|HAO HATA KAMA MARIAUS009772330B2 DEL MAT MAN AT KA NA MALO MATTER (12 ) United States Patent (10 ) Patent No. : US 9 ,772 ,330 B2 Nussbaum ( 45 ) Date of Patent : Sep . 26 , 2017 (54 ) NUCLEIC ACID APTAMER -BASED WO 2007024676 A2 3 / 2007 DIAGNOSTIC METHODS WITH NOVEL Wo 2007117444 A2 10 / 2007 TECHNIQUES FOR SIGNAL WO 2009070640 A2 6 / 2009 ENHANCEMENT OTHER PUBLICATIONS (71 ) Applicant : APTATECK BIO LTD . , Ness Ziona ( IL ) Zeeshan M , et al .“ Comparison of different phenotypic methods of detection of methicillin resistance in Staphylococcus aureus with (72 ) Inventor: Ofer Nussbaum , Rehovot ( IL ) the molecular detection of mec - a gene ” J . Coll . Physicians. Surg . Pak . 17 :666 -70 (2007 ) . ( 73 ) Assignee : APTATECK BIO LTD ., Rehovot ( IL ) Edmonds, M . “ Branched RNA ” BioEssays . 6 :212 - 216 ( 1987 ). Lin ( I ) ( Clinical Chemistry 2009 vol . 55 , p . 1686 - 1693 ) . ( * ) Notice : Subject to any disclaimer , the term of this Goddard et al. ( Prog Polym Sci 2007 vol. 32 , p . 698 -725 ) . patent is extended or adjusted under 35 Lisa R . Paborsky et al . “ The Single - stranded DNA Aptamer -binding U . S . C . 154 (b ) by 0 days . Site of Human Thrombin ” The Journal of Biological Chemistry . 268 :20606 - 20811 ( 1993 ) . ( 21) Appl. No. : 14 / 565 ,759 Mandona Sassanfar, Jack W . Szostak . “ An RNA motif that binds ATP ” Letters to Nature . 364 : 550 - 553 ( 1993 ) . ( 22 ) Filed : Dec. 10 , 2014 Monica Mir, Ioanis Katakis . “ Aptamers as elements of bioelectronic devices " The Royal Society of Chemistry . 3 :620 -622 (2007 ) . (65 ) Prior Publication Data Matthew Levy et al . “ Quantum - Dot Aptamer Beacons for the Detection of Proteins ” ChemBioChem . 6 :2163 -2166 (2005 ) . US 2015 /0160207 A1 Jun . 11, 2015 Williams James . “ Aptamers” Encyclopedia of Analytical Chemis try . 4848 -4871 ( 2000 ) . Supriya S . Pai, Andrew D . Ellington . “ Using RNA Aptamers and the Related U . S . 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C12Q 1 / 706 ( 2013 .01 ) ; GOIN 33 /54306 (Continued ) ( 2013 . 01 ) ; C12N 2310 / 16 (2013 .01 ) ; C12N 2320 / 10 ( 2013 .01 ) ; GOIN 2333 /49 (2013 .01 ) ; GOIN 2333 /585 ( 2013 .01 ) ; GOIN 2469 / 10 Primary Examiner — Jacob Cheu ( 2013 .01 ) ( 74 ) Attorney , Agent, or Firm — Browdy and Neimark , ( 58 ) Field of Classification Search PLLC None See application file for complete search history . (57 ) ABSTRACT ( 56 ) References Cited The present invention concerns methods for the detection f U . S . PATENT DOCUMENTS target molecules in a sample including several steps of signal amplification allowing the detection of a very low number of 3 , 951, 748 A * 4 / 1976 Devlin .. .. .. A61K 39 /44 target molecules in the tested sample . The detection assay is 435 / 28 based on the use of a universal probe which enables the 5 , 925 , 533 A 7 / 1999 Doth et al . 435 / 7 . 94 signal amplification . The specific recognition of the target 6 ,458 ,543 B1 10 / 2002 Gold et al . 2009 /0203028 AL molecule is achieved by using a specific binding agent, 8 / 2009 Yamamoto preferably an aptamer . The invention further concerns kits FOREIGN PATENT DOCUMENTS and methods for the diagnosis of pathological conditions . wo 0179562 Al 10 / 2001 Wo 2005113817 A2 12 / 2005 15 Claims, 13 Drawing Sheets US 9 ,772 , 330 B2 Page 2 References Cited Yong Huang “ Electrochemical immunosensor of platelet - derived ( 56 ) growth factor with aptamer -prmed polymerase amplification ” Ana lytical Biochemistry 382 : 16 - 22 ( 2008 ) . OTHER PUBLICATIONS Jun Wang et al. “ In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection " Program in Tao Chen et al. “ Evaluation of Quantitative PCR and Branched Infectious Diseases and Immunity 6 :571 - 583 ( 2000 ) . Chain DNA Assay for Detection of Hepatitis B virus DNA in Sera Dianah Barrett et al. “ Kinetic Characterization of the from Hepatocellular Carcinoma and Liver Transplant Patients ” Glycosyltransferase Module of Staphylococcus aureus PBP2 ” Jour Journal of Clinical Microbiology . 