JP4884715B2 - WT1 mRNA detection probe and leukemia blast detection reagent - Google Patents

Description

  The present invention relates to a WT1 mRNA detection probe and a leukemia blast detection reagent. More particularly, the present invention relates to the detection of leukemia blasts utilizing the detection of WT1 mRNA by FRET (fluorescence resonance energy transfer).

  For the diagnosis and treatment of leukemia, analysis of chimeric chromosomes of leukemia blasts (cancerous leukocytes) is important. In order to perform an accurate analysis, it is desirable that the ratio of leukemia blasts in the sample (the ratio of the total number of leukocytes) is high, but usually the ratio is only a few percent, which is insufficient for accurate analysis. It is. Thus, although various methods have attempted to concentrate leukemia blasts, there is no sufficient method that satisfies the requirements of clinical practice.

JP 2001-286285 A Inoue K et al., Blood, vol. 84, pp. 3071-3079 (1994).

  If an excellent method for concentrating or separating leukemia blasts from the leukocyte fraction can be developed, leukemia can be diagnosed more accurately and treated more appropriately. Therefore, an object of the present invention is to provide a reagent for specifically detecting leukemia blasts, which can be applied to enrichment or separation of leukemia blasts.

  In order to specifically detect leukemia blasts, the inventors focused on the WT1 gene. The WT1 gene was initially found as a causative gene for Wilms tumor, which is a childhood kidney cancer, but has subsequently been found to be highly expressed in leukemia blasts (Non-patent Document 1). The present inventors thought that leukemia blasts can be detected by specifically detecting cells expressing WT1 mRNA, and leukemia blasts can be separated by combining with a cell sorter or the like.

  Then, as a method for specifically detecting cells expressing WT1 mRNA, a method using FRET (Patent Document 1) was applied, and a FRET probe for detecting WT1 mRNA was developed.

That is, the present invention provides a WT1 mRNA detection probe (FRET probe; one is a donor probe and the other is an acceptor probe) comprising the following oligonucleotide pairs.
(1) 5′-ggcggcgggggtggcg-3 ′ and 5′-gatgaaggagtgaggcggc-3 ′.
(2) 5'-gacagtgaaggcgctcaggc-3 'and 5'-gtgaactggccggaaaagtg-3'.
(3) 5'-gtggttggggaactgc-3 'and 5'-gatcctcatgcttgaatg-3'.
(4) 5'-ccatttcactgagctggag-3 'and 5'-tggttgctctgcccttctgt-3'.
(5) 5'-attgggctccgcagaggatg-3 'and 5'-caccgtgcgtgtgtattctgt-3'.
(6) 5'-ggtatggtttctcaccagtg-3 'and 5'-acagtccttgaagtcacact-3'.
(7) 5′-tacctgtatgagtcctggtg-3 ′ and 5′-gaaggcttttcgcttgttt-3 ′.
Here, an energy donor fluorescent dye is introduced into one oligonucleotide of the oligonucleotide pair (donor probe), and an energy acceptor fluorescent dye is introduced into the other oligonucleotide (acceptor probe). Further, in the hybrid of the oligonucleotide pair and WT1 mRNA, the energy donor fluorescent dye and the energy acceptor fluorescent dye are present at positions separated by 2 to 20 bases.

  Since the FRET probes of the above (1) to (7) hybridize to WT1 mRNA with high efficiency, WT1 mRNA can be detected with high sensitivity.

  The FRET probes (1) to (7) are deoxyribonucleotides, ribonucleotides, 2′-O-methyl-ribonucleotides and 2′-O-4′-C-bridged nucleotides (2′-O-4). Any of the nucleotides selected from '-C-methylene bridged nucleotides, 2'-O-4'-C-ethylene bridged nucleotides, etc.) or any combination of these nucleotides. Oligonucleotide. Further, the oligonucleotide may be a phosphorothioate type instead of a normal phosphodiester type.

