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Ep 2811298 A1 (19) TZZ __ _T (11) EP 2 811 298 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 10.12.2014 Bulletin 2014/50 G01N 33/53 (2006.01) G01N 33/542 (2006.01) (21) Application number: 13002949.9 (22) Date of filing: 07.06.2013 (84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • Hall, Jonathan GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 4143 Dornach (CH) PL PT RO RS SE SI SK SM TR • Pradere, Ugo Designated Extension States: 8046 Zürich (CH) BA ME •Roos,Martina 8050 Zürich (CH) (71) Applicant: ETH Zurich 8092 Zurich (CH) (54) FRET-Method for identifying a biomolecule-modulating compound (57) The present invention relates to a method for the excitation energy spectrum of the fluorophore accep- identifying a compound modulating an interaction be- tor or the energy spectrum absorbed by the dark quench- tween two biomolecules or two domains of one biomol- er overlap at least partially. Preferably, the first and sec- ecule, the first biomolecule or first domain comprising at ond biomolecules are selected from the group consisting least one fluorophore donor and the second biomolecule of polypeptides, sugars, polynucleotides, polyamines or second domain comprising at least one fluorophore and lipids, and more preferably the first biomolecule is acceptor or a dark quencher, wherein the fluorophore selected from the group consisting of polypeptides inter- donor and fluorophore acceptor or the fluorophore donor acting with polynucleotides and the second biomolecule and dark quencher are spectrally paired such that the is selected from the group consisting of polynucleotides, energy spectrum emitted by.the fluorophore donor and preferably microRNAs (miRNA). EP 2 811 298 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 811 298 A1 Description [0001] The present invention relates to a method for identifying a compound modulating an interaction between two biomolecules or two domains of one biomolecule, the first biomolecule or first domain comprising at least one fluorophore 5 donor and the second biomolecule or second domain comprising at least one fluorophore acceptor or a dark quencher, wherein the fluorophore donor and fluorophore acceptor or the fluorophore donor and dark quencher are spectrally paired such that the energy spectrum emitted by the fluorophore donor and the excitation energy spectrum of the fluorophore acceptor or the energy spectrum absorbed by the dark quencher overlap at least partially. Preferably, the first and second biomolecules are selected from the group consisting of polypeptides, sugars, polynucleotides, 10 polyamines and lipids, and more preferably the first biomolecule is selected from the group consisting of polypeptides interacting with polynucleotides and the second biomolecule is selected from the group consisting of polynucleotides, preferably4 microRNAs (miRNA). Background of the invention 15 [0002] The field of the invention relates to the use of Förster resonance energy transfer (FRET), which is a mechanism describing energy transfer between two chromophores. When both chromophores are fluorescent, i.e. so-called fluor- ophores, the term "fluorescence resonance energy transfer" is often used instead. In order to avoid an erroneous interpretation of the phenomenon that is always a non-radiative transfer of energy (even when occurring between two 20 fluorophores), the name "Förster resonance energy transfer" is preferred. A fluorophore donor, initially in its electronically excited state, may transfer energy to a fluorophore acceptor through non-radiative dipole-dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor making FRET extremely sensitive to small distances. [0003] Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance 25 of each other, typically in the proximity of 1 to 10 nm. Such measurements are used as research tools in biology and chemistry. FRET is typically determined by measuring the variation in acceptor emission intensity. When the donor and acceptor are in proximity the acceptor emission will increase because of the FRET from the donor to the acceptor (sensitized emission). FRET efficiencies can also be inferred from the photobleaching rates of the donor in the-presence and absence of an acceptor. 