U.S. patent application number 10/556679 was filed with the patent office on 2010-12-23 for transcription chip.
Invention is credited to Miwa Bando, Yoji Fukuda, Mineyoshi Hiyoshi, Hiroshi Kido, Moritoshi Kinoshita, Hiroshi Mizuguchi.
Application Number | 20100323904 10/556679 |
Document ID | / |
Family ID | 33447291 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100323904 |
Kind Code |
A1 |
Kido; Hiroshi ; et
al. |
December 23, 2010 |
Transcription chip
Abstract
A transcription chip comprising a substrate and, immobilized
thereon, at least one polynucleotide including an element sequence
to which a transcription factor can be bound.
Inventors: |
Kido; Hiroshi; (Tokushima,
JP) ; Hiyoshi; Mineyoshi; (Tokushima, JP) ;
Bando; Miwa; (Tokushima, JP) ; Kinoshita;
Moritoshi; (Tokushima, JP) ; Fukuda; Yoji;
(Tokushima, JP) ; Mizuguchi; Hiroshi; (Tokushima,
JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Family ID: |
33447291 |
Appl. No.: |
10/556679 |
Filed: |
May 13, 2004 |
PCT Filed: |
May 13, 2004 |
PCT NO: |
PCT/JP04/06796 |
371 Date: |
June 29, 2007 |
Current U.S.
Class: |
506/9 ;
506/17 |
Current CPC
Class: |
C12Q 1/6837 20130101;
C12Q 2565/531 20130101; C12Q 1/6837 20130101 |
Class at
Publication: |
506/9 ;
506/17 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/08 20060101 C40B040/08 |
Foreign Application Data
Date |
Code |
Application Number |
May 16, 2003 |
JP |
2003-138395 |
Claims
1. A transcription chip wherein at least one polynucleotide
comprising one or more element sequences to which a transcription
factor can be bound is immobilized on a substrate.
2. The chip according to claim 1 wherein at least two
polynucleotides comprising one or more element sequences to which
transcription factors can be bound respectively are bound.
3. The chip according to claim 1 wherein said polynucleotide has a
partial sequence of a promoter in the upstream (5') side of each
gene.
4. The chip according to claim 1 wherein said substrate is made by
forming a diamond thin film on a support.
5. The chip according to claim 1 wherein said polynucleotide is
bound to the diamond thin film through an optionally appropriate
spacer.
6. A method for assaying a binding of a transcription factor
comprising a step of interacting a sample capable of comprising the
transcription factor with the transcription chip according to claim
1.
7. The method according to claim 6 for evaluating an effect of a
subject substance on the transcription factor, wherein said sample
is a cell lysate of cells cultured in the presence of the subject
substance.
8. The method according to claim 7 wherein the binding of the
transcription factor is detected by an antibody against the
transcription factor or a method utilizing mass spectrometry.
9. The method according to claim 8 wherein the detection using the
antibody is an ELISA and the method utilizing mass spectrometry is
a peptide mass fingerprint method.
Description
TECHNICAL FIELD
[0001] The present invention relates to a transcription chip and a
method for measuring an activity of a transcription factor using
the chip.
BACKGROUND ART
[0002] A DNA chip is a tool to profile gene polymorphism and
variance of mRNA expression levels, and is currently used in wide
research fields. Proteins which are final products obtained by
translating mRNA which transfer genomic information bear
momentarily changing vital phenomena. Thus, it has been believed
that more useful information than the information obtained by
profiling the mRNA is obtained from the protein, and techniques for
proteome analysis which analyzes the protein exhaustively have been
actively developed.
[0003] In such an actual circumstance, if a system where a protein
is captured on a substrate and efficiently characterized is
completed, it is estimated that the system will be a new technology
which is superior to conventional analytical methods for the
proteome using two dimensional electrophoresis or liquid
chromatography in terms of scale and cost. Up to now, antibody
chips have been developed as the chip which targets the proteome
analysis. The antibody chip is the tool where many antibodies are
immobilized on chip surfaces and amounts of antigens which interact
with the immobilized antibodies, respectively are profiled. The
antibody chips were developed later than DNA chips, but their
practical application in the wide fields such as drug discovery,
toxicity tests or pathological diagnosis in the future is
anticipated because the variance of the proteins can be directly
analyzed.
[0004] A transcription factor is deeply involved in cellular
inflammatory response, carcinogenesis and transcription controlling
factor activation because it is the protein which is bound to a
region called an element sequence in an upstream region of each
gene and regulates the expression of various genes. Therefore, it
is very important to, at a protein level, profile various
transcription factors which keep biological activity.
[0005] Conventionally, the transcription factor has been analyzed
by a technique using acrylamide gel electrophoresis using a
radioisotope called a gel shift assay, but the technique is
problematic in throughput and safety, and it is believed that it is
difficult to apply to future proteomic techniques.
[0006] The present invention intends to provide a technology to
efficiently profile the transcription factor.
DISCLOSURE OF INVENTION
[0007] The present inventor has made a transcription chip where
single or double-stranded DNA comprising one or more element
sequences of transcription factors are immobilized on a substrate,
and has found that expression levels of the transcription factor or
a substance which interacts with the factor can be profiled by the
chip based on a biological function such as DNA binding
capacity.
[0008] The present invention provides the following transcription
chips and assay methods of the transcription factor by the use
thereof.
[0009] [1] A transcription chip wherein at least one polynucleotide
comprising one or more element sequences to which a transcription
factor can be bound is immobilized on a substrate.
[0010] [2] The chip according to [1] wherein at least two
polynucleotides comprising one or more element sequences to which
transcription factors can be bound respectively are bound.
[0011] [3] The chip according to [1] wherein the above
polynucleotide has a partial sequence of a promoter in the upstream
(5') side of each gene.
[0012] [4] The chip according to [1] wherein the above substrate is
made by forming a diamond thin film on a support.
[0013] [5] The chip according to [1] wherein the above
polynucleotide is bound to the diamond thin film through a
optionally appropriate spacer.
[0014] [6] A method for assaying a binding of a transcription
factor comprising a step of interacting a sample capable of
comprising the transcription factor with the transcription chip
according to any of [1] to [5].
[0015] [7] The method according to [6] for evaluating an effect of
a subject substance on a transcription factor, wherein the above
sample is a cell lysate of cells cultured in the presence of the
subject substance.
[0016] [8] The method according to [6] or [7] wherein the binding
of the transcription factor is detected using an antibody against
the transcription factor or a method utilizing mass
spectrometry.
[0017] [9] The method according to [8] wherein the detection using
the antibody is an ELISA and the method utilizing the mass
spectrometry is a peptide mass fingerprint method.