  The present invention also provides a leukemia blast detection reagent comprising the FRET probe. Since leukemia blasts highly express WT1, it is possible to identify cells that have been confirmed to be highly expressing WT1 mRNA using the FRET probe as leukemia blasts.

  The WT1 mRNA detection probe of the present invention can specifically detect WT1 mRNA with high sensitivity, thereby detecting leukemia blasts. Therefore, leukemia blasts can be specifically separated using the leukemia blast detection reagent of the present invention, and can be applied to diagnosis and treatment of leukemia.

  The WT1 mRNA detection probe of the present invention comprises a pair of oligonucleotides (FRET probe pair) each having the base sequence shown in Table 1 and labeled with one molecule of a fluorescent dye.

  In Table 1, a, t, g, and c represent nucleotide bases of adenine, thymine, guanine, and cytosine (provided that t is appropriately read as u (uracil)). The position represents a relative position in the WT1 mRNA when the adenine position of the start codon of the WT1 mRNA is 1.

  The introduction position of the fluorescent dye is determined so that FRET is generated from the energy donor fluorescent dye to the energy acceptor fluorescent dye with a significant efficiency in the hybrid formed by the donor probe, the acceptor probe, and the WT1 mRNA. It is clear in the prior art literature that these conditions are satisfied when the distance between the two fluorescent dyes is 2-20 bases on the hybrid (Tsuji et al., Biophys. J., vol. 78, pp. 3260-3274 (2000) and Tsuji et al., Biophys. J., vol. 81, pp. 501-515 (2001)). Further, the introduction position of the fluorescent dye may be any of 5 ′ end, 3 ′ end and in the chain (Tsuji et al., Biophys. J., vol. 78, pp. 3260-3274 (2000) and Tsuji et al. Biophys. J., vol. 81, pp. 501-515 (2001), etc.). In order to generate FRET with high efficiency, the distance between the two fluorescent dyes is preferably 2 to 4 bases.

  The energy donor fluorescent dye is bound directly or via a linker such as a hexylamino group. As the energy donor fluorescent dye, an energy donor fluorescent dye usually used in FRET can be used, and examples thereof include the following fluorescent dyes: 4,4-difluoro-1,3,5,7-tetramethyl-4-bora- 3a, 4a-diaza-s-indacene-8-propionic acid (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene-8-propionicacid) and Its derivatives (these are available from Molecular Probes as the Bodipy493 / 503 series); tetramethylrhodamine isothiocyanate (may be a mixture of 5-isothiocyanate and 6-isothiocyanate) (tetramethylrhodamine-5- (and- 6) -isothiocyanate) and its derivatives (these are available from Molecular Probes as TRITC series); 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4 Diaza-s-indacene-3-propionic acid (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionicacid) and its derivatives (these are Bodipy FL Series available from Molecular Probes); Alexa Fluor series such as Alexa Fluor 488 (available from Molecular Probes) and the like.

  The energy acceptor fluorescent dye is also bound directly or through a linker such as a hexylamino group. As the energy acceptor fluorescent dye, an energy acceptor fluorescent dye usually used for FRET can be used, and examples thereof include the following fluorescent dyes: 1,1′-bis (ε-carboxypentyl) -3,3,3 ′. , 3'-Tetramethylindodicarbocyanine-5,5'-disulfonic acid potassium salt (1,1'-bis (ε-carboxypentyl) -3,3,3 ', 3'-tetramethylindodicarbocyanine-5,5' -disulfonatepotassium salt) and its derivatives (these are available from Amersham Biosciences as the Cy5 series); 1,1′-bis (ε-carboxypentyl) -3,3,3 ′, 3′-tetramethylindocarbocyanine -5,5'-disulfonic acid potassium salt (1,1'-bis (ε-carboxypentyl) -3,3,3 ', 3'-tetramethylindocarbocyanine-5,5'-disulfonatepotassium salt) and its derivatives (these are Amersham as Cy3 series X-rhodamine isothiocyanate (may be a mixture of 5-isothiocyanate and 6-isothiocyanate) (X-rhodamine-5- (and-6) -isothiocyanate) and its derivatives (available from Io Science) These are available from Molecular Probes as the XRITC series); 6-(((4,4-Difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indacene-3-yl ) Styryloxy) acetyl) aminohexanoic acid (6-(((4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoicacid) and its derivatives (these are available from Molecular Probes as the Bodipy 630/650 series); 6-(((4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza -S-indasen-3-yl) styri Xyl) acetyl) aminohexanoic acid (6-(((4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoicacid ) And derivatives thereof (these are available from Molecular Probes as the Bodipy650 / 665 series); Alexa Fluor series (available from Molecular Probes) such as Alexa Fluor 647, and the like.