38 : 5 : 1977 - 1980 (2000 ) . nal of Bacteriology 187 : 6 :2215 -2217 ( 2005 ) . P . Maruna et al . “ Physiology and Genetics of Procalcitonin ” Physi Anna Farra et al. “ Role of outer membrane protein OprD and ology Research . 49 : S57 -561 (2000 ) . penicillin -binding proteins in resistance of Pseudomonas aeruginosa Paresh Dandona et al . “ Porcalcitonin Increase after Endotoxin to imipenem and meropenem ” International Journal of Antimicro Injection in Normal Subjects ” 79 : 5 : 1605 - 1608 ( 1994 ). bial Agents 31 :427 - 433 ( 2008 ) . Chien -Hung Chen . “ Quantitative detection of hepatitis B virus Monika Olempska et al. “ Detection of tumor stem cell markers in DNA in human sera by branched - DNA signal amplification ” Journal pancreatic carcinoma cell lines” Hepatobiliary and Pancreatic Dis of Virological Methods. 53: 131 - 137 ( 1995 ) . eases Intemational. 6 : 1 : 92 - 97 ( 2007 ) . Peter Birner et al . “ Signal -Amplified Colorimetric In Situ Hybrid Ulla Balslev et al . “ An Outbreak of Borderline Oxacillin - Resistant ization for Assessment of Human Papillomavirus Infection in Cer Staphylococcus aureus (BORSA ) in a Dermatological Unit ” Micro vical Lesions” Modern Pathology 14 : 7 : 702 -709 ( 2001) . bial Drug Resistance 11 : 1 :78 - 81 ( 2005 ) . Hilla Giladi et al. “ Small Interfering RNA Inhibits Hepatitis B Virus Thierry Delair et al . “ Synthesis and Characterization of Nucleic Replication in Mice ” Molecular Therapy 8 : 5 : 769 -776 (2003 ) . Acid - ( Co ) Polymer Conjugates : Application to Diagnostics. ” Poly Lorena Pardo et al . “ Characteristics of Community - Associated mers for Advanced Technologies 9 : 349 - 361 ( 1998 ) . Methicillin - Resistant Staphylococcus aureus (CA -MRSA ) Strains Julien Bernard et al. “ Water -Soluble Dendrigrafts Bearing Sac Isolated from Skin and Soft - Tissue Infections in Uruguay ” Inter charidic Moieties: Elaboration and Application to Enzyme Linked national Journal of Microbiology 2009 : 472126 : 1 - 5 ( 2009 ) . OligoSorbent Assay ( ELOSA ) Diagnostic Tests ” Bioconjugate Yuan - Yuan Li “Ultrasensitive Densitometry Detection of Cytokines Chemistry 17 : 6 - 14 (2006 ) . with Nanoparticle -Modified Aptamers " Clinical Chemistry 53: 6 : 1061 - 1066 (2007 ) . * cited by examiner U . S . Patent Sep. 26 , 2017 Sheet 1 of 13 US 9 ,772 , 330 B2 Coating substance . Magnetic : beads ws Aptamers . Figure 1A Coating substance : - . » Magnetic RO itinuuting beads O Aptamers .5 Figure 1B U . S . Patent Sep. 26 , 2017 Sheet 2 of 13 US 9 , 772 , 330 B2 biotin [ X - Y - Z -] n -SE . Figure 2A biotin . 2 . .. d * S T : *: : M ASEvlaga Figure 2B atent Sep . 26 , 2017 Sheet 3 of 13 US 9 ,772 , 330 B2 *** WW T7 promoter * L iberia ds- DNA biotin Aptamers Figure 3 U . S . Patent Sep. 26 , 2017 Sheet 4 of 13 US 9 , 772 , 330 B2 Cleaved substrate Color / fluorescence surface Alkaline phosphatase or HRP Native substrate Streptavidin Biotin ** * ** Biotinuna -ds DNA cu DOMA. Figure 4A Ds -DNA with T7 RNA polymerase promoter - SE wi w NTPs my T7 RNA polymerase biotin - UTP with W Invitro transcription . Biotin labeled RNA Figure 4B U . S . Patent _ Sep . 26 , 2017 _ Sheet 5 of13 US 9 ,772 , 330 B2 ss - DNA ss -DNA Succinimidyls Streptavidin Aptamer Figure5A Figure 5B Figure 50 HRPlilii Branch!? ?unit ?????????? ??iqfir?????????? P * SE - ss - DNA ? TMB Substrate ???? 8 Figure 5D U . S . Patent Sep. 26 , 2017 Sheet 6 of 13 US 9 , 772 , 330 B2 Protein coated Magnetic beads DMP blocked 3 5 ' labeled Aptamer AntigenAndigen E n Succinimidyl- dsDNA E who WO ER Magnetic bar Figure 6 Protein coated Magnetic 5 ' labeled capture beads DMP blocked Aptamer Antigen WomeAptamer /SA /bDNA nga complex bare E Egmore ?? MagneticMagnetic barbar Figure 7 U . S . Patent Sep. 26 , 2017 Sheet 7 of 13 US 9 , 772 , 330 B2 1 ) 1236 B10 . sri. .* . '. t V :. * " * .: .: : . .: . ?. * .