BRIEF DESCRIPTION OF DRAWINGS
[0018] FIG. 1 shows an antibody chip and a transcription chip in
comparison.
[0019] FIG. 2 shows immobilized genes.
[0020] FIG. 3 shows chips to which FITC-labeled NF.kappa.B response
sequence (PRDII-1) has been immobilized.
[0021] FIG. 4 shows a result of a competition experiment of the
NF.kappa.B response sequence.
[0022] FIG. 5 shows a result of examining immobilized
double-stranded DNA.
[0023] FIG. 6 shows a result of examining blocking agents.
[0024] FIG. 7 shows results of a simultaneous reproducibility test
and a reproducibility test between measurements (different lot
competition: DLC).
[0025] FIG. 8 shows comparison of GeneDia.TM. with DLC chip.
[0026] FIG. 9 shows a result of sensitivity test at a significant
level of 5%.
[0027] FIG. 10 shows results of NF.kappa.B detection in HeLa cell
extracts (A), and expression profiling of inflammatory-related
transcription factors in HeLa cells using GeneDia.TM. (B).
[0028] FIG. 11 shows microarrayed double-stranded DNA.
[0029] FIG. 12 shows a result of specific detection of NF.kappa.B
by a fluorescent scanner.
[0030] FIG. 13 shows a peptide map of NF.kappa.B by on-chip
digestion.
[0031] FIG. 14 shows a peptide map of NF.kappa.B obtained by
trypsin digestion in a solution.
[0032] FIG. 15 shows results of database search by PMF.
[0033] FIG. 16 shows results of the on-chip digestion.
[0034] FIG. 17 shows VDR peptide mass fingerprint obtained by
trypsin digestion in a solution.
[0035] FIG. 18 shows results of direct detection of VDR from the
transcription chip of the present invention.
[0036] FIG. 19 shows one example of a DLC plate for MALDI-TOF
MS.
[0037] FIG. 20 shows a method for modifying a substrate having a
diamond thin film.
[0038] FIG. 21 shows peptide mass fingerprint of NF.kappa.B (A) and
secondary spectra 1701.10 m/z (B), 1808.01 m/z (C), and 2413.35 m/z
(D).
[0039] FIG. 22 shows results of identified NF.kappa.B obtained by
MASCOT database search.
[0040] The present invention will be described in more detail
below.
[0041] The transcription chip of the present invention is different
from both the antibody chip and the DNA chip (FIG. 1). The
transcription factor is deeply involved in cellular inflammatory
response, carcinogenesis and transcription controlling factor
activation because it is the protein which is bound to a region
called an element sequence in an upstream region of each gene and
regulates the expression of various genes. Therefore, it is very
important to, at a protein level, profile various transcription
factors which keep biological activity.
[0042] The transcription factor is a factor which is bound to a DNA
and is involved in transcription regulation such as transcription
initiation. For example, NF.kappa.B (e.g., p50, p65), NFATc1, CREB,
ATF-2, c-Jun, c-Rel, c-Fos, AP1, AP-2, RBP-J, Nrf2, KLF5, BTEB2,
NF-AT, MITF, RUNX family, GATA-1, GATA-2, HIF1.alpha., HLF, Brn-3,
EBP, CDP, c-Myb, c-Myc, E2F, EGR, Ets, Etsl/PEA3, FAST-1, BRCA1,
HNF-4, GATA, NF-1, Max, IRF-1, NFATc, NF-E1, NF-E2, 1-Oct, MEF-1,
MEF-2, Myc-Max, p53, Pax-5, Pbxl, TR, AhR, ER, GR, MR, AR, VDR,
RAR, RXR, LXR, FXR, PPAR, ERR, ROR, SXR, PXR, USF-1, Sp1, Stat1,
Stat3, Stat4, Stat5, Stat6, COUP-TF, Ftz-F1, TFIIB, TFIID, TBP,
TFIIE, TFIIF, TFIIH, TAF, Poll, PolII, PolIII, ELL, TFIIS, Elongin,
P-TEFb, DSIF, CBP/p300, p160 (SRC-1, TIF2, AIB1) TRRAP/GCN5, NcoR,
SMRT, HDAC, DRIP/TRAP, Smad, and the like are exemplified.
[0043] As the transcription factor, the mammalian transcription
factor is preferable, and in particular, the human transcription
factor is preferable.
[0044] The element sequences are publicly known, and a
polynucleotide comprising one or two or more of various element
sequences can be appropriately formed on the chip. Specific element
sequences include sequences (GGAATTTCCC and GGGAAATTCC; GGGGATCCC
and GGGATCCCC; TGACTCAT and ATGAGTCA; AGGTCA and TGACCT; AGGTCA and
TGACCT; AGGTCA and TGACCT; AGGTCATGACCT; AGAACA and TGTTCT;
TGACGTCA) underlined in primers in the following Example 1. Other
element sequences are described in for example, Sagroves et al.'s
report (Cancer Cell 1:211-212 (2002) and Ishii et al.'s report
(Science 232:1410-1413 (1986)).
[0045] As the element sequence, the element sequence to which the
mammalian transcription factor can be bound is preferable, and in
particular, the element sequence to which the human transcription
factor can be bound is preferable.
[0046] It is preferable that oligonucleotides corresponding to
multiple transcription factors whose dynamics is required to be
figured out are immobilized on the chip of the present invention
because all of the transcription factors can be profiled on one
chip. Of course, the oligonucleotide corresponding to only one
transcription factor may be immobilized on one array, and the
transcription factors may be profiled using a required number of
the chips.
[0047] At least one element sequence can be contained in one
polynucleotide, one or more of the same element sequences may be
contained, and two or more element sequences may be contained in
one polynucleotide.
[0048] The polynucleotide comprising the element sequence, which is
bound onto the substrate may be artificially made by binding
appropriate oligonucleotides or polynucleotides to each end of the
element sequence known publicly, but it is possible to preferably
use a partial sequence of a promoter in the upstream (5') side of
each gene. A length of the polynucleotide is not particularly
limited, but, for example, is the length composed of about 10 to
500, preferably about 15 to 300, and more preferably about 20 to
100 nucleotides.
[0049] The polynucleotide composed of excessively long base pairs
is difficult to be prepared, produces self-complementary base
pairs, is hybridized at a different site from the target site, and
thus no objective oligonucleotide is likely to be obtained.
[0050] As the polypeptide comprising the element sequence, the
polynucleotides in SEQ ID NOS:1 to 22 are exemplified.