  In the present invention, it is preferable to use Bodipy 493/503 or Alexa Fluor488 as the energy donor fluorescent dye, and it is preferable to use Cy5 or Alexa Fluor 647 as the energy acceptor fluorescent dye.

  The above FRET probes are deoxyribonucleotides, ribonucleotides, 2′-O-methyl-ribonucleotides and 2′-O-4′-C-bridged nucleotides (2′-O-4′-C-methylene bridged nucleotides, 2 Any oligonucleotide selected from the group consisting of '-O-4'-C-ethylene-bridged nucleotides, etc.) or any combination of these nucleotides.

  A probe composed of deoxyribonucleotides is a DNA probe, and a probe composed of ribonucleotides is an RNA probe.

  In 2′-O-methyl-ribonucleotide, the hydroxyl group at the 2′-position of ribose of the ribonucleotide is methylated, and the probe composed of the oligonucleotide is more stable against nucleases than DNA probes and RNA probes. Etc. have improved.

  2'-O-4'-C-bridged nucleotide is a nucleotide in which 2'-O and 4'-C of ribose of ribonucleotide are bridged with an alkylene chain (methylene, ethylene, etc.), and Locked Nucleic Acid Also called (LNA). Cross-linking restricts the flexibility of the ribose ring, thereby improving the hybridization ability (increasing Tm value) and improving the stability against nucleases.

  The oligonucleotide may be a phosphorothioate type instead of a normal phosphodiester type, and such an oligonucleotide is referred to as an S-oligo. In an oligonucleotide, riboses are usually bonded to each other via a phosphodiester bond, but S-oligos in which the bond is replaced with a phosphorothioate group have improved stability against nucleases. In addition to oligonucleotides in which deoxyribonucleotides and ribonucleotides are linked by phosphorothioate groups, 2RET-O-methyl-ribonucleotides and oligonucleotides in which 2'-O-4'-C-bridged nucleotides are linked by phosphorothioate groups Can function as a probe.

  The oligonucleotide according to the present invention can be synthesized according to a known method (eg, phosphoramidite method, β-cyanoethylamidite method, phosphorothioate method). Depending on the type of the target oligonucleotide, necessary reagents (phosphoramidite reagents and the like) can be used properly, and such reagents are commercially available.

  The WT1 mRNA detection probe of the present invention has been described above. A method for detecting leukemia blasts using the WT1 mRNA detection probe will be described below.

  First, the WT1 mRNA detection probe of the present invention is introduced into living cells (leukocytes). As the introduction method, known methods such as microinjection method, electroporation method, lipofection method and the like can be applied. Further, a method of perforating the membrane with a bacterial toxin such as streptolysin O can also be applied.

  After introducing the WT1 mRNA detection probe into the cell, the donor probe and the acceptor probe are hybridized to the WT1 mRNA. The hybridization condition may be, for example, by incubating the cell into which the leukemia blast detection reagent has been introduced for several minutes at room temperature.

  When membrane perforation treatment is performed with streptricin O or the like, it is preferable to re-encapsulate the membrane (incubate with a medium containing serum for several minutes) after hybridization.

