US 2002O177218A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/0177218 A1 Fang et al. (43) Pub. Date: Nov. 28, 2002 (54) METHODS OF DETECTING MULTIPLE DNA Publication Classification BINDING PROTEIN AND DNA INTERACTIONS IN A SAMPLE, AND (51) Int. Cl." ....................................................... C12N 1/20 DEVICES, SYSTEMS AND KITS FOR (52) U.S. Cl. .......................................................... 435/252.3 PRACTICING THE SAME (57) ABSTRACT (76) Inventors: Yu Fang, Fremont, CA (US); Methods for detecting the presence of at least one, usually Xiao-Yang Wang, Mountain View, CA a plurality of, DNA binding proteins, e.g., transcription (US); Pierre Turpin, San Francisco, factors, in a Sample, both qualitatively and quantitatively, are CA (US) provided. In the Subject methods, a Substrate having one or Correspondence Address: more DNA probes immobilized on a surface thereof, one for each DNA binding protein of interest, is contacted with a BOZICEVIC, FIELD & FRANCIS LLP Sample under conditions Sufficient for binding complexes of 200 MIDDLEFIELD RID the probes and their respective DNA binding proteins to be SUTE 200 produced. The Sample may be purified with respect to one or MENLO PARK, CA 94025 (US) more DNA binding proteins or be a cellular/nuclear extract. (21) Appl. No.: 10/113,877 Resultant binding complexes on the Surface of the Substrate are then detected and related to the presence of the DNA (22) Filed: Mar. 29, 2002 binding protein-DAN interations of interest in the Sample. Also provided are devices and Systems for use in practicing Related U.S. Application Data the subject methods. The subject methods find use in a variety of different applications, e.g., detecting the presence (60) Provisional application No. 60/280,658, filed on Mar. of a transcription factor in a Sample, the Study of transcrip 30, 2001. Provisional application No. 60/314,330, tion factor profiles in response to a given Stimulus, Screening filed on Aug. 20, 2001. for therapeutic agents, and the like. Patent Application Publication Nov. 28, 2002. Sheet 1 of 13 US 2002/0177218 A1 Patent Application Publication Nov. 28, 2002. Sheet 2 of 13 US 2002/0177218 A1 Figure 2 33... : Add callysates: : 33 33r & 3. Extract 3. -- iii-inding Targon sing & antibody and ash E:: resilts 33: 3. i-C Patent Application Publication Nov. 28, 2002 Sheet 3 of 13 US 2002/0177218A1 Figure 3a - Eli » (« EMSA O. 10 COO NFkB p50 concentration, uM Patent Application Publication Nov. 28, 2002 Sheet 4 of 13 US 2002/0177218A1 Figure 3b 1 2 3 4 5 6 7 8 9 O 11 12 13 -& & 8.33. 48 88: Patent Application Publication Nov. 28, 2002 Sheet 5 of 13 US 2002/0177218 A1 Figure 4a i. .8 O hutant oligo . i. s f:3. i.2 3.1 a 5 sag 10 g 20 g 30 ug 25 g 5 g. 100 ng 200ng - t Dose of Helge NPt. NE Dose of competition oligo 30 g of HeLa NFa. NE Patent Application Publication Nov. 28, 2002. Sheet 6 of 13 US 2002/0177218 A1 Figure 4b Wild-type oligo f O Mutant 1.4 5 gig is 20 g 3 g 25 ng 5 ng 1 ng 200ng l- - Dose of Jurka NE Dose of competition oligo 30 g of Jurkat NE Patent Application Publication Nov. 28, 2002 Sheet 7 of 13 US 2002/0177218A1 Figure 4c a Wild-type oligo Mutant oligo ... 3 i 1.8- . D O.0 a. l org 5 pig 10 pg 20g 30 g 25 mg 50 fig 100 g 200ng l- l Dose of HeLaf PMANE Dose of competition oligo 30g of HeLat PMANE Patent Application Publication Nov. 28, 2002 Sheet 8 of 13 US 2002/0177218 A1 Figure 5 NFkB p50 dsDNA-coated ATF-2 dsDNA-coated c-Fos dsDNA-coated Patent Application Publication Nov. 28, 2002 Sheet 9 of 13 US 2002/0177218 A1 Figure 6 0.84 6 . NFke p50 NFkB pe;5 e-Rei c-ress CR-3- AF-2 Patent Application Publication Nov. 28, 2002. Sheet 10 of 13 US 2002/0177218 A1 Figure 7 2.2. O5 O to a lar its to - i. Transcription Factor Patent Application Publication Nov. 28, 2002. Sheet 11 of 13 US 2002/0177218 A1 Figure 8 1.25 Wies O, 7 5 O. 5 O 0.25 OOO p53 c-Myb c-Myc Max USF1 USF2, Transcription Factor Raji U-937 Nucl. Extr. Patent Application Publication Nov. 28, 2002 Sheet 12 of 13 US 2002/0177218 A1 Figure 9: 48 DBP Transfactor Glass Array (format3.0) 48 DBP TransFactor Glass Array (Format3.0) A - B Each chamber: P50 SMAD4 ISGF-3g P50 SMAD4 ISGF-3g c-Fos Pit SA3 cos Pi Si A3 Oct-1 ERHSF-1 Oct-1 ERHSF-1 MYO) Pax-5 P. A MYO Pax-5 P.I. -2x6 wt & mut oligos -6 Ab. cocktail -fluorescent 2nd Ab. 55 c-Myth MA) P65 c-Myb MA) REB-E2F. SA: CREB-1 E2F-1 SA GATA TRb1 CTCF A GATA TRb1 croF JunB RXRb ETS-1 A JunB RXRb ETS-1 c-Re MAX STAT6 e-Rei AXSAT& ATF2 prbSTAT5 ATF2 prb STAT5 160 