylation modification binding protein, which specifically includes the following steps:

(1) Preparation of probe-magnetic bead complex: use BSA and tRNAs to block streptavidin-labeled magnetic beads; combine pretreated magnetic beads with biotin-labeled DNA probes;

(2) Extraction and pretreatment of total cell protein: extract the total protein sample with lysis buffer, then add non-pretreated magnetic beads to it for incubation, remove the magnetic beads, and collect the supernatant;

(3) Capture binding protein: add the supernatant of the total cell protein sample prepared in step 2 to the probe-magnetic bead complex prepared in step 1, add the binding buffer and incubate, collect the magnetic beads, and wash the magnetic beads three times with the binding buffer , to obtain protein-probe-magnetic bead complexes.

Further, the mass volume percent concentration of BSA in step (1) is 0.05-0.5%.

Preferably, the mass volume percent concentration of the BSA is 0.2%.

Further, the concentration of the tRNAs is 30-60 μg/mL.

Preferably, the concentration of said tRNAs is 40 μg/mL.

Further, the lysis buffer formula in step (2) is: 10mM Tris-Cl (pH7.5), 10mM NaCl, 2mM EDTA and 0.5% TritonX-100.

Further, the binding buffer formula in step (3) is: 10mM Tris-Cl (pH7.5), 1.5mM MgCl2, 150mM KCl, 0.5mM DTT and 0.05% NP-40.

In order to better simulate the unique methylation structure of fetal DNA in the present invention, we combined some insertion sequences with the surrounding sequence of the unique methylation modification site on the fetal gene, so that the secondary structure of the sequence is in the shape of a stem loop .

Features and beneficial effects of the present invention are as follows:

1) The present invention makes it easier for the modification on the DNA probe to enrich the binding protein by simulating the fetal-specific DNA methylation modification structure naturally existing in the cell.

2) The present invention uses BSA and tRNAs to block magnetic beads in advance, which can respectively reduce the binding of magnetic beads to non-specific proteins and genomic DNA without affecting the binding of probes to target proteins.

3) In the present invention, the magnetic beads are first incubated with the probe, and then incubated with the total protein after removing the redundant probe. Compared with the traditional probe, it is incubated with the protein first and then with the magnetic beads or the probe, protein and beads are incubated at the same time. Great savings The amount of magnetic beads used is reduced, and the experimental cost is saved. The present invention uses magnetic beads instead of agarose beads, saves the step of centrifugation, saves time and reduces non-specific precipitation. At the same time, the step of agarose bead centrifugation is replaced by magnetic bead washing, which can remove the supernatant more thoroughly and improve the specificity of the reaction.

In summary, the specific DNA methylation modification probe sequence and secondary structure of the present invention can effectively capture fetal-specific DNA methylation modification binding proteins, significantly improving the enrichment of specific binding proteins.

【Description of drawings】

Fig. 1 is a schematic diagram of the design of the probe of the present invention.

Fig. 2 is a map of binding proteins captured by the probe of the present invention.

【Detailed ways】

The principles and features of the present invention are described in conjunction with the following examples, which are only used to explain the present invention, and are not intended to limit the scope of the present invention.

Embodiment 1 A probe design method for capturing fetal-specific DNA methylation modification binding protein, comprising the following steps:

1) The base sequence of the methylation probe:

5′-biotin-TGCC(5-mC)GGACTCTGC(5-mC)GCTCTTTGG(5-mC)GAGGGCAGG(5-mC)GGCCAGGAG(5-mC)GTCCGCATT(5-mC)GCCCCGGGT(5-mC)GGCAGTGCC(5 -mC)GGACTCTGC(5-mC)GCTCTTTGG(5-mC)GAGGGCAGG(5-mC)GGCCAGGAG(5-mC)GTCCGCATT(5-mC)GCCCCGGGT(5-mC)GGCAG-3';

The base sequence of the control non-methylated probe:

5′-biotin-TGCCCGGACTCTGCCGCTCTTTGGCGAGGGCAGGCGGCCAGGAGCGTCCGCATTCGCCCCGGGTCGGCAGTGCCCGGACTCTGCCGCTCTTTGGCGAGGGCAGGCGGCCAGGAGCGTCCGCATTCGCCCCGGGTCGGCAG-3′;

2) The insertion sequence between the 5' end of the probe and the first methylation site, and between the 3' end and the second methylation site is a human genome irrelevant sequence, and the insertion sequence is not related to the probe. Complementary to other sequences in the needle.

The probe is composed of a base stem-loop structure, and the 3' or 5' of the probe is labeled with biotin, which can combine with the avidin on the magnetic beads.

The length of the probe is 140bp.

The length from the 5' end of the probe to the first methylation site is 4 bp.

The length from the 3' end of the probe to the fourteenth methylation site is 5 bp.

The number of methylation modification sites of the probe is 14.

The methylation probe contains 14 cytosine (C) methylation modification sites, and the cytosine methylation (5-mC) refers to coupling methylation at the 5' carbon position of cytosine grooming.

The 5'-biotin refers to a biotin label coupled to the 5' end of the sequence.

The specific schematic diagram is shown in Figure 1.