30 [0004] Alternatively, FRET can be measured between a fluorophore donor and a dark quencher. A dark quencher is a family of substances that absorbs emission energy from a fluorophore donor and dissipates the energy as non-UV- visible light or heat, whereas a typical "fluorescent quencher" i.e. fluorophore acceptor re-emits much of the "donated" energy as light. Black hole quencher (BHQ™) dyes from Biosearch Technologies, Inc., Novato, California. USA) are examples of members of the dark quencher family. Dark quenchers such as BHQ dyes are used in molecular biology 35 in conjunction with fluorophores. When the two are close together, e.g. 10-100 A, such as in a molecule, e.g. a protein, the donor’s emission is at least partially suppressed by the quencher. This effect can be used to study molecular geometry and motion. [0005] MicroRNAs (miRNA) are a large class of small non-coding RNAs which modulate protein translation as negative gene regulators by post-transcriptionally repressing gene expression. They are involved in cell differentiation, develop- 40 ment and metabolism. MiRNAs can function as tumor suppressors and oncogenes. The dysregulation of miRNA ex- pression has been linked to various human malignancies, in particular human cancers, and therefore miRNAs represent a new class of potential drug targets. Mature miRNAs are produced from long primary miRNA transcripts (pri-miRNAs) through sequential cleavages by the Micro-processor and Dicer complexes to release pre-miRNA and mature miRNA species, respectively. MiRNAs regulate the expression of a large part of the human genome by binding to partially 45 complementary sites in the 3’ UTRs of mRNAs and inhibiting protein translation or inducing deadenylation and degra- dation. [0006] Lin28, also called Lin28a and its homologue Lin28b are one of many thousands of RNA binding proteins (RBPs, see list in appended Table 1) which bind to a specific motif (GGAG, or GNNG where N is any ribonucleotide) in pri- and pre-let-7 miRNAs inhibiting their processing and depleting cells of mature let-7. This mechanism appears to be quite 50 general and occurs in about 15% of tumors of different histology and Lin28 and Lin28b activation is associated with advanced disease and poor clinical prognosis (see e.g. Viswanathan, et al., Nat Gen, 2009, 41, 843). [0007] Roos et al. (Poster by ETH Zürich, Institute of Pharmaceutical Sciences, Department of Applied Sciences: "Antisense oligonucleotides inhibit LIN28 binding to pre-let-7" Keystone Conference, "Noncoding RNAs in Development and Cancer", January 20-25, 2013, Vancouver, Canada) designed and tested methoxy antisense oligonucleotides (ASO) 55 to specifically antagonize Lin28 from binding to the terminal loop region of pre-let-7 in order to elevate processing by Dicer and Drosha in order to prevent the cell from mature let-7 loss. These ASOs were tested by an RNA-based competition ELISA. Kd-values of a selection of tested ASO were determined by Surface Plasmon Resonance (SPR) measurements. The two best ASOs were tested in a biochemical Dicer assay using HPCL-MS for analysis to ensure a 2 EP 2 811 298 A1 proper pre-let-7a-2 processing by Dicer in presence of ASO. [0008] Yeom et al. (EMBO REPORTS, 12:7, 690-696, 2011) teaches a method for integrating single-molecule fluo- rescence microscopy and immunopurification.to investigate Lin28-mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which also regulates let-7 microRNA biogenesis. TUT4 immunoprecipitates 5 together with fluorescent Cy5-labelled miRNA pre-let-7. The real-time analysis of the uridylation by the TUT4 immuno- precipitates suggests that Lin28 functions as a processivity factor of TUT4 in the Lin28/TUT4/let-7 complex. [0009] WO 2009/048935 A2 discloses a method for promoting miRNA processing of pri-let-7 miRNA to mature miRNA in a human cancer cell, the method comprising contacting a cell with an agent inhibiting the activity or expression of Lin-28. [0010] It is the objective of the present invention to provide an improved and high throughput screening (HTS)-suitabie- 10 assay for identifying a compound modulating an interaction between two biomolecules such as e.g. polypeptides, sugars, polynucleotides, polyamines and lipids or between two domains of the same biomolecule. In particular it is an objective of the present invention to provide an improved HTS-suitable assay for identifying a compound modulating an interaction between polypeptides and polynucleotides, preferably microRNAs(miRNA). [0011] This objective is solved according to the present invention by a method for identifying a compound modulating 15 an interaction between two biomolecules or two domains of one biomolecule, the first biomolecule or first domain com- prising at least one fluorophore donor and the second biomolecule or second domain comprising at least one fluorophore acceptor or a dark quencher, wherein the fluorophore donor and fluorophore acceptor or the fluorophore donor and dark quencher are spectrally paired such that the energy spectrum emitted by the fluorophore donor and the excitation energy spectrum of the fluorophore
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