ARPPAR P160 ARPPAR SRF-1 GR RF-5 SRF-1 GRIRF-5 -24 DBP-DNA 1 side C-1 SAT C-ta PSA Sp1 PS3 NF-ATc1 Sp1 P53 NF-ATc1 -2 conditions/slide AP-2 YY1PR AP-2 YY1PR C-Myc EGR C/EBPa AC-Myc EGRC/EBPa -less extracts -less Ab. A369RAORES PARYINGEN BOT Patent Application Publication Nov. 28, 2002. Sheet 13 of 13 US 2002/0177218 A1 Figure 10 3000 Comparison of Single Antibody versus Mixing Antibodies 2so g2 9 200 CD 2 c 1500 C 9) O I 1000 500 Single Mix Single Mix Single Mix Single Mix Single Mix Single Mix Antibody NF-kBp50 EGR c-Rei Max SRF-1 ATF2 Transcription Factor Hela-TNFO K562+PMA Raji Raji U937 Jurkat Nuclear Extract A B C D E F US 2002/0177218 A1 Nov. 28, 2002 METHODS OF DETECTING MULTIPLE DNA with a biotinylated dis-DNA probe and an antibody for the BINDING PROTEIN AND DNA INTERACTIONS IN transcription factor. The resultant mixture is then transferred A SAMPLE, AND DEVICES, SYSTEMS AND KITS to anti-lgG coated microWells, and the presence of DNA/ FOR PRACTICING THE SAME transcription factor/antibody complexes are detected chro mogenically with AP conjugated Streptavidin. There are CROSS-REFERENCE TO RELATED disdvantages with this method, including the fact that reac APPLICATIONS tions are performed in more than one container and the lack 0001 Pursuant to 35 U.S.C. S 119 (e), this application of Specificity with respect to the detection of active V. claims priority to the filing date of U.S. Provisional Patent inactive transcription factor, leading to decreased Sensitivity. Application Serial No. 60/280,658 filed Mar. 30, 2001 and 0008. Yet another transcription factor assay is reported in U.S. Provisional Patent Application Serial No. 60/314,330 Renard et al., Nucleic Acids Res. (Feb. 15, 2001) 29:E21, filed Aug. 20, 2001; the disclosures of which are herein which article describes a calorimetric assay that employs a incorporated by reference. substrate bound oligonucleotide which includes an NFKB consensus binding Sequence, where both purified Samples INTRODUCTION and cell extracts are assayed. In the assays reported in this document, only a Single transcription factor, NFKB, is FIELD OF THE INVENTION assayed. 0002 The field of this invention is DNAbinding proteins, 0009. There is continued interest in the development of particularly transcription factors. new ELISA based assays for transcription factors and analo gous DNA binding proteins. Of particular interest would be BACKGROUND OF THE INVENTION the development of Such an assay that could be employed to detect multiple transcription factors in a single Sample, 0003) The study of the interactions between a DNA including a cellular or nuclear extract, where the assay is binding protein and DNA is one of the most rapidly growing more sensitive than EMSA. areas of molecular biology. Transcription factors, a Subset of DNA binding proteins, are at the heart of the regulation and 0010 Relevant Literature control of gene expression, replication, and recombination. 0011 Benotmane, et al. (1997) Analytical Biochemistry Because of their important roles, inhibition and Stimulation 250:181-185; Gubler et al., Biotechniques (1995) 18:1011 of transcription factor binding to DNA is of great interest in 1014; Hibma et al., Nucleic Acids Res. (1994) 22: 3806 the discovery of potential targets for new drugs. 3807; McKay et al., Anal. Biochem (1998) 1:28:34; Mollo, 0004 Several different protocols have been developed to Methods Mol. Biol. (2000) 130:235-246; Renard et al., Nuc. study DNA-protein interactions, such as DNA-protein pho Acids Res. (Feb. 15, 2001) 29:E21; and Revzin, Biotech tocrosslinking, South-western blotting, in Vivo reporting niques (1989) 7:346-355. See also: (a) U.S. Pat. Nos. system and the electrophoretic mobility shift assay (EMSA). 4.963,658; 4.978,608; 5,011,770; (b) WO95/30026; WO EMSA is one of the most powerful tools to study the 98/08096; WO 99/19510; WO 01/73115; and (c) EPO 620 functional relationship between a DNA binding protein and 439 and EP 1 136567. its cognate DNA site. However, the EMSA has some intrin sic disadvantages Such as radioactive usage, limitation of SUMMARY OF THE INVENTION Sample numbers and long assay time. These disadvantages 0012 Methods for detecting, both qualitatively and quan and the need for a high throughput format have led to the titatively, the presence of at least one, usually a plurality of, development of an enzyme-linked DNA-protein binding DNA binding proteins, e.g., transcription factors, in a assay to complement the traditional EMSA.