  Next, cells in which a hybrid of the donor probe / acceptor probe and WT1 mRNA is present are detected. Leukemia blasts can be detected by irradiating the cells with excitation light of a fluorescent dye bound to the donor probe and observing fluorescence from the fluorescent dye bound to the acceptor probe based on FRET.

  The excitation light irradiation simultaneously excites the energy donor fluorescent dye of the donor probe hybridized with the mRNA and the energy donor fluorescent dye of the donor probe not hybridized with the mRNA. FRET occurs only when the ceptor probe is hybridized, and at this time, fluorescence is generated from the energy acceptor fluorescent dye of the acceptor probe. That is, observation of fluorescence from the energy acceptor fluorescent dye means that the donor probe and the acceptor probe are close to each other, and it can be seen that WT1 mRNA is expressed in living cells.

  The living cells expressing the WT1 mRNA detected in this manner are selectively separated. Although there is no restriction | limiting in particular regarding the isolation | separation method, While using the cell sorter (Fluorescence Activated Cell Sorter, FACS), it is preferable to isolate | separate selectively, while detecting the living cell which is expressing WT1 mRNA.

  The cell sorter is equipped with a flow cytometer and a cell sorting device. By irradiating individual cells stained with a fluorescently labeled probe with a specific substance with laser light in the middle of a narrow channel, scattered light ( Forward scattered light and side scattered light) and fluorescence signal information is measured for each cell, and the result is displayed, for example, as a frequency distribution (dot plot), and specific signal information is emitted. The cells can be gated to sort the desired cells. Such a method is called flow cytometry.

  For example, live cells expressing WT1 mRNA can be isolated by applying the method described below. That is, the relative fluorescence intensity of the fluorescent dye when irradiated with a laser that excites an energy donor fluorescent dye (for example, Bodipy 493/503) in an individual living cell by a cell sorter, and an energy acceptor fluorescent dye based on FRET ( For example, signal information on the relative fluorescence intensity of Cy5) is obtained, and, for example, dot plot is performed with the former as the horizontal axis and the latter as the vertical axis, and a cell group in which the latter has a high value is selected, and the region is designated as R2. . Furthermore, the signal information of the forward scattered light based on the cell size of the living cell to be measured and the side scattered light based on the complexity of the internal structure of the living cell is obtained, for example, with the former as the horizontal axis and the latter as the vertical axis. Dot plotting is performed, a cell group considered to represent a living cell to be measured is selected, and an area is designated as R1. Then, by setting a cell sorter so that only living cells satisfying both selection conditions of R1 and R2 (belonging to R1 and R2) can be sorted, only cells expressing WT1 mRNA are selectively separated. Can do.

  (Synthesis of FRET probe)

  Here, in the hybrid formed by the three of the donor probe, the acceptor probe, and the WT1 mRNA, the distance between the fluorescent dyes is 4 bases, but the distance between the fluorescent dyes is not limited to 4 bases. Moreover, the introduction position of the energy donor fluorescent dye in the donor probe is the 5 ′ end, and the energy acceptor fluorescent dye in the acceptor probe is in the chain of 3 or 4 bases from the 3 ′ end, but the introduction position is limited to these. It is not something. The three-component hybrid is formed such that the 5 ′ end of the donor probe is adjacent to the 3 ′ end of the acceptor probe. However, the present invention is not limited to this form, and the acceptor probe 5 ′ end is the donor. It may be formed adjacent to the 3 ′ end of the probe. That is, for example, in Table 2 above, x may be an energy acceptor fluorescent dye such as Cy5, and y may be an energy donor fluorescent dye such as Bodipy 493/503.

  Donor probes having the sequences described in Table 2 were synthesized according to the method described in Patent Document 1. More details are as follows. 2.5 mg NHSS (N-Hydroxysulfosuccinimidesodium salt) dissolved in 30 μL sterilized water, 5 mg EDAC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) dissolved in 50 μL sterilized water, and 1 mg A solution obtained by dissolving Bodipy493 / 503 propionic acid in 50 μL of DMF was mixed and reacted at room temperature for 30 minutes.