[0051] The substrate is not particularly limited as long as it can
bind the DNA, and it is possible to use those in which carboxyl
group is introduced by binding an aqueous polymer such as
carboxymethyl dextran and PEG modified terminally with the carboxyl
group to the substrate surface, but preferably, the chips having
diamond thin film where numerous carboxyl residues are formed on
the surface, e.g., GeneDia.TM. (registered trade mark), DLC chip
are exemplified. The substrate having the diamond thin film is
preferable, and in particular GeneDia.TM. is preferable because
numerous double-stranded DNA can be bound thereto at high density.
The diamond thin film can be formed on any of supports, e.g., a
silicon substrate.
[0052] An outline of a method for introducing a substituent such as
amino group and carboxyl group onto the diamond thin film is shown
in FIG. 20.
[0053] The binding of the DNA and the substrate can be performed by
a coupling reagent. The coupling reagent includes reagents used for
forming ordinary peptide bonds, and for example, carbodiimides
(DCC, WSC) and carbonyldiimidazole and the like are exemplified.
Alternatively, COOH terminal group on the substrate can be made
into active ester such as N-hydroxysuccinic acid imide and
1-hydroxybenzotriazole using the above carbodiimides, particularly
water-soluble carbodiimide, and bound to the DNA having a binding
group such as amino group at the 5' or 3' terminus. Furthermore,
the distance between the substrate and the DNA may be appropriately
extended by binding the DNA through an appropriate spacer, and a
binding sequence of the transcription factor may be estranged from
the substrate of the transcription chip by placing an appropriate
sequence (e.g., sequence derived from the promoter) between the
binding site of the DNA and the binding site of the transcription
factor. By the use of the appropriate promoter, it becomes possible
to immobilize the binding sequence for the transcription factor
with keeping a certain space from the surface, and the assay of the
transcription factor can be performed with assuring mobility of the
transcription factor.
[0054] In Examples of the present invention, the polynucleotide
having NH.sub.2(CH.sub.2).sub.12 at the 5' terminus was used. Such
a modified oligonucleotide comprising the substituent (e.g.,
NH.sub.2(CH.sub.2).sub.12) having the binding group such as amino
group at the 5' or 3' terminus can be produced in accordance with
publicly known methods, e.g., the methods disclosed below and in JP
HEI-3-74239 B.
http://www.eurogentec.be/upload/Oligocatalogue/oligo.sub.--10.pdf
http://www.glenres.com/ProductFiles/10-1912.html
http://www.fasmac.co.jp/The
[0055] The chip of the present invention is used for detecting a
transcription factor by binding double-stranded DNA on the
substrate, binding the transcription factor to an element sequence
of the double-stranded DNA and detecting the bound transcription
factor using mass spectrometry or an antibody in accordance with an
appropriate detection method.
[0056] The double-stranded DNA may be bound by binding single
strand DNA (binding the polynucleotide through this onto the
substrate) having a reactive group such as amino group at the 5' or
3' terminus onto the substrate and subsequently hybridizing the
polynucleotide complementary thereto, or by directly binding the
double-stranded DNA having at least one chain binding group (e.g.,
NH.sub.2) onto the substrate.
[0057] The transcription chip made in this way is then contacted
with a sample which can comprise a transcription factor to bind the
transcription factor onto the chip. As such a sample, cell lysate
of mammalian cells is preferable, and in particular the cell lysate
of human cells is preferable. For example, effect of various
culture conditions and subject substances on the transcription
factor can be quantitatively evaluated by profiling transcription
factors in cell lysate from various human cell lines cultured under
various culture conditions or in the presence of the subject
substance using the chip of the present invention.
[0058] The transcription factor can be quantified by
immunologically measuring using an antibody against the
transcription factor and a publicly known method such as ELISA or
measuring using mass spectrometer. For example, the transcription
factor may be directly measured by mass spectrometer, but it is
preferable to identify and quantify the bound transcription factor
by digesting the transcription factor bound to the chip with an
appropriate protease such as trypsin, analyzing the resulting
peptide fragments by mass spectrometer and comparing with a mass
spectrum pattern (mass fingerprint) of the protease-digested
transcription factor. As the mass spectrometer, MALDI-TOF is
preferably exemplified.
[0059] In ELISA, various blocking agents such as tris-, skim milk-,
gelatin- and ethanolamine-based blocking agents can be used to
enhance the specificity.
[0060] It is preferable to label the oligonucleotide of the
invention with a fluorescent dye such as fluorescein such as FITC,
rhodamine, cy2, cy3 and cy5 or a luminescent substance such as
acridium ester, luminol and luminescent adamantane.
BEST MODES FOR CARRYING OUT THE INVENTION
[0061] The present invention will be described in more detail with
reference to the following Examples.
Example 1
(1) Experimental Materials and Methods
(i) Experimental Apparatuses
[0062] Fluorescent scanner: CRBIO II (Hitachi Software Engineering
Co., Ltd.), microarrayer: SPBIO (Hitachi Software Engineering Co.,
Ltd.), microplate reader: .mu. Quant (BIO-TEK), plate shaker: Micro
mixer MX-4 (Sanko Junyaku Co., Ltd.), CO.sub.2 incubator: HERAcell
(Kendro), mass spectrometer: ABI4700 Proteomics Analyzer (Applied
Biosystems), and Voyager-DESTR (Applied Biosystems).
(ii) Experimental Materials
[0063] Chips, plates: DLC chips (3 mm, 3 mm) (Toyo Kohan Co.,
Ltd.), GeneDia.TM. C4 (3 mm, 3 mm) (Toyo Kohan Co., Ltd.), 96-well
U-bottom microplate (Greiner), NF.kappa.B (p50) (Promega),
antibody: anti-NF.kappa.B p50 antibody (Active Motif),
anti-NF.kappa.B p50 antibody (Rockland, #100-4164), POD-labeled
anti-rabbit IgG (Active Motif), AP-labeled anti-rabbit IgG (Nacalai
Tesque Inc.), FITC-labeled anti-rabbit IgG (Nacalai Tesque Inc.),
BD Mercury TransFactor kit Inflammation 1 (BD Biosciences).
