US 20190142882A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2019 /0142882 A1 Shepherd et al . ( 43 ) Pub . Date : May 16 , 2019 ( 54 ) MICROBIAL PRODUCTION OF PURE (52 ) U . S . CI. SINGLE STRANDED NUCLEIC ACIDS CPC . .. .. A61K 35 / 76 (2013 .01 ) ; C12N 15 / 11 ( 2013. 01 ) ; A61K 47 /6901 ( 2017 .08 ) ; CO7K (71 ) Applicant : Massachusetts Institute of 14 / 005 ( 2013 .01 ) ; CO7K 7 /06 ( 2013 .01 ) ; C12N Technology , Cambridge, MA (US ) 2810 /60 (2013 .01 ) ; C12N 15 / 1096 ( 2013 .01 ) ; C12N 15 /65 ( 2013 . 01 ) ; C12N 2795 / 14122 (72 ) Inventors : Tyson Shepherd , Arlington , MA (US ) ; ( 2013 .01 ) ; C12N 2795 / 14142 (2013 .01 ) ; C12N Rebecca Du , Cambridge , MA (US ); 15 / 1044 ( 2013 .01 ) Mark Bathe, Cambridge , MA (US ) ( 21 ) Appl. No .: 16 / 189 ,418 (57 ) ABSTRACT (22 ) Filed : Nov. 13 , 2018 Methods and compositions for bacterial production of pure single - stranded DNA ( ssDNA ) composed of custom Related U . S . Application Data sequence and size have been developed . The methods enable ( 60 ) Provisional application No . 62 / 584 , 664, filed on Nov . scalability and bio - orthogonality in applications of scaf 10 , 2017 folded DNA origami, offering one- step purification of large quantities of pure ssDNA amendable for immediate folding Publication Classification of DNA nanoparticles . The methods produce pure ssDNA (51 ) Int. CI. directly from bacteria . In some embodiments the E . coli A61K 35 / 76 ( 2006 . 01 ) helper strain M13cp combined with a phagemid carrying C12N 15 / 11 ( 2006 .01 ) only an f1 -origin allows for , without the need for additional A61K 47 /69 ( 2006 . 01 ) purification from contaminating dsDNA . This system is CO7K 14 /005 ( 2006 .01 ) useful for generalized circular ssDNA synthesis , and here is CO7K 7 / 06 ( 2006 .01 ) applied to the assembly of DNA nanoparticles folded both in C12N 15 / 10 ( 2006 .01 ) vitro and direct from phage . C12N 15 / 65 (2006 .01 ) Specification includes a Sequence Listing . p15A -ori CMR oll replication pX _ replication Helper Plasmid 8180 bp PIV assembly OV replication DVIl minorCoatProtein pix _ minorCoatProtein ÞVill_ majorCoalProtein pXl_ assembly pl assembly pill minorCoatProtein pVi minorCoatProtein Patent Application Publication May 16 , 2019 Sheet 1 of 14 US 2019 /0142882 A1 p15A -ori Cmo pil replication px replication Helder Plasmid 8180 bp PIV assembly pV _ replication pVil_ minorcoatProtein pix _ minorCoatProtein PVII majorCoatProtein pXl_ assembly plassembly pill minorcoatprotein pVi minorCoatProtein FIG . 1 11 ori Mini Phagemid 1676 bp APR FIG . 2 Patent Application Publication May 16 , 2019 Sheet 2 of 14 US 2019 /0142882 A1 EN RF phagemid coli si M13cp DNA nanoparticle 03cloning SSDNA production - 6188isolation folding FIG . 