US 2016.0076021A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0076021 A1 Stojanovic et al. (43) Pub. Date: Mar. 17, 2016 (54) APTAMIER METHODS AND COMPOSITIONS (60) Provisional application No. 61/798,079, filed on Mar. 15, 2013. (71) Applicants: Milan N. Stojanovic, Fort Lee, NJ (US); Tilla S. Worgall, New York, NY (US); Kyung-Ae Yang, New York, NY (US) Publication Classification (51) Int. C. (72) Inventors: Milan N. Stojanovic, Fort Lee, NJ (US); CI2N 5/10 (2006.01) Tilla S. Worgall, New York, NY (US); GOIN33/53 (2006.01) Kyung-Ae Yang, New York, NY (US) CI2N 15/I 15 (2006.01) (73) Assignee: THE TRUSTEES OF COLUMBIA (52) U.S. C. UNIVERSITY IN THE CITY OF CPC .......... CI2N 15/1048 (2013.01); C12N 15/115 NEW YORK, NEW YORK, NY (US) (2013.01); G0IN33/5308 (2013.01); C12N 23 10/16 (2013.01); C12N 2320/13 (2013.01); (21) Appl. No.: 14/855,171 CI2N 2320/10 (2013.01) (22) Filed: Sep.15, 2015 (57) ABSTRACT Related U.S. Application Data Methods of selecting an aptamer that specifically binds to a (63) Continuation-in-part of application No. PCT/US14/ target molecule complexed with a derivatization agent. Also 29281, filed on Mar. 14, 2014. disclosed are specific aptamers and methods of use thereof. Patent Application Publication Mar. 17, 2016 Sheet 1 of 17 US 2016/0076021 A1 FIG. 1A - FIG. 1C A. receptor target receptor target B. w W, w s & 8 ww. ---O. “. 8:XXX Y targe Aptamer eaaceti organometallic receptors (metal, on eft, complexed to organic ligand, on right C. 8 *-*.s 8 k NY( ) 8 :g 8. 8: f G C & C YC. G1 3. A --- c.s d ( - || /> ciC A (s K d Ali d is C-C-GAG. A G-c G-d c.c.GAGA Gé & d; G-? N->->G G cT cT c-G d --G-G-c-f-c-f-c-de C- YPCR Amplification and Poo Regeneration V --- Patent Application Publication Mar. 17, 2016 Sheet 2 of 17 US 2016/0076021 A1 FIG. 1 D - FIG. 1E 1883$ CiA38isties asci roy: Stegesarakia. Patent Application Publication Mar. 17, 2016 Sheet 3 of 17 US 2016/0076021 A1 FIG. 2 -"olo s x8. Cp*R*AA Cp*RhAA isypii....- ... 3: ixitxii: :: *::::::ii Patent Application Publication Mar. 17, 2016 Sheet 4 of 17 US 2016/0076021 A1 FIG. 3A - FIG. 3F A. C. D. A. t-c -T c Sensor Sensitivity & A (tyrosine-Gprh sensor : 3 800 -1 NH -C *. 600 -g -t 40000 B. -- -é 200 --- - 8 500 1s -6 tyrosine conc. ut E. F. Sensor Selectivity (Tyrosine-CpRh sensor) 800 *000 x- Tyrosire re. Asis 80030 So exa. “*-. A-2-sites ArqiAt aga :: - - Cyster,C. 60000 40000: t 3ycits ise - Lysine 4000 26 -- Relhorite x 8 ------- --- Periyalarine 20038 8 tree- -s-- Metitionis:tryptophan ryosine healthy subject serum 0. 29 30 o 00 200 300 400 CpRh conc. (iv) a Dution times Patent Application Publication Mar. 17, 2016 Sheet 5 of 17 US 2016/0076021 A1 FIG. 4A - FIG. 4F A. B. C. NH C GC S is G T-c g-A cC-T A HN OH Ag a-G-G-A-d-G-G-A NH2 t cg A. Af G - C D. Sensor Sensitivity (Arginine-Cprh sensor) 100000 80000 s 6000 : 40000 20000 9 29 43 630 Arginine conc. (uN) E. F. Sensor Selectivity (Arginine-CpRh sensor) 0000 10000 r-8- Arginine r-- Cru: 80000 80000 -8-M-8 -- Garine -- Glycine o 60000 r0m. Hisine d 60000 l re- Methions:e : 2 - Ornithine 40000 40000 rar Tryptophar, 20000; -- tyrosipew 20000 0 3 2 40 80 O 100 200 360 400 CpRh conc. (uR) Dilution tinnes Patent Application Publication Mar. 17, 2016 Sheet 6 of 17 US 2016/0076021 A1 FIG. 4G - FIG. 4 G. H. *: it cofactor Kid 6 rv 3:N3:388x3:33 . J. 88: S388 88. 883 ic s : w 88: 23:38: 8 8 & 3& 8, 283 8 488 A88 8:38:::::::::3883 : &isio acid concertraict 388 Patent Application Publication Mar. 17, 2016 Sheet 7 of 17 US 2016/0076021 A1 FIG. 5A - FIG. 5F -8. 3. 88: & 83 3xxxxix. 888 &cs. 8:8 8. s& s: & Enex58 is 3. S. i.e. ii. Patent Application Publication Mar. 17, 2016 Sheet 8 of 17 US 2016/0076021 A1 FIG. 5G - FIG. 5N Sensor Selectivity Sensor Sensitivity (Giucose-oranic acid sensor) Glucose-Bororic acid sensor) 880 80s --------------. ---8 60000- *w8. Wax. ex 60s - f --&--. Gligosa -x^ s 8 -- iticss me - 400 rise toss : 40 -- Scaric acxd only aw (Saactoss --&-- 8sics & Succuse 200 2 000 X - - - 20 3. 4. so 2 49 s Sugar conc. (m) Boronic acid conc. (uM) - & 8 & s I. M-8 ---------------------------------------- s 8 4. s: & s Sixx xxx... x8 S&s six. 8 Patent Application Publication Mar. 17, 2016 Sheet 9 of 17 US 2016/0076021 A1 FIG. 6A - FIG. 6B 250 8. -* 2008 ^- *8* Glucose 15000 Y s8-: Goose ran Ficise . -- Ficose order Warfose '40000 g -- Aarose ---. Gaiacose --8e Gaciose 20 4. 