On the other hand, by using a DNA / RNA synthesizer (model 394 manufactured by PerkinElmer or model 8909 manufactured by Perceptive Biosystems), an oligo DNA having a base sequence excluding x of the base sequences shown in Table 2 by β-cyanoethylamidite method. Synthesized. A hexylamino group was introduced into the 5 ′ end of the synthesized oligo DNA using 6- (trifluoroacetylamino) hexyl- (2-cyanoethyl)-(N, N-diisopropyl) -phosphoramidite and lyophilized. . This was dissolved in 200 μL of 0.5 M Na ₂ HCO ₃ / NaH ₂ CO ₃ buffer (pH 9.3).

  These were mixed and reacted overnight under light-shielding conditions. After gel filtration of the reaction solution to remove unreacted dye, reversed-phase high-performance liquid chromatography using CAPCELL PACK18 (manufactured by Shiseido Co., Ltd., column size: 6 mm inner diameter x 250 mm total length) : 40 ° C, mobile phase composition: 5% acetonitrile aqueous solution containing 5 mM triethylammonium acetate (A) and 40% acetonitrile aqueous solution containing 5 mM triethylammonium acetate (B), concentration gradient: 30-80% B (0-20 minutes)) Fractions having absorption at 260 nm and 493 nm were collected and lyophilized.

  Next, the acceptor probes described in Table 2 were synthesized according to the method described in Patent Document 1. More details are as follows. One tube of Cy5 dye (Amersham, Fluorolink Cat. No. PA25001) was dissolved in 100 μL of sterilized water.

On the other hand, an oligo DNA in which a hexylamino group was introduced into a portion corresponding to y in the base sequence shown in Table 2 was synthesized using Uni-Link AminoModifier (manufactured by Clontech) and lyophilized. This was dissolved in 200 μL of Na ₂ HCO ₃ / NaH ₂ CO ₃ buffer (0.5 M, pH 9.3).

  These were mixed and reacted overnight under light-shielding conditions. After gel filtration of the reaction solution to remove unreacted dye, reversed-phase high performance liquid chromatography using CAPCELL PACK18 (flow rate: 1 mL / min, column temperature: 40 ° C., mobile phase composition: 5 mM containing triethylammonium acetate 5 A fraction having an absorption at 260 nm was fractionated with a 40% acetonitrile aqueous solution (B) containing 5% acetonitrile aqueous solution (A) and 5 mM triethylammonium acetate (concentration gradient: 15-60% B (0-20 minutes)). The absorption spectrum of the obtained fraction was measured in the range of 220-700 nm, and after confirming the absorption maximum of Cy5 at 650-700 nm, the obtained fraction was freeze-dried.

(Preparation of WT1 mRNA)
Plasmid DNA containing human WT1 cDNA (pUCWT1; purchased from RIKEN BioResource Center) was cleaved with restriction enzymes EcoRI and HincII to obtain a WT1 cDNA fragment. The WT1 cDNA fragment was ligated using a ligation kit (manufactured by Takara Bio Inc.) so that the fragment was located downstream of the T7 promoter at the digestion site of RNA synthesis vector pBluescriptII KS + with EcoRI / HincII. The obtained recombinant plasmid was introduced into competent cells (manufactured by Takara Bio Inc.) of Escherichia coli JM109 strain, and the resulting transformant was cultured to replicate the recombinant plasmid.

  The replicated plasmid was linearized with HincII and then treated with Proteinase K (manufactured by Takara Bio Inc.) and phenol / chloroform for denaturation and deproteinization. Using the purified gene fragment as a template, RNA was synthesized using an in vitro transcription kit (Megascript T7 Kits: Ambion). DNase I (manufactured by Takara Bio Inc.) was added to the RNA solution to digest plasmid DNA, RNA was precipitated by adding an equal volume of lithium chloride solution and twice the amount of ethanol, washed with 70% ethanol, and then dried. . RNA was dissolved in distilled water containing no RNase and used in the following experiments.