[0064] Reagents: WSC (Dojin), NHS (Wako), POD color development
substrate TMBZ (Sumitomo Bakelite Co., Ltd., #ML-1120T), activation
buffer 1 (10 mM WSC, 0.1 M MES, pH 4.5), activation buffer 2 (100
mM WSC, 20 mM NHS, 0.1 M NaPB, pH 6.0), blocking buffer (1 M
Tris-HCl (pH 8.0), 150 mM KCl, 0.1% Tween 20), binding buffer (1%
BSA, 10 mM HEPES buffer, pH 7.5, 10 .mu.g/ml salmon sperm DNA, 2 mM
DTT, diluted (.times.100) protease inhibitor cocktail (SIGMA)),
washing buffer (50 mM NaCl, 10 mM NaPB (pH 7.5), 0.1% Tween 20),
Protein Assay Reagent (BioRad), TNF.alpha. (Wako #203-15263),
phorbol 12-myristate 13-acetate (PMS)(SIGMA #P1585), sinapic acid
solution (1 mg/mL sinapic acid, 50% acetonitrile, 0.1%
Trifluoroacetic acid (TFA)), .alpha.-Cyano-4-hydroxycinnamic acid
(.alpha.-CHCA) solution (1 mg/mL .alpha.-CHCA, 50% acetonitrile,
0.1% TFA), low salt concentration buffer (5 mM Tris-HCl (pH 8.0), 1
mM NaCl), trypsin reaction solution (50 mM ammonium bicarbonate,
0.1-4 .mu.g/mL trypsin), GFX purification kit (Amersham,
#27-9602-01), NE-PER Nuclear and Cytoplasmic extraction reagents
(PIERCE #78833) ProteoMass.TM. Peptide MALDI-MS calibration
kit.
[0065] Oligonucleotides: The following primers and oligonucleotides
were synthesized. Underlines indicate recognition sequences
(element sequences) of transcription factors. The oligonucleotide
was mixed with a complementary chain thereof so that a final
concentration of double-stranded DNA was 50 .mu.M, treated with
heat at 95.degree. C. for 5 min, and gradually cooled.
TABLE-US-00001 (PCR primers) PRD F primer
5'-NH.sub.2(CH.sub.2).sub.12-CCTCACAGTTTGTAAATCTTTTTCCC-3' PRD R I
primer 5'-FITC-GGCCTATTTATATGAGATGGTCCTC-3' PRD R II primer
5'-FITC-AGAGGAATTTCCCACTTTCACTTC-3 (Immobilized oligonucleotides)
PRD II:
5'-NH.sub.2(CH.sub.2).sub.12-GGGAGCTGAGTAGGGAAATTCCATGCATGCGGGA
AATTCCCATG -3' 5'-CATGGGAATTTCCCGCATGCATGGAATTTCCCTACTCAGCTCCC-3'
5'-FITC-CATGGGAATTTCCCGCATGCATGGAATTTCCCTACTCAGC TCCC-3'
Ig.kappa.B:
5'-NH.sub.2(CH2).sub.12-GGGAGCTGAGTAGGGGATCCCATGCATGCGGGGA
TCCCCATG-3' 5'-FITC-CATGGGGATCCCCGCATGCATGGGATCCCCTACTCAGCTCC C-3'
API:
5'-NH.sub.2(CH.sub.2).sub.12-GGGAGCTGAGTATGACTCATATGCATGCTGACTCA
TCATG-3' 5'-CATGATGAGTCAGCATGCATATGAGTCATACTCAGCTCCC-3' VDRE :
5'-NH.sub.2(CH.sub.2).sub.12-GGGAGCTGAGTAAGGTCAAGGAGGTCAATGCATGC
AGGTCAAGGAGGTCACATG-3'
5'-CATGTGACCTCCTTGACCTGCATGCATTGACCTCCTTGACCTTACT CAGCTCCC-3' RARE:
5'-NH.sub.2(CH.sub.2).sub.12-GGGAGCTGAGTAAGGTCACCAGGAGGTCAATGCAT
GCAGGTCACCAGGAGGTCACATG-3'
5'-CATGTGACCTCCTGGTGACCTGCATGCATTGACCTCCTGGTGACCT TACTCAGCTCCC-3 '
ERE:
5'-NH.sub.2(CH.sub.2).sub.12-GGGAGCTGAGTAAGGTCACAGTGACCTATGCATGC
AGGTCACAGTGACCTCATG-3'
5'-TCAGGTCACAGTGACCTGATCTCAGGTCACAGTGACCTTTCACGAG GTAC-3' TRE:
5'-NH.sub.2(CH.sub.2).sub.12-GGGAGCTGAGTAAGGTCATGACCTATGCATGCAGG
TCATGACCTCATG-3' 5'-CATGAGGTCATGACCTGCATGCATAGGTCATGACCTTACTCAGCTC
CC-3' GRE:
5'-NH.sub.2(CH.sub.2).sub.12-GGGAGCTGAGTAAGAACACTGTGTTCTATGCATGC
AGAACACTGTGTTCTCATG-3'
5'-CATGAGAACAGACTGTTCTGCATGCATAGAACAGACTGTTCTTACT CAGCTCCC-3' CRE:
5'-NH.sub.2(CH.sub.2).sub.12-GGGAGCTGAGTATGACGTCAATGCATGCTGACGTC
ACATG-3' 5'-CATGTGACGTCAGCATGCATTGACGTCATACTCAGCTCCC-3'
(iii) Experimental Methods i. Preparation of Chips for
Immobilization of Element Sequences (for ELISA)
[0066] Chips (DLC or GeneDia.TM.) were set with coating side up in
a 96-well U-bottom microplate made from polypropylene.
Subsequently, 50 .mu.L of the activation buffer 1 comprising
double-stranded DNA element sequences at various concentration was
added, and the plate was sealed. Then, the plate was shaken on a
plate shaker at room temperature for one hour. The plate was washed
twice with the washing buffer. Then, 200 .mu.L of the blocking
buffer was added, the plate was shaken at room temperature for one
hour, and subsequently stored at 4.degree. C.
ii. On-Chip Enzyme-Linked Immunosorbent Assay (on Chip ELISA)
[0067] The plate was washed twice with the washing buffer,
NF.kappa.B at various concentrations diluted with the binding
buffer was added, and the plate was shaken at room temperature for
one hour. The plate was washed three times with the washing buffer,
anti-NF.kappa.B antibody diluted 2000 times with antibody diluting
buffer was added, and the plate was shaken at room temperature for
one hour. The plate was washed three times with the washing buffer,
POD-labeled anti-rabbit IgG secondary antibody diluted 2000 times
with the antibody diluting buffer was added, and the plate was
shaken at room temperature for one hour. The plate was washed five
times with the washing buffer, and subsequently absorbance at a
wavelength of 450 nm based on the absorbance of TMBZ which was a
color development substrate of POD was measured by a microplate
reader.
iii. Preparation of HeLa Cell Extract
[0068] HeLa cells was cultured up to 70% confluence in 25 mL of
DMEM medium to which 10% Fetal Bovine Serum (FBS) had been added
using 250 cm.sup.2 dish in a CO.sub.2 incubator at 37.degree. C.