Example 2 A method of using the probe in Implementation 1 to capture fetal-specific DNA methylation modification binding proteins

Specific steps are as follows:

(1) Preparation of probe-magnetic bead complex: Wash 80 μL streptavidin-labeled magnetic beads with 1 mL 1× TBST, remove the TBST washing solution, and then add 1 mL 1× TBST containing BSA and tRNAs to the magnetic beads, After incubating at room temperature for 1 hour, remove the supernatant of the TBST solution; add 2-5 μg DNA probes to the pretreated magnetic beads, and add 100 μL 1×TBST, mix gently at 4°C for 30-60 minutes, remove the supernatant; use 1×TBST Wash the beads twice to remove TBST.

Wherein, the formula of the 1×TBST is: 10mM Tris-HCl, 10mM NaCl, 0.1% Tween-20; the concentration of BSA in the 1×TBST is 0.05-0.5%, and the concentration of tRNAs is 20-70 μg/mL.

(2) Extraction and pretreatment of total cell protein: Wash 2×107 cells twice with pre-cooled 1×PBS, centrifuge at 4°C and 1200 rpm for 5 min each time, discard the supernatant; add 800 μL of pre-cooled lysis buffer, 8 μL of 10 mg/mL PMSF (phenylmethylsulfonyl fluoride) solution, 8 μL protease inhibitor cocktail and 4 μL 100 mM DTT (dithiothreitol) solution, vigorously vortex for 20 seconds to fully mix and resuspend the precipitate, ice bath for 30 minutes; Vortex for 15-30 seconds to mix thoroughly; centrifuge at 12,000g-16,000g at 4°C for 15 minutes, discard the precipitate, and the supernatant is the total protein extract of cells; add 40 μL of unpretreated magnetic beads to the total protein extract, 4 Rotate gently at ℃ for 60 min; the supernatant is the pretreated total protein. The formula of the lysis buffer is: 10mM Tris-Cl (pH7.5), 10mM NaCl, 2mM EDTA and 0.5% TritonX-100;

(3) Capture binding protein: Add the total protein prepared in step 2 to the probe-magnetic bead complex, and add 200 μL binding buffer, 5 μL protease inhibitor cocktail, 5 μL 10 mg/mL PMSF solution and 5 μL 0.5 MEDTA solution; 4 °C Rotate and mix and incubate for 60-120 min, collect the magnetic beads, add 1000 μL binding buffer, 10 μL 10 mg/mL PMSF solution and 10 μL protease inhibitor cocktail at 4 °C, wash the magnetic beads, and repeat the washing three times to form the probe-protein complex. The formula of the combined Buffer is: 10mM Tris-Cl (pH7.5), 1.5mM MgCl2, 150mM KCl, 0.5mMDTT and 0.05% NP-40;

The above probe-protein complex was boiled and denatured at 100°C for 10 minutes with 6×protein loading buffer to prepare electrophoresis samples, and the pulldown samples were separated by electrophoresis with 5%-25% gradient polyacrylamide gel, and then separated by silver The method of staining showed protein electrophoresis bands (as shown in Figure 2), and the results showed that, compared with non-methylated probes, methylated probes could effectively capture DNA specifically bound to fetal-specific DNA methylated probes. protein (as shown in Figure 2), and then these specifically bound protein bands were tapped and recovered for mass spectrometry identification to obtain the binding protein to the fetal-specific DNA methylation modification probe.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention.

Claims (2)

1. A method of probe design for capturing a fetal-specific DNA methylation-modified binding protein, comprising the steps of:

1) Base sequence of methylation probe:

5′-biotin-TGCC(5-mC)GGACTCTGC(5-mC)GCTCTTTGG(5-mC)GAGGGCAGG(5-mC)GGCCAGGAG(5-mC)GTCCGCATT(5-mC)GCCCCGGGT(5-mC)GGCAGTGCC(5-mC)GGACTCTGC(5-mC)GCTCTTTGG(5-mC)GAGGGCAGG(5-mC)GGCCAGGAG(5-mC)GTCCGCATT(5-mC)GCCCCGGGT(5-mC)GGCAG-3′；

base sequence of control unmethylated probe:

5′-biotin-TGCCCGGACTCTGCCGCTCTTTGGCGAGGGCAGGCGGCCAGGAGCGTCCGCATTCGCCCCGGGTCGGCAGTGCCCGGACTCTGCCGCTCTTTGGCGAGGGCAGGCGGCCAGGAGCGTCCGCATTCGCCCCGGGTCGGCAG-3′；

2) The insertion sequence between the 5 'end of the probe and the first methylation site and between the 3' end and the second methylation site is a human genome unrelated sequence, and the insertion sequence is not complementary with other sequences in the probe;

wherein the probe consists of a base stem-loop structure, and 3 'or 5' of the probe is provided with a biotin label and can be combined with avidin on the magnetic beads; the length of the probe is 10-490bp;

the length from the 5' end of the probe to the first methylation site is 1-70bp; the length from the 3' end of the probe to the fourteenth methylation site is 1-70bp;

the number of methylation modification sites of the probe is 1-49;

the methylation probe comprises 14 cytosine (C) methylation modification sites, wherein cytosine methylation (5-mC) refers to coupling methylation modification at a 5' -carbon position of cytosine; the 5'-biotin refers to coupling biotin marks at the 5' -end of the sequence.

2. The method of designing a probe for capturing a fetal-specific DNA methylation modified binding protein of claim 1, wherein the probe is 140bp in length, 4bp in length from the 5 'end of the probe to the first methylation site, 5bp in length from the 3' end of the probe to the fourteenth methylation site, and 14 methylation modification sites.

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