(Measurement of fluorescence spectrum change)
Changes in the fluorescence spectrum due to FRET accompanying the close hybridization of the donor probe and the acceptor probe were measured. Add donor probe and acceptor probe to a final concentration of 1 μM in 150 μL of 1 × SSC (150 mM sodium chloride, 17 mM sodium citrate, pH 7.0) solution, let stand at room temperature for 15 minutes, and then use a four-sided quartz cuvette. The fluorescence spectrum was measured in the range of excitation wavelength 488 nm and fluorescence wavelength 500-750 nm with a fluorescence spectrophotometer (F4500; manufactured by Hitachi, Ltd.). Next, 50 pmol of WT1 mRNA was added to this solution and allowed to stand for 30 minutes, and then the fluorescence spectrum at room temperature was measured to observe changes in the fluorescence spectrum accompanying hybridization. After measuring the fluorescence spectrum, 50 pmol of WT1 mRNA was further added, and the fluorescence spectrum was measured again. The same operation was repeated until the amount of WT1 mRNA reached 200 pmol, and the fluorescence spectrum was measured.

Changes in the fluorescence spectra of the probes listed in Table 2 are shown in FIGS. When the WT1Ex1c probe was used (FIG. 3), no change in the fluorescence spectrum was observed, but when any other probe was used (FIGS. 1-2 and 4-8), a change in the fluorescence spectrum was observed. It was. Table 3 summarizes the ratio of the fluorescence intensity at 670 nm to the fluorescence intensity at _{515 nm} (I _{670 nm} / I _{515 nm} ) in the presence of the FRET probe alone and in the presence of WT1 mRNA (200 pmol).

(Investigation of FRET probe introduction into cells)
As a method for introducing a FRET probe into a living cell, membrane perforation with streptocrine O (SLO) and re-encapsulation with a serum medium were used. SLO treatment was performed by referring to the method described in Giles RV etal., Nucleosides & Nucleotides, vol. 16, 1155-1163 (1997).

  As a conditional search for introducing the FRET probe, first, the introduction efficiency and cytotoxicity of the SLO method were examined using FITC-Dextran (molecular weight 9300; manufactured by Sigma).

  25000 Units of SLO (manufactured by Sigma) was dissolved in 25 mL of PBS (phosphate buffered saline) containing 0.05% BSA (bovine serum albumin; manufactured by Sigma) to prepare a 1000 Unit / mL SLO solution. Prior to membrane perforation, DTT (dithiothreitol) at a final concentration of 5 mM was added and left at 37 ° C. for 2 hours.

RPMI containing 10% FBS (fetal calf serum) and 1% Glu (glutamate) K562 cultured cell line (JCRB0019) reported to constitutively express WT1 -1640 medium (all manufactured by Invitrogen), and the cells are collected by centrifugation (800 g, 5 minutes, room temperature; the following centrifugation is performed under the same conditions) and suspended in RPMI-1640 medium (without serum). The serum component was removed by centrifugation again. Cells were suspended in RPMI-1640 medium and dispensed to contain 4 × 10 ⁶ cells per centrifuge tube. After centrifugation, the supernatant was discarded, and the pelleted cell mass was suspended in 200 μL of RPMI-1640 medium containing FITC-Dextran (final concentration 10 μM).

To this 200 μL cell suspension, add SLO solution so that the final number of units is 0, 1, 2, 4, 6, 8, 10, 12, 14 Unit / 10 ⁶ cells. Incubation was performed for a minute. After incubation, add 1 mL of RPMI-1640 medium containing 10% FBS and 1% Glu to the cell suspension and incubate for 30 minutes at 37 ° C. Add PI (PropidiumIodide; Sigma) and add ice. Incubated for 10 minutes at temperature. PI is a reagent that stains DNA in the nucleus as an intercalator. Since PI cannot normally permeate the cell membrane, it is used to confirm whether the cell membrane punctured by SLO has been re-encapsulated by the addition of serum medium.