After changing the medium to serum free medium, TNF.alpha. was
added at a final concentration of 100 ng/mL, and then the cells
were cultured in the CO.sub.2 incubator at 37.degree. C. for 30
minutes. The cells detached with a cell scraper were centrifuged
(.times.1000 g, 5 minutes) to remove the medium. FBS was added and
mixed, and then the mixture was centrifuged (.times.1000 g, 5
minutes) again to remove a supernatant. The cell lysis buffer was
added to a cell pellet to lyse the cells. A cell lysate was
centrifuged (.times.1000 g, 5 minutes), then the supernatant was
collected, and a protein concentration therein was measured. The
on-chip ELISA was performed using 60 .mu.g of the protein derived
from the cell lysate per chip. Meanwhile, for profiling the
transcription factor, nuclear extracts derived from the HeLa cells
stimulated with TNF.alpha. (100 ng/mL) or PMA (1 .mu.g/mL), or
unstimulated control were used. The nuclear extracts were prepared
using a kit from Pearce. The on-chip ELISA was performed using 20
.mu.g of the protein derived from the nuclear extract per chip.
iv. Preparation of Arrayed Double-Stranded DNA on Chip
[0069] DLC chips were set with coating side up in a 96-well
U-bottom microplate made from polypropylene. Subsequently, 50 .mu.L
of the activation buffer 2 was added, and the plate was sealed and
shaken on the plate shaker at room temperature for 30 minutes. The
plate was washed twice with distilled water, and dried by
centrifugation. The activated DLC chip was set in a microarrayer,
and then 25 .mu.M 30% double-stranded oligonucleotide prepared in
glycerol was spotted on the activated DLC chip using a 150 .mu.m
pin. The chips were incubated at 50.degree. C. for 12 hours,
blocked using the blocking buffer, and then stored at 4.degree.
C.
v. Fluorescence Detection of Transcription Chip Array
[0070] After washing twice with the washing buffer, NF.kappa.B
diluted at 100 ng/mL with the binding buffer was added, and shaken
at room temperature for one hour. After washing three times with
the washing buffer, anti-NF.kappa.B antibody (RCK) diluted 100
times with antibody diluting buffer was added, and shaken at room
temperature for one hour. After washing twice with the washing
buffer, FITC-labeled anti-rabbit IgG antibody diluted 50 times with
the antibody diluting buffer was added, and shaken at room
temperature for one hour. After washing twice with the washing
buffer, at a fluorescence wavelength of 535 nm based on
Fluorescence of FITC excited with laser at a wavelength of 473 nm,
fluorescent intensity was detected by CRBIO II microarray
scanner.
vi. Detection of Transcription Factor Captured onto Transcription
Chip by MALDI-TOF MS
[0071] Chips (DLC or GeneDia.TM.) were set with coating side up in
a 96-well U-bottom microplate made from polypropylene.
Subsequently, 50 .mu.L of the activation buffer 1 comprising
double-stranded DNA element sequences at various concentrations was
added, and the plate was sealed. Then, the plate was shaken on the
plate shaker at room temperature for one hour. The plate was washed
twice with the washing buffer. Then, 200 .mu.L of the blocking
buffer was added, the plate was shaken at room temperature for one
hour, and subsequently stored at 4.degree. C. After washing twice
with the washing buffer, NF.kappa.B diluted with the binding buffer
at various concentrations was added, and shaken at room temperature
for one hour. After washing three times with the washing buffer,
the plate was washed with the low salt concentration buffer.
Subsequently, the chips were dried by centrifugation. The chip was
fixed with a MALDI plate using two sided tape, then, 1 .mu.L of 1
mg/mL sinapic acid solution was added as a matrix to the chip, and
was dried.
vii. On-Chip Digestion and Peptide Mass Fingerprint
[0072] The chips were transferred to a new ELISA plate, 1 .mu.L of
a trypsin reaction solution was added to the chip surface, the
chips were incubated at 37.degree. C. for 2 to 6 hours, and the
reaction was terminated by adding 1 .mu.L of 0.1% TFA.
Subsequently, 1 .mu.L of 1 mg/mL .alpha.-CHCA solution was added as
a matrix to the chip, and was dried. In that, 0.5 .mu.L was spotted
on an MALDI plate. A peptide map was analyzed using MS-Fit
Search.
viii. Identification of Captured Molecule by Tandem Mass
Spectrum
[0073] Using the aforementioned method, NF.kappa.B was captured
onto DLC, and on-chip trypsin digestion was performed. A peptide
mass fingerprint was acquired by ABI4700proteomicsanalyzer using
ProteoMass.TM. Peptide MALDI-MS Calibration kit for mass
calibration. Obtained PMF of NF.kappa.B was used for tandem mass
analysis.
(3) Experiment Results
(A) Investigation of Basic Conditions
[0074] i. Immobilization of Double-Stranded DNA onto GeneDia.TM.
and DLC Chips
[0075] A fragment of the region including an element region present
in an upstream sequence of interferon f31 gene among the element
sequences which NF.kappa.B recognized was acquired by PCR method
using PRD F primer and PRD R1 primer (PRD II-1, FIG. 2). A PCR
product was purified using GFX purification kit, and immobilized.
Consequently, it was confirmed that double-stranded DNA was bound
to both DLC and GeneDia.TM. chips (FIG. 3).
ii. Investigation of Optimal Condition for on-Chip ELISA (DLC) a.
Competitive Inhibition Experiment
[0076] In order to investigate whether it is possible to capture
the transcription factor by the double-stranded DNA immobilized on
the chip and specifically detect it by ELISA, it was investigated
whether PRD II-1 competes with the immobilized PRD II-1 for the
binding NF.kappa.B by adding the PRD II-1 into the binding buffer.
