US 20130317087A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0317087 A1 Maquat et al. (43) Pub. Date: Nov. 28, 2013 (54) METHODS AND COMPOSITIONS RELATED Publication Classification TO STAUFEN 1 BINDING SITES FORMED BY DUPLEXING ALU ELEMENTS (51) Int. Cl. Inventors: Lynne E. Maquat, Rochester, NY (US); CI2O I/68 (2006.01) (75) (52) U.S. Cl. Chenguang Gong, Rochester, NY (US) CPC .................................... CI2O I/6883 (2013.01) (73) Assignee: UNIVERSITY OF ROCHESTER, USPC ............. 514/44A: 436/501; 506/9: 435/6.12: Rochester, NY (US) 536/24.5 (21) Appl. No.: 13/984,709 (22) PCT Fled: Feb. 9, 2012 (57) ABSTRACT (86) PCT NO.: PCT/US12A24534 Disclosed are compositions and methods for identifying S371 (c)(1), binding sites of targets of Staul-mediated mRNA decay; (2), (4) Date: Aug. 9, 2013 methods and compositions for treating Subjects with condi tions resulting from Staul-mediated mRNA decay, and Related U.S. Application Data method of screening for therapeutic agents. Also disclosed is (60) Provisional application No. 61/440,967, filed on Feb. the new pathway as a means for cells to down-regulate the 9, 2011. expression of Staul-binding mRNAs. Patent Application Publication Nov. 28, 2013 Sheet 1 of 39 US 2013/0317087 A1 SSS: : we & SRN & sire Sv$3's S&S $. 3:Sys, was SS on- &S sys’s were {SS $$. water SSS S. Ya. sees &C., SSX . A. se OASL mRN F.G. 1-1 Patent Application Publication Nov. 28, 2013 Sheet 2 of 39 US 2013/0317087 A1 W. SyrSw six e S&NA ses a. Y G - s s S RNs, CX S&N & s 3. CXCL1 RN. 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X8. 888 & 8x s'i s i -- STAl-As (NB: a-HA -- Cainexit (S. c-Cainexin) T. SERNE RNA SERPINE mRNA (9%) st SERPINE mRNA S is g|After IPBefore IP (%) -- 28, YRNA o St. F.J2870 mRNA (%) see, FS Sys Nis 3 After Pi Before P 6 s : S: Ss iss -...s. X--> w-XX. en- assir is (N --SSRNA s g After P. Before P (%) SG RNA - E. coii ACY RNA FG. 7 Patent Application Publication Nov. 28, 2013 Sheet 20 of 39 US 2013/0317087 A1 Siri aa.sa sa ss was RAS Niš see Sis RNA 3 3 Rastrip mRNA (6) se s r g Sir s i re s ww. ww.S. s N as: vee S abara % sa yearis is SERPINE1 mRNA (%) · - Fifi RA Fi21870 in RNA is s: - RASFP RNA RAB, FIP RNA (i. 8 × 8 is is six sess --Sfig7 rRNA FG. 8 Patent Application Publication Nov. 28, 2013 Sheet 21 of 39 US 2013/0317087 A1 sts sixà, issisi is Siria SS SSSSŠ SS. f.i.28$ $ g siRNA&S Rås is sisal S. FG. 8 Patent Application Publication Nov. 28, 2013 Sheet 22 of 39 US 2013/0317087 A1 S RNA AG ei 393 sis s S $sS$Š ss S 3 WSW& SS&S& S.S. S.ŠS&sSS S . .................. $............ &s. S. WYY S. s' S S.Š S. S S.Š S. SS & S S S SSis SS$ Š$$$$. S&S SS - AeaelSsss V Y Y YY Šssysa. SS Ss SS 432 SSSir &SSRNA FIG. 9 Patent Application Publication Nov. 28, 2013 Sheet 23 of 39 US 2013/0317087 A1 --- S. s g ema e xeer O r m t 5(f) -- 3i. : s 5 - S is9 <2 - 25 S I C () tWNSqS-74 WN&SCS-24 ZWNSGS-24 WNASGS-7, do ss WIS OJuOO Patent Application Publication Nov. 28, 2013 Sheet 24 of 39 US 2013/0317087 A1 % wnaesqs„“*…! -%),~ **vnºsas s Patent Application Publication Nov. 28, 2013 Sheet 25 of 39 US 2013/0317087 A1 s - s S &Y s as: era &W, . 8-v- 8 Y x - 8 Patent Application Publication Nov. 28, 2013 Sheet 26 of 39 US 2013/0317087 A1 C , s s -- 3.22 mg/ml 3 s r 2.12 mg/ml 0. 4. ww. s - 1.04 mg/ml. s \\ - 0.35 mg/ml 3 w8 , 2 8s s s3 8s s' (Svedbergs) FIG 10 Patent Application Publication Nov. 28, 2013 Sheet 27 of 39 US 2013/0317087 A1 RNase A is sy s: & S. 3 : SR s S3 is s S sy s E. s. S s s pEGR-STA.SSS S is & YW is s a. * : * r * r * is SSS S.: S:C. , S: , E.S: pnik we iswres Ali & Sr Sai - Ss - a ^ p 8 xxx wax. XXXX 888 S. & X is s &is S & Saxx. & ej-, op. 'RB'sE. "E. " city - ini-SS-R'sS * RER.'RB's (8 a-RFP) rippMiss 'RED's RBD's ice,- exposure hSIAU1 (WB: OSIA1) SIAU rt exposureDorker --Carexit 8 x-Conexis F.G. 10 Patent Application Publication Nov. 28, 2013 Sheet 28 of 39 US 2013/0317087 A1 ****************************************************************************************** ~~~~€Œ-~~~~€Œ---- IIº‘DILI sy SeDice ea SepiceJehu Patent Application Publication Nov. 28, 2013 Sheet 29 of 39 US 2013/0317087 A1 --s - SS wsa 8x s & S-3 stXFs is ww. ************ II“OICH 9,088, s E3. 3. & & S. Patent Application Publication Nov. 28, 2013 Sheet 30 of 39 US 2013/0317087 A1 s S. S. & S S *s y & s Patent Application Publication Nov. 28, 2013 Sheet 31 of 39 US 2013/0317087 A1 Structure css-complexotion signifigance score FIG. 13 Patent Application Publication Nov. 28, 2013 Sheet 32 of 39 US 2013/0317087 A1 A-2 PSA Rest structure 1 structure 2 Selection range Class Symmetry operation X. Š x^ Number of atons interface surface tot Number of residues interface surface total Solvent-accessible area.A. interface Selection range i Cess Protein Symmetry operation ------, -g- if Symmetry ID 8885 Number ofinterface atoms surface total Number of residues interface surface it total Solvent-accessible area.A. interface total Patent Application Publication Nov.