  After incubation, the cell suspension was centrifuged, the supernatant was discarded, suspended in PBS, and washed by centrifugation. The precipitated cell mass was suspended in 1 mL of PBS, and the presence or absence of cell damage in the process of membrane perforation and re-encapsulation by SLO was confirmed from the transmission image and fluorescence image of the cell group by observation with a fluorescence microscope. At the same time, FACS (FACSCalibur; manufactured by Becton Dickinson) enables cell size (FSC-Height: forward scattered light), internal structure complexity (SSC-Height: side scattered light), and FITC- The amount of Dextran (as fluorescence intensity FL1) and the number of incompletely encapsulated cells (as PI fluorescence intensity FL3) were measured.

FIG. 9 shows the result of fluorescence microscope observation, and FIG. 10 shows the result of FACS measurement. From these results, it was considered that the optimal concentration of SLO, which has the highest FRET probe introduction efficiency by membrane perforation treatment and hardly causes incomplete re-encapsulation, is 10 Unit / 10 ⁶ cells.

Next, re-encapsulation conditions (temperature and time) by incubation in serum medium were examined. A final concentration of 10 Unit / 10 ⁶ cells SLO and a final concentration of 10 μM FITC-Dextran were added to 40 μL of PBS containing 2 × 10 ⁶ K562 cells and incubated at 37 ° C. for 10 minutes. After completion of the incubation, 1 mL of RPMI-1640 medium containing 10% FBS and 1% Glu was added to the cell suspension, and incubation was performed at 37 ° C. or ice temperature. 10, 20, 30, 40 and 60 minutes after the addition of the medium, PI was added and incubated at ice temperature for 10 minutes.

  In FIG. 11, the ratio of the cell into which FITC-Dextran and PI were taken in in each condition was shown. As is clear from FIG. 11, no significant difference was observed in the effect of re-encapsulation regardless of whether re-encapsulation was performed at 37 ° C. or ice temperature. Also, no significant difference was observed between 10 minutes and 60 minutes in terms of treatment time.

(Introduction of FRET probe into K562 cells and separation of cells by FRET signal)
K562 cells were collected by centrifugation, suspended in RPMI-1640 medium, and then the cell suspension was dispensed so that 2 x 10 ⁶ cells of K562 cells were contained in each centrifuge tube and centrifuged. . 10 μL of 100 μM donor probe (final concentration 25 μM), 10 μL of 100 μM acceptor probe (final concentration 25 μM) and 20 μL of SLO solution (1000 Unit / mL) are suspended in the pelleted cell mass that has settled ( 40 μL in total) and incubated at 37 ° C. for 10 minutes. RPMI-1640 medium containing 1 mL of 10% FBS and 1% Glu was added and incubated at ice temperature for 10 minutes. After centrifugation, it was washed once with PBS and centrifuged. After suspending in 1 mL of PBS, analysis was performed by FACS.

  As the FRET probe, a WT1Ex1b probe whose fluorescence spectrum changed remarkably and a WT1Ex1c probe whose fluorescence spectrum did not change at all were used. The analysis result of FACS is shown in FIG. In the cells into which the WT1Ex1b probe was introduced, an increase in the fluorescence of the acceptor probe by FRET was observed, whereas in the cells into which the WT1Ex1c probe was introduced, an increase in the fluorescence by FRET was not observed.