As a control, the experiment was performed by adding the
double-stranded oligonucleotide which did not contain the
immobilized PRD II-1 sequence into the binding buffer. As a result,
the capture of NF.kappa.B onto the chip was strongly inhibited in a
concentration dependent manner when the PRD II-1 had been added
into the binding buffer whereas almost no inhibitory effect was
observed when the control sequence was used (FIG. 4). From the
above, it has been suggested that NF.kappa.B specifically
recognizes and binds the PRD II-1 sequence on the DLC chip.
b. Investigation of Immobilized DNA Sequence
[0077] In order to investigate an ELISA condition for NF.kappa.B,
the double-stranded DNA comprising the element DNA sequence
different in length and copy number were synthesized by the PCR
method or a synthetic method, and analyzed. When PRD II-1
comprising one copy of the PRD II sequence was compared with PRD
II-2, the immobilized PRD II-2 was more excellent in efficiency of
trapping NF.kappa.B (FIG. 5). Even when PRD II-2 was compared with
the PCR product (PRD II.times.6) comprising 6 copies of the same
element sequence, the immobilized PRD II-2 was more excellent in
immobilization efficiency. Next, without using the PCR method, it
was investigated whether NF.kappa.B could be efficiently captured
using the oligonucleotide artificially synthesized. When using the
double-stranded oligonucleotide comprising two copies of
NF.kappa.B-binding element sequence Ig.kappa.B derived from
immunoglobulin and the oligonucleotide PRD II comprising two copies
of the PRD II sequence, the trapping efficiency of NF.kappa.B
equivalent to or more than that of PRD II-2 was observed (FIG.
5).
[0078] From the above results, it has been suggested that it is
possible to efficiently trap NF.kappa.B by the recognition sequence
close to the NF.kappa.B recognition sequence at a peripheral end
side (liquid phase side opposite to an immobilized site) of the PCR
product or the double-stranded oligonucleotide, an
NF.kappa.B-antibody complex is unlikely to access to the internal
recognition sequence due to steric hindrance, and the copy number
is not important for trapping NF.kappa.B in the ELISA detection
system. However, it has been revealed that it is necessary to
investigate the blocking condition to enhance the specificity
because in the negative control where an AP1 sequence was
immobilized or no DNA was immobilized, about 30 to 50% color
development was observed.
c. Investigation of Blocking Condition
[0079] In order to enhance the specificity of the ELISA, various
blocking agents were examined. As blocking buffers, tris-based (1 M
Tris-HCl (pH 8.0), 150 mM KCl, 0.1% Tween 20), skim milk-based (5%
skim milk, 150 mM KCl, 0.1% Tween 20), BSA-based (5% BSA, 150 mM
KCl, 0.1% Tween 20), gelatin-based (2.5% gelatin, 150 mM KCl, 0.1%
Tween 20), and ethanolamine-based (150 mM ethanolamine, 150 mM KCl,
0.1% Tween 20) buffers were used. As a result, no large difference
in specificity was observed among them (FIG. 6). Thereafter,
tris-based buffer was used as the non-protein blocking agent.
Reproducibility among measurements could be enhanced by adding a
step at 4.degree. C., O/N subsequent to a blocking reaction time
period at room temperature for one hour.
iii. Investigation of Reproducibility
[0080] Using the condition optimized in the previous section,
simultaneous reproducibility and the reproducibility among the
measurements of the DLC chips were examined. As a result, favorable
results were obtained in the simultaneous reproducibility and the
reproducibility among the measurements (FIG. 7).
iv. Comparison of GeneDia.TM. (Diamond Chip) and DLC Chip a.
Comparison of Quantitativity by Concentration Change
[0081] In order to compare the performances of DLC chip and
GeneDia.TM., the comparison was performed under the same condition
using NF.kappa.B at various concentrations. As a result, they
exhibited the nearly same quantitativity, but at a lower
concentration, GeneDia.TM. was more excellent in NF.kappa.B
detection (FIG. 8).
b. Comparison of Detection Limit
[0082] In order to analyze the profiles at the low concentration
range (10 ng/mL or less) in detail, the detection limit in each
chip was attempted to be calculated. As a result, for GeneDia.TM.,
the PRD II-immobilized chip exhibited significant difference from
the AP1 sequence-immobilized chip and the no DNA-immobilized chip
at a significant level of 5% at both concentrations of 1.0 ng/mL
and 0.33 ng/mL, whereas for DLC, the PRD II-immobilized chip
exhibited the significant difference from both the AP1
sequence-immobilized chip and the no DNA-immobilized chip only at a
concentration of 1.0 ng/mL (20 pM) (FIG. 2.1. 3-9). When using AP1
as a control, it was revealed that the detection limit was 0.33
ng/mL (6.7 pM) or less because the significant difference was
observed only for the AP1-immobilized chip at a concentration of
0.33 ng/mL. Therefore, it has been suggested that it is possible to
detect NF.kappa.B in the sample at a low concentration such as pM
order. From these, it seems that it is also possible to construct
the similarly highly sensitive on-chip ELISA system for the other
transcription factor.
(B) Practical Application of Transcription Chips
[0083] i. Detection of Transcription Factor in Culture Liquid
[0084] The on-chip ELISA was performed to examine whether the
transcription factor NF.kappa.B p50 can be captured from an extract
of cultured HeLa cells. As a result, when using the extract of the
cells stimulated with TNF.alpha. (100 ng/mL), stronger color
development on the PRD II-immobilized chip was observed compared to
the case of using the extract of unstimulated control cells (FIG.
10A).
[0085] For the other transcription factors, it was examined whether
the expression levels of various transcription factors were changed
by the stimulation with TNF.alpha. or PMA (10 nM). In this method,
an AP-labeled secondary antibody was used. As a result, many
transcription factors (NF.kappa.B p65, c-Rel, c-Fos, c-Jun, ATF2,
CREB) in addition to NF.kappa.B p50 could be specifically captured
(FIG. 10B).
ii. Microarrayed Transcription Chip
[0086] Until now, it has been attempted that the double-stranded
DNA chains are immobilized on a solid phase to capture a
DNA-binding protein, which is then detected using the ELISA method.
However, problems as the technology for proteome analysis have not
been solved because throughput property and the number of samples
simultaneously detected in multiple parameters are limited. In this
section, the development of transcription microarray chips using
the DLC chips as the technology in place of these methods will be
reported.
a. Investigation of Making Arrayed Double-Stranded DNA
[0087] The DLC chip on which the FITC-labeled PRD II
oligonucleotide had been immobilized at 9 spots was detected using
CRBIO II fluorescence scanner (FIG. 11). It was revealed that the
double-stranded DNA could be also immobilized similarly by spotting
using an arrayer.
b. Specific Detection of NF.kappa.B from Transcription Microarray
Chip
[0088] The oligonucleotides comprising 9 element sequences, PRD II,
AP1, p53, VDRE, RARE, CRE, TRE, GRE and ERE which were not labeled
with FITC were similarly immobilized on the DLC chip. NF.kappa.B
(100 ng/mL) was added to the DLC chip on which these 9
transcription factors had been arrayed, and detected by
fluorescence immunoassay. As a result, the fluorescence was
detected only from the oligonucleotide comprising PRD II which was
the NF.kappa.B recognition sequence. From the above, it has been
revealed that NF.kappa.B can be specifically detected on the DLC
chip on which the element sequence has been arrayed (FIG. 12).