6 O 5 53 surga conc. (m) Sugar conc. (ni) Patent Application Publication Mar. 17, 2016 Sheet 10 of 17 US 2016/0076021 A1 FIG. 7A - FIG. 7D x-Y. Tyr g Y. v A --- grrrrris ; : Corpex-bidingR -. r p motif1 on which& aptamer ---8 - 8. is built &- & Citruline C s'. Nor-Seitective e k Yx...' Cross-reactive - i rp SY.W --- : 8-- - 8 : : k a a. *k Patent Application Publication Mar. 17, 2016 Sheet 11 of 17 US 2016/0076021 A1 FIG. 7E - FIG. 7H --- 3 8 8 Patent Application Publication Mar. 17, 2016 Sheet 12 of 17 US 2016/0076021 A1 FIG. 8A - FIG. 8C sciective: Patent Application Publication Mar. 17, 2016 Sheet 13 of 17 US 2016/0076021 A1 FIG. 9A - FIG. 9C A B C g s A & & s & s & : AS: 3:38:::::8: $8388:w8 as ::::::&- . ; as 3. : Patent Application Publication Mar. 17, 2016 Sheet 14 of 17 US 2016/0076021 A1 FIG. 1 OA - FIG. 1 OB i.e. 8-8 8S 3. Sciectivey 3.fk Y---------g-r-g-g-a-S.--- -A- -s -4 overie 'N' W w i.e. Seisective ower is: & 8 : Y-------. Patent Application Publication Mar. 17, 2016 Sheet 15 of 17 US 2016/0076021 A1 FIG. 11A - FIG. 11B x PW Patent Application Publication Mar. 17, 2016 Sheet 16 of 17 US 2016/0076021 A1 FIG. 12A - FIG. 12B & &'s *... : A 60000 x Periyaarie rx tyrosie « tryptophare men glycine & XS.S.S.S.S.S.S.S.SYYYYY." o 1000 B Amino acid concentraton (u) Patent Application Publication Mar. 17, 2016 Sheet 17 of 17 US 2016/0076021 A1 FIG. 12C - FIG. 12D " e-o-o: 8. x. s -- Peya are or ryptopian -- tyrosite soo food * D Arrino acid concentration (iii) US 2016/0076021 A1 Mar. 17, 2016 APTAMER METHODS AND COMPOSITIONS isolating an eluted aptamer having high affinity for the target complex. In some embodiments, the method includes system CROSS-REFERENCE TO RELATED atic evolution of ligands by exponential enrichment APPLICATIONS (SELEX). 0001. The present application is a Continuation in Part of 0009. In some embodiments, the aptamer does not sub International Application No. PCT/US14/29281 filed 14 Mar. stantially bind the non-complexed target molecule. In some 2014, which claims the benefit of U.S. Provisional Applica embodiments, the aptamer does not substantially bind the tion Ser. No. 61/798,079 filed 15 Mar. 2013, each of which is non-complexed derivatization agent. In some embodiments, incorporated herein by reference in its entirety. the method includes counter-selecting an aptamer against the derivatization agent alone or against the target molecule STATEMENT REGARDING FEDERALLY alone. SPONSORED RESEARCH ORDEVELOPMENT 0010. In some embodiments, the oligonucleotide library includes randomly generated oligonucleotide sequences of a 0002 This invention was made with government support fixed length flanked by a constant 5' end and a constant 3' end, under grant numbers CBET-1033288 and CBET-1026592 the constant 5' end and the constant 3' end functioning as a awarded by National Science Foundation. The government primer. has certain rights in the invention. 0011. In some embodiments, the aptamer is a DNA, RNA, or XNA molecule. In some embodiments, the aptamer com MATERIAL INCORPORATED-BY-REFERENCE prises at least about 15 oligonucleotides up to about 100 0003. The Sequence Listing, which is a part of the present oligonucleotides. In some embodiments, the aptamer has an disclosure, includes a computer readable form comprising equilibrium constant (Kd) of about 1 pMup to about 10.0LM: nucleotide and/or amino acid sequences of the present inven about 1 pM up to about 1.0 uM; about 1 pMup to about 100 tion. The Subject matter of the Sequence Listing is incorpo nM; about 100 pM up to about 10.0 uM; about 100 pM up to rated herein by reference in its entirety. about 1.0 uM; about 100 pMup to about 100 nM; or about 1.0 nM up to about 10.0 uM; about 1.0 nM up to about 1.0 uM; BACKGROUND OF THE INVENTION about 1 nMup to about 200 nM; about 1.0 nMup to about 100 nM; about 500 nM up to about 10.0 uM; or about 500 nM up 0004 Systematic evolution of ligands by exponential to about 1.0 uM. enrichment (SELEX) is a combinatorial technique in molecu 0012. In some embodiments, the target molecule com lar biology for producing oligonucleotides of either single prises a small molecule, a protein, or a nucleic acid. In some stranded DNA or RNA that specifically bind to a target ligand embodiments, the target molecule comprises a small mol or ligands.