CN110964073B

CN110964073B - Probes and methods for capturing fetal-specific DNA methylation-modified binding proteins

Info

Publication number: CN110964073B
Application number: CN201911188858.4A
Authority: CN
Inventors: 金莉萍; 郑青亮
Original assignee: Shanghai First Maternity and Infant Hospital
Current assignee: Shanghai First Maternity and Infant Hospital
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2023-06-02
Anticipated expiration: 2039-11-28
Also published as: CN110964073A

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

Probes and methods for capturing fetal-specific DNA methylation-modified binding proteins

[ field of technology ]

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

[ background Art ]

Clinically, the detection of circulating cell-free fetal DNA (cff-DNA) in maternal plasma is currently an important method for achieving noninvasive prenatal diagnosis. As early as 1997, professor Luming et al reported the presence of cff-DNA in maternal plasma, showing that fetal-derived DNA could be detected in maternal plasma 2 weeks after pregnancy and cleared and disappeared rapidly from maternal plasma within 2 hours after delivery (loym, cobetta N, chamberlain PF, et al present of fetal DNA in maternal plasma and service.Lancet 1997; 350:485-7). Thus, cff-DNA of maternal plasma has become an important target for non-invasive prenatal diagnosis. However, cff-DNA represents only a small fraction (about 10%) of maternal plasma, the remainder all being derived from maternal blood cells; the current noninvasive prenatal diagnosis using cff-DNA is based mainly on paternal genetic markers such as SYR genes (Chiu RW, lo YM. Non-invasive prenatal diagnosis by fetal nucleic acid analysis in maternal plasma: the com of age. Semin Fetal Neonatal Med 2011; 16:88-93.).

Recently, researchers have attempted to develop fetal DNA polymorphism markers that are not related to fetal genetics. One important method is to detect epigenetic markers of the fetus in maternal plasma, indicating the presence of different DNA methylation patterns between fetal tissue and maternal blood cells (Chan KC, ding C, gerovassili A, et al hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnostics. Clin Chem 2006; 52:2211-8.). DNA methylation generally refers to the presence of methylation at the 5' carbon of cytosine nucleotides followed by guanine nucleotides, which constitute a CpG dinucleotide. Currently, 485,577 CpG modification sites exist in fetal tissues through methylation chips, and are present in UTR regions, promoter regions or gene coding regions. There is evidence that fetal circulating DNA in maternal peripheral blood is mainly derived from placenta, as it is the only channel of nutrient transport between mother and child. Thus, it is possible to develop specific molecular markers between maternal and fetal tissue based on markers of different DNA methylation patterns.

At present, no report on design of a DNA methylation probe with fetal related specificity is seen, so that development and design of a DNA methylation probe special for a fetus and an operation method for capturing DNA methylation modified binding protein special for the fetus are very important, and a new drug target is found for mechanism research and clinical intervention of pregnancy related diseases such as premature birth, repeated spontaneous abortion and the like of the fetus.

[ invention ]

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

The object of the invention is achieved by:

the invention provides a design method of a probe for capturing a fetal specific DNA methylation modified binding protein, which is characterized by comprising the following 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 to other sequences in the probe.

Further, 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.

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 1-70bp, preferably 4bp.

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

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

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

Further, the 5'-biotin means that biotin is coupled to the 5' -end of the sequence.

The invention also provides a method for capturing a fetal-specific DNA methylation-modified binding protein, comprising the steps of:

(1) Preparation of probe-magnetic bead complexes: blocking streptavidin-labeled magnetic beads with BSA and tRNAs; binding the pretreated magnetic beads to biotin-labeled DNA probes;

(2) Extracting and pre-treating cell total protein: extracting a total protein sample by using a lysis Buffer, adding untreated magnetic beads into the total protein sample for incubation, removing the magnetic beads, and collecting a supernatant;

(3) Capturing a binding protein: and (3) adding the supernatant of the cell total protein sample prepared in the step (2) into the probe-magnetic bead compound prepared in the step (1), adding a binding Buffer, incubating, collecting magnetic beads, and washing the magnetic beads three times by using the binding Buffer to obtain the protein-probe-magnetic bead compound.

Further, the BSA in the step (1) has a mass-volume percentage concentration of 0.05-0.5%.

Preferably, the BSA has a mass-volume percentage concentration of 0.2%.

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

Preferably, the concentration of tRNAs is 40. Mu.g/mL.

Further, the formula of the cleavage buffer in the step (2) is as follows: 10mM Tris-Cl (pH 7.5), 10mM NaCl, 2mM EDTA and 0.5% Triton X-100.

Further, in the step (3), the formula of the combined buffer is as follows: 10mM Tris-Cl (pH 7.5), 1.5mM MgCl2, 150mM KCl, 0.5mM DTT and 0.05% NP-40.

In order to better simulate the special methylation structure of the fetal DNA in cells, the invention combines a plurality of insertion sequences according to the special methylation modification site peripheral sequences on the fetal genes, so that the secondary structure of the sequences is in a stem loop shape.

The invention has the following characteristics and beneficial effects:

1) The invention allows the modification on the DNA probe to be more easily enriched for binding proteins by mimicking the fetal-specific DNA methylation modification structure naturally occurring in the cell.