Therefore, it is possible to construct the system in which multiple
transcription factors are simultaneously detected in a sample in a
small amount by combining antibodies which specifically recognize
the transcription factors which is bound to the other element
sequences.
iii. Detection of Transcription Factor by MALDI-TOF MS
[0089] The method of detecting the transcription factors by the
on-chip ELISA is excellent in throughput as the system in which
multiple transcription factors are simultaneously analyzed using
specific antibodies, compared with the ELISA and the gel shift
assay. However, in this system, the known transcription factor can
be detected, but it is impossible to detect an unknown protein. In
the proteome analyses at present, it is a very important
proposition to detect the unknown protein. Thus, it seems to be
highly useful to develop the technology of detecting the protein
using the transcription chip. In this section, the development of
detection method of the transcription factor using MALDI-TOF MS
mass spectrometric system will be reported.
a. Peptide Mass Fingerprint (PMF) of NF.kappa.B by On-Chip
Digestion
[0090] It is generally difficult to digest with trypsin on the
chip. Causes thereof include inactivation of the enzyme on the
hydrophobic surface, masking the substrate site due to steric
hindrance, and the like. However, the double-stranded DNA are
dissociated at a low salt concentration on the transcription chip,
and thus DNA-protein interaction is lost at a low salt
concentration to easily liberate the protein in the enzyme reaction
solution. Also, the chip surface appears to exhibit hydrophilicity
because the DNA are covalently bound thereto. Therefore, the
transcription chip appeared to be the ideal chip for the on-chip
digestion. After 2 pmol/chip of NF.kappa.B was added and reacted,
the on-chip trypsin digestion was performed. Subsequently, the
peptides present on the chip were purified by Zip Tip purification
chip, and detected by an MALDI plate or directly detected from the
transcription chip.
[0091] As a result, an ideal peptide map was observed when the
trypsin digestion was performed on the chip, the peptides were
purified by Zip Tip, and the sample was added to the MALDI plate
(FIG. 13). This well conformed to the peptide map observed by
digesting NF.kappa.B in a solution with trypsin (4 .mu.g/mL) (FIG.
14). Database search was performed by MS-FIT Search based on this
peptide mass fingerprint information, and consequently, NF.kappa.B
was hit at a very high score. Therefore, it was confirmed that many
peptides derived from NF.kappa.B were captured (FIG. 15).
b. Detection and Identification of Vitamin D Receptor (VDR) by PMF
Method
[0092] Next, the on-chip digestion of 500 fmol/chip of vitamin D
receptor was performed using trypsin (1 .mu.g/mL) on the chip on
which the element sequence VDRE which was the recognition sequence
of the vitamin D receptor had been immobilized. Subsequently, the
sample was added to the MALDI plate, and consequently, the same
peptide map as that obtained by trypsin digestion of the sample in
the solution was obtained as is the case with NF.kappa.B (FIGS. 16
and 17).
[0093] Meanwhile, it was examined whether the peptide map could be
directly observed or not by directly irradiating the laser on the
transcription chip. As a result, the peptide map of VDR could be
directly observed on the chip (FIG. 18).
c. Identification of Transcription Factor by Tandem Mass Spectrum
Analysis
[0094] The peptide mass fingerprint (PMF) method is used for the
identification of the protein contained in the sample by mass
spectrometry, but it is difficult to identify the objective protein
contained in the sample when the number of spectral peaks obtained
is low. Thus, when the objective protein is identified in the
sample containing much impurities, the identification by tandem
mass spectrum analysis is often performed. However, the tandem mass
spectrum analysis has been generally inferior in sensitivity
compared to the peptide mass fingerprint method.
[0095] For the peaks of 1701.10 m/z, 1808.01 m/z and 2413.35 m/z
from the peptide mass fingerprint (FIG. 21A) obtained by
calibrating the mass from the transcription chip to which
NF.kappa.B had been immobilized, the tandem mass analysis was
performed (FIGS. 21B, C and D). A secondary spectrum could be
obtained for each peak, and thus, these analysis results were
applied to MASCOT database search. As a result, all of the peptides
well conformed to mass data of NF.kappa.B in the database (FIG.
22). The above results suggest that the transcription chip can
efficiently capture NF.kappa.B through the element DNA and
consequently the tandem mass analysis can be applied.
(4) Applicability of the Present Invention
[0096] (A) Quantitative Detection of Transcription Factor with High
Sensitivity Using Transcription Chip
[0097] It is the first large subject of this study that an
experimental system with high sensitivity and specificity is
constructed to establish a high sensitive quantitative detection
system by allowing the chip (transcription chip) where the element
sequence has been immobilized on the DLC chip or GeneDia.TM. to
capture the protein (transcription factor). For solving this
subject, it has been important to make the DNA-immobilized chip
having a high capturing capacity of the transcription factor and
establish an assay system in which non-specific binding of the
transcription factor and the antibody can be minimized. The above
subject was responded by examining binding reaction conditions such
as blocking condition and examining the immobilized DNA
sequences.
[0098] For the recognition sequence (element sequence) of the
transcription factor NF.kappa.B, the results in which the location
in the immobilized double-stranded DNA seemed to be more important
than the copy number were obtained (FIG. 5). The capacity of
capturing NF.kappa.B was higher in the case of locating the element
sequence closer to a liquid layer in the immobilized
double-stranded DNA. It was suggested that the inner recognition
sequence located far from the surface side of the oligonucleotide
hardly formed the transcription factor-antibody complex due to
steric hindrance. Therefore as the general method for realizing the
highly sensitive detection, it was suggested that it was important
for the capacity of capturing the transcription factor to design
the element sequence close to the surface side (opposite side to
the immobilized site) of the double-stranded oligonucleotide. It
was revealed that both the oligonucleotide and the PCR product
could be immobilized as the immobilized DNA. Thus, a transcription
initiation complex in a certain gene region will become possible in
the future by acquiring the certain gene region by PCR and
immobilizing in addition to ordinary assays using the
oligonucleotides.
[0099] The investigation of the blocking condition was important
for decreasing non-specific bindings in the assay. The BSA
concentration at 1% in the binding buffer was the optimal
condition. When the experimental system was constructed, increased
assay values in the negative controls probably due to the effect of
non-specific bindings were often observed, but the experimental
system with high reproducibility could be constructed by using the
tris buffer and prolonging the blocking time period (4.degree. C.,
0/N) (FIG. 7).