FIG. 1 is a graph showing changes in fluorescence spectrum when using the WT1Ex1a probe. FIG. 2 is a graph showing changes in fluorescence spectrum when the WT1Ex1b probe is used. FIG. 3 is a graph showing changes in fluorescence spectrum when the WT1Ex1c probe is used. FIG. 4 is a graph showing changes in fluorescence spectrum when the WT1Ex2 probe is used. FIG. 5 is a graph showing changes in fluorescence spectrum when the WT1Ex5 probe is used. FIG. 6 is a graph showing changes in fluorescence spectrum when the WT1Ex6 probe is used. FIG. 7 is a graph showing changes in fluorescence spectrum when the WT1Ex8 probe is used. FIG. 8 is a graph showing changes in fluorescence spectrum when using the WT1KTS probe. FIG. 9 is a diagram showing the results of fluorescence microscope observation of K562 cells after membrane perforation treatment and re-encapsulation treatment. The numbers in the figure represent the number of SLO units (Unit / 10 ⁶ cell). FIG. 10 is a graph showing the results of FACS analysis of K562 cells after membrane perforation treatment and re-encapsulation treatment. Open triangles correspond to FL1 (+) and FL3 (-) cells, and represent FITC-Dextran cells that are not stained with PI. That is, it represents a cell in which the probe has been introduced into the cell and has been successfully re-encapsulated. Black triangles correspond to FL1 (−) and FL3 (−) cells, and represent cells that are not transfected with FITC-Dextran and that are not stained with PI. That is, it represents an untreated cell in which the cell is not membrane perforated. Black circles correspond to FL1 (−) and FL3 (+) cells, and represent cells that are not introduced with FITC-Dextran and stained with PI. That is, it represents a dead cell in which the cell membrane cannot be re-encapsulated and the cell membrane is broken. Open squares correspond to FL1 (+) and FL3 (+) cells, but are considered dead cells that failed to be re-encapsulated. FIG. 11 is a graph showing the percentage of cells that have taken up FITC-Dextran and PI under various re-encapsulation conditions. FIG. 12 is a diagram showing FACS analysis results of cells into which the WT1Ex1b probe and WT1Ex1c probe have been introduced. (A) No probe added (b) WT1Ex1b probe added (c) WT1Ex1c probe added.

Claims (10)

A first oligonucleotide represented by 5′-ggcggcgggggtggcg-3 ′ introduced with a first fluorescent dye and a second oligonucleotide represented by 5′-gatgaaggagtgaggcggc-3 ′ introduced with a second fluorescent dye Consisting of oligonucleotides,
Of the first and second fluorescent dyes, one is an energy donor fluorescent dye, the other is an energy acceptor fluorescent dye,
In the hybrid of the first and second oligonucleotides and WT1 mRNA, the first and second fluorescent dyes are present at positions separated by 2 to 20 bases.
WT1 mRNA detection probe. The WT1 mRNA detection probe according to claim 1, wherein the oligonucleotide is composed of deoxyribonucleotides. The WT1 mRNA detection probe according to claim 1, wherein the oligonucleotide is composed of ribonucleotides. The WT1 mRNA detection probe according to claim 1, wherein the oligonucleotide is composed of 2'-O-methyl-ribonucleotide. The WT1 mRNA detection probe according to claim 1, wherein the oligonucleotide is composed of 2'-O-4'-C-bridged nucleotides. The WT1 mRNA detection probe according to claim 5 , wherein the 2'-O-4'-C-bridged nucleotide is a 2'-O-4'-C-methylene bridged nucleotide. The WT1 mRNA detection probe according to claim 5 , wherein the 2'-O-4'-C-bridged nucleotide is a 2'-O-4'-C-ethylene bridged nucleotide. Chimera oligo composed of two or more nucleotides selected from the group consisting of deoxyribonucleotides, ribonucleotides, 2′-O-methyl-ribonucleotides and 2′-O-4′-C-bridged nucleotides The WT1 mRNA detection probe according to claim 1, which is a nucleotide. The WT1 mRNA detection probe according to any one of claims 2 to 8 , wherein the oligonucleotide is a phosphorothioate type nucleotide. A leukemia blast detection reagent comprising the WT1 mRNA detection probe according to any one of claims 1 to 9 .

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