2) The invention can respectively reduce the combination of the magnetic beads with nonspecific proteins and genomic DNA by using BSA and tRNAs to block the magnetic beads in advance, and can not influence the combination of the probes and target proteins.

3) According to the invention, the magnetic beads are firstly incubated with the probes, and then incubated with the total protein after the redundant probes are removed, so that compared with the traditional probes which are incubated with the proteins and then incubated with the magnetic beads or the probes, the proteins and the beads are incubated simultaneously, the use amount of the magnetic beads is greatly reduced, and the experimental cost is saved. The invention adopts magnetic beads to replace agarose beads, saves the step of centrifugation, saves time and reduces non-specific precipitation. Meanwhile, the step of agarose bead centrifugation is replaced by the step of magnetic bead washing, so that the supernatant can be removed more thoroughly, and the specificity of the reaction is improved.

In conclusion, the specific DNA methylation modified probe sequence and the secondary structure can effectively capture the specific DNA methylation modified binding protein of the fetus, and the enrichment of the specific binding protein is obviously improved.

[ description of the drawings ]

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

FIG. 2 is a graph of binding proteins captured using a probe of the present invention.

[ detailed description ] of the invention

The principles and features of the present invention are described in connection with the following examples, which are intended to be illustrative of the invention and are not intended to limit the scope of the invention.

Example 1a method of probe design for capturing fetal-specific DNA methylation-modified binding proteins comprising the steps of:

1) Base sequence of methylation probe:

base sequence of control unmethylated probe:

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 probe length was 140bp.

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

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

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

The methylation probe comprises 14 cytosine (C) methylation modification sites, and 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.

A specific schematic diagram is shown in fig. 1.

Example 2A method for capturing fetal-specific DNA methylation-modified binding proteins Using the probe of example 1

The method comprises the following specific steps:

(1) Preparation of probe-magnetic bead complexes: 80. Mu.L of streptavidin-labeled beads were washed with 1mL of 1 XTBE, the TBST wash was removed, 1mL of 1 XTBE containing BSA and tRNAs was added to the beads, and the supernatant of the TBST solution was removed after incubation for 1h at room temperature; adding 2-5 μg of DNA probe into the pretreated magnetic beads, adding 100 μl of 1 XTBE, mixing at 4deg.C with gentle rotation for 30-60min, removing supernatant; the beads were washed twice with 1 XTBST to remove TBST.

Wherein the formula of the 1 XTBST is as follows: 10mM Tris-HCl,10mM NaCl,0.1%Tween-20; the BSA concentration in 1 XTBST is 0.05-0.5% and the tRNAs concentration is 20-70. Mu.g/mL.

(2) Extracting and pre-treating cell total protein: washing 2X 107 cells twice with pre-chilled 1 XPBS, centrifuging at 4deg.C for 5min at 1200rpm, and discarding the supernatant; add 800. Mu.L of pre-chilled lysis Buffer, 8. Mu.L of 10mg/mL PMSF (phenylmethylsulfonyl fluoride) solution, 8. Mu. L protease inhibitor cocktail and 4. Mu.L of 100 mM DTT (dithiothreitol) solution, vortex vigorously for 20 seconds to mix well and re-suspend the pellet, ice bath for 30min; the high-speed violent vortex is fully and evenly mixed for 15 to 30 seconds every 2 minutes; centrifuging at 4deg.C for 15min at 12,000g-16,000g, discarding the precipitate, and collecting the supernatant as total cell protein extract; adding 40 μl of non-pretreated magnetic beads to the total protein extract, and gently rotating at 4deg.C for 60min; the supernatant is the pretreated total protein. The formula of the cracking Buffer is as follows: 10mM Tris-Cl (pH 7.5), 10mM NaCl, 2mM EDTA and 0.5% TritonX-100;

(3) Capturing a binding protein: adding the total protein prepared in the step 2 to the probe-magnetic bead complex, and adding 200. Mu.L of binding Buffer, 5. Mu. L protease inhibitor cocktail, 5. Mu.L of a PMSF solution of 10mg/mL and 5. Mu.L of a 0.5M EDTA solution; and (3) rotating, mixing and incubating for 60-120min at 4 ℃, collecting magnetic beads, adding 1000 mu L of binding Buffer, 10 mu L of PMSF solution with concentration of 10mg/mL and 10 mu L protease inhibitor cocktail at 4 ℃, washing the magnetic beads, and repeating washing for three times to obtain the probe-protein complex. The formula of the combined Buffer is as follows: 10mM Tris-Cl (pH 7.5), 1.5mM MgCl2, 150mM KCl, 0.5mM DTT and 0.05% NP-40;

the probe-protein complex is subjected to sample cooking denaturation by using a 6 x protein loading buffer at 100 ℃ for 10min, an electrophoresis sample is prepared, a pulldown sample is subjected to electrophoresis separation by using 5% -25% gradient polyacrylamide gel, a protein electrophoresis strip is displayed by a silver staining method (shown in figure 2), and the result shows that compared with a non-methylation probe, a methylation probe can effectively capture a protein specifically bound with a DNA methylation probe specific to a fetus (shown in figure 2), and then the specific bound protein strips are subjected to tapping recovery for mass spectrum identification, so that the binding protein with the DNA methylation modified probe specific to the fetus is obtained.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims

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:

base sequence of control unmethylated probe:

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.