SG11201806879SA - A simple one-step real-time molecular system for microrna detection - Google Patents

Abstract

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property -, Organization M1111101110101011111 HO 11111 01101011111011011101100 111110 ill OEN 11 International Bureau .. .... ..Yejd (10) International Publication Number (43) ..... .....1 International Publication Date WO 2017/146653 Al 31 August 2017 (31.08.2017) WIPO I PCT (51) International Patent Classification: Molecular and Cell Biology, 61 Biopolis Drive, Proteos, C12Q 1/68 (2006.01) Singapore 138673 (SG). TANG, Melissa Xue Mei; c/o In- (21) International Application Number: stitute of Molecular and Cell Biology, 61 Biopolis Drive, PCT/SG2017/050086 Proteos, Singapore 138673 (SG). (74) Agent: SPRUSON & FERGUSON (ASIA) PTE LTD; (22) International Filing Date: P.O. Box 1531, Robinson Road Post Office, Singapore 24 February 2017 (24.02.2017) 903031 (SG). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (30) Priority Data: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, 10201601384T 24 February 2016 (24.02.2016) SG DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (71) Applicant: AGENCY FOR SCIENCE, TECHNOLOGY HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, AND RESEARCH [SG/SG]; 1 Fusionopolis Way, #20-10 KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, Connexis North Tower, Singapore 138632 (SG). MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, (72) Inventors: AW, Sherry; c/o Institute of Molecular and RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, Cell Biology, 61 Biopolis Drive, Proteos, Singapore TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, 138673 (SG). TEO, Yin Nah; c/o Institute of Molecular ZA, ZM, ZW. and Cell Biology, 61 Biopolis Drive, Proteos, Singapore _ 138673 (SG). COHEN, Stephen Michael; c/o Institute of (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, [Continued on next page] = = (54) Title: A SIMPLE ONE-STEP REAL-TIME MOLECULAR SYSTEM FOR MICRORNA DETECTION o 0 (57) : The present application discloses an isolated nucleic acid mo- lecule for detecting the presence of a target nucleic acid sequence and uses t• thereof, wherein said molecule is capable of forming bimolecular interactions = Niev,, with a target nucleic acid sequence and forms a ternary complex stabilising a detection agent when bound. The detection agent is preferably DFHBI ((Z)-4- Variable (3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethy1-1 H-imidazol-5(4H)-one. = Variable Stem-loop P4 The target nucleic acid is preferably a miRNA sequence. Preferably, the isol- = Stem-loop ,-)3 ated nucleic acid sequence comprises two domains that complementarily bind to the 5' and 3' ends of the target nucleic acid sequence. The isolated nucleic acid sequence also comprises regions of nucleic acids that do not bind with the target nucleic acid sequence and are capable of forming secondary struc- = — = .. tures, such as stems, stem loops, t-RNA type structures, cloverleaves, tetra- loops and pseudoknots. The application further discloses corresponding meth- ods of determining a disease in a subject by using the isolated nucleic acid se - quence to detect one or more disease-specific target nucleic acids. = = — G G - C Stem P2 u'll 9 — € 3 —c. - . _ 1-1 Stem- en ir) loop P1 ,tD ,tD 41 G G 7r 11 --.... Figure 8 IN Il © ei O WO 2017/146653 Al MIDEDIMOMOIDEIR010130101fillEINUMEMOIMIE GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, Declarations under Rule 4.17: TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, DE, LT, of inventorship (Rule 4.17w)) Published: LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). SE, GA, with international search report (Art. 21(3)) — with sequence listing part of description (Rule 5.2(a))