[0100] As a result of examining the above, eventually, the
detection limit down to 0.33 ng/mL and 1.00 ng/mL were obtained for
the case using GeneDia.TM. and the case using the DLC chip,
respectively. Using HeLa cells, it became possible to perform
profiling of inflammatory transcription factors activated by the
stimulation with TNF.alpha. and PMA at protein levels.
(B) Detection of Transcription Factor Using Highly Integrated
(Microarrayed) Double-Stranded DNA Chip
[0101] As the intended use for small scale studies, the
conventional technology has accomplished profiling and
quantification of the transcription factors. However, considering
the throughput property and consumed quantity as a supporting
system for diagnosis and drug discovery in the light of proteome
analyses in the future, the conventional technology has not
actually come into practical use.
[0102] As shown in the present results of the on-chip ELISA, a
quantitative measurement on a size of 3 mm.times.3 mm was enabled.
Thus, it is possible to perform the quantitative and highly
sensitive measurement on the size of approximately two glass slides
equivalent to that in one 96-well microplate.
[0103] Furthermore, in the on-chip fluorescence immunoassay, it
could be monitored that NF.kappa.B was specifically bound to the
PRD II sequence on the chip onto which nine spots of the
double-stranded DNA had been immobilized at a size of 3 mm.times.3
mm by making the transcription chip having the microarrayed
double-stranded DNA (FIG. 12).
(C) Detection and Identification of Transcription Factor Using
MALDI-TOF MS
[0104] It becomes possible to detect and identify the unknown
protein or analyze post-translational modification, which can not
be detected by ELISA, by detecting the protein using the mass
spectrometer.
[0105] At present, a protein chip system using SELDI-TOF MS from
Ciphergen is commercially available, and the protein chip to which
the antibody has been immobilized using this system has been
developed, but it is not sufficient in terms of throughput property
and accuracy of mass analysis.
[0106] Meanwhile, with respect to the transcription chip to which
the DNA has been immobilized, generally, the molecular weights of
the transcription factors are often about 50,000, and it has been
difficult to identify from the mass information by the current
accuracy of the mass spectrometer. Thus, it was speculated that if
a system using a diamond (DLC) chip capable of immobilizing the DNA
at high density and MALDI-TOF MS using the peptide mass fingerprint
method by trypsin digestion was used, the mass information of the
transcription factor molecule could be directly acquired from the
chip, and the detection system was made. As a result, the
transcription factor could be detected and identified directly on
the chip for the first time (FIG. 18). In addition, by combining
with the tandem mass spectrum analysis, it became possible to
detect and identify the transcription factor captured on the chip
(FIG. 21).
[0107] According to the present invention, the profiling of many
transcription factors is realized by the on-chip ELISA using the
highly sensitive transcription chip, it is possible to sensitively
detect and quantify the transcription factor in the culture cell
extract and the tissue, and it is possible to use the above as
tools for screening, predicting the toxicity at the drug discovery
or diagnostic information.
[0108] Also, it is possible to directly identify the transcription
factor on the chip using the peptide mass fingerprint method.
Furthermore, the tandem mass spectrum analysis was possible, and
thus, it is possible to identify the transcription factor in the
sample comprising the impurities.
[0109] It is possible to detect and identify the transcription
factor with keeping sensitivity and accuracy equivalent to or more
than those of the conventional MALDI plate by the use of the
transcription chip (FIG. 19) corresponding to a focus position of
ionized laser.
[0110] According to the present invention, it is possible to
efficiently profile the transcription factor.
Sequence CWU 1
1
25126DNAArtificial SequenceTranscription Factor 1cctcacagtt
tgtaaatctt tttccc 26225DNAArtificial SequenceTranscription Factor
2ggcctattta tatgagatgg tcctc 25324DNAArtificial
SequenceTranscription Factor 3agaggaattt cccactttca cttc
24444DNAArtificial SequenceTranscription Factor 4gggagctgag
tagggaaatt ccatgcatgc gggaaattcc catg 44544DNAArtificial
SequenceTranscription Factor 5catgggaatt tcccgcatgc atggaatttc
cctactcagc tccc 44644DNAArtificial SequenceTranscription Factor
6catgggaatt tcccgcatgc atggaatttc cctactcagc tccc
44742DNAArtificial SequenceTranscription Factor 7gggagctgag
taggggatcc catgcatgcg gggatcccca tg 42842DNAArtificial
SequenceTranscription Factor 8catggggatc cccgcatgca tgggatcccc
tactcagctc cc 42940DNAArtificial SequenceTranscription Factor
9gggagctgag tatgactcat atgcatgctg actcatcatg 401040DNAArtificial
SequenceTranscription Factor 10catgatgagt cagcatgcat atgagtcata
ctcagctccc 401154DNAArtificial SequenceTranscription Factor
11gggagctgag taaggtcaag gaggtcaatg catgcaggtc aaggaggtca catg
541254DNAArtificial SequenceTranscription Factor 12catgtgacct
ccttgacctg catgcattga cctccttgac cttactcagc tccc
541358DNAArtificial SequenceTranscription Factor 13gggagctgag
taaggtcacc aggaggtcaa tgcatgcagg tcaccaggag gtcacatg
581458DNAArtificial SequenceTranscription Factor 14catgtgacct
cctggtgacc tgcatgcatt gacctcctgg tgaccttact cagctccc
581554DNAArtificial SequenceTranscription Factor 15gggagctgag
taaggtcaca gtgacctatg catgcaggtc acagtgacct catg
541650DNAArtificial SequenceTranscription Factor 16tcaggtcaca
gtgacctgat ctcaggtcac agtgaccttt cacgaggtac 501748DNAArtificial
SequenceTranscription Factor 17gggagctgag taaggtcatg acctatgcat
gcaggtcatg acctcatg 481848DNAArtificial SequenceTranscription
Factor 18catgaggtca tgacctgcat gcataggtca tgaccttact cagctccc
481954DNAArtificial SequenceTranscription Factor 19gggagctgag
taagaacact gtgttctatg catgcagaac actgtgttct catg
542054DNAArtificial SequenceTranscription Factor 20catgagaaca
gactgttctg catgcataga acagactgtt cttactcagc tccc
542140DNAArtificial SequenceTranscription Factor 21gggagctgag
tatgacgtca atgcatgctg acgtcacatg 402240DNAArtificial
SequenceTranscription Factor 22catgtgacgt cagcatgcat tgacgtcata
ctcagctccc 402310DNAArtificial SequenceTranscription Factor Element
Sequence 23ggaatttccc 102410DNAArtificial SequenceTranscription
Factor Element Sequence 24gggaaattcc 102512DNAArtificial
SequenceTranscription Factor Element Sequence 25aggtcatgac ct
12
* * * * *
References