3 Bacterial colonies Colonies selected streaked onto agar and isolated into Quantitative PCR of plates individualwells of a clarified media 96 -well plate FIG . 4 Colonies selected Capillary Bacterial colonies and isolated into Clones grown in electrophoresis to streaked onto agar individual wells of a media and spun plates 96 - or 384- well down test for ssDNA from ????? ?? plate each well FIG . 5 Patent Application Publication May 16 , 2019 Sheet 3 of 14 US 2019 /0142882 A1 lox Synthetic Fragment cre Synthetic Phagemid Fragment lox AMPR FIG . 6 Pump Fion www FIG . 7 Patent Application Publication May 16 , 2019 Sheet 4 of 14 US 2019 /0142882 A1 FIG . SA - - - - - FIG . 8B Patent Application Publication May 16 , 2019 Sheet 5 of 14 US 2019 /0142882 A1 071103 3000bp 200g , uncoboties . .. FIG . 9A + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Patent Application Publication May 16 , 2019 Sheet 6 of 14 US 2019 /0142882 A1 PF1Cv3 1568 nt Synthetic Sequence FIG . 10A DigitalInformation PF1A - Cv3 2775 nt --** * * * ** * * * * ** * * * * * * ** * * ** digital information ier TORFIG . 10B Patent Application Publication May 16 , 2019 Sheet 7 of 14 US 2019 /0142882 A1 ALMOLDALUAMALAN 74nm 28.6mm 28.6nm UN www AAAA AAN Drdl(296) memesamPSil(377) 461)Noel( Mmel(274) -Bmrl(729). Bpml(759) meAll(837) www 689)(AndlAlp 1000Bsal(750) BsaW(980) 10C.FIG Nael(146)Drall252 HOME Eael(1077)puul(1059) Xmnl(1288) NgOMIV(144)Banil178 500 1077)Eael( 4 V ht AmpR We WWWWWW (2519)Pst" 2520nt 2500FOR PPB84amp SIGN. Smil1309)(Apal!Acul-1356)( CARMEN XXKA AmpR (2143)BmgBl2273BluAl-BspM2337()Pcil ( 2241)Spel(2284)Bsu361 2000-Y 11MSpA)1324(/158014BineBaeGit)1360( PROMOTER XXX MET MY :X Why 1500TAatil1611)( 10 (1677)EcoRI(1609)Zral 3 (1775)Kas|- (2091)Pmll (2009)Snabl (1939)AWN (1851)SexAl*(1793)Hindi (1779)Pull(1777)Sfol (1776)Nari Eco0191)1665( (1493)Sspi Patent Application Publication May 16 , 2019 Sheet 8 of 14 US 2019 /0142882 A1 (3228 ) Psti 3000 mon TEXNOLWENN ARUNONNONDA F1 ORI ST S VI OROKONCE umumanPX TETRAHEDRON 66 WOWOWKURW KAMAS .ACARINHO JUNIONOWO B PF1A -PXtet66 PXTETRAHEDRON60bpINSERT www.AShow ww 3229 nt WWMWAN Doner Meninas w AmpR NCHI AP KONNA AmpR PROMOTER SIGN .. FONN UM 2000 CU CATHARINA NAVODI NOUVEUHAEUSCORO ( 1677) ECORI 1500 FIG .& 10D Patent Application Publication May 16 , 2019 Sheet 9 of 14 US 2019 /0142882 A1 RNASTAPLEGENES CAT PROMOTER BINDING PHAGEMID 12, 000 SITE SCAFFOLD FIORI CMR 015A NEMUNON PACYC184 ORI WMtb VECTOR Fath REMOM 77 PROMOTER A 2000 blalampR ) RNA STAPLES T7 UNIVERSAL SEPARATED BY / mano T7 PROMOTER MINPRIMER SEQUENCE X PROMOTER RIBOZYMES/ (NEB # S12489 ) 10 ,000 che - T7 PROMOTER MULTIPLE TOMORA CLONING 6xHis AFFINITY TAG ALL -IN -ONE PLASMID SITE 12, 548 bp Www MCS1 LINKER T7 PROMOTER RBS (Ndel TERMINATOR FOR ÉCORI) RIBOSOME EXPRESSION con CEN N A BINDING SITE COS INNON M13 GENEVOxHis \ M13 GENE IL PROMOTER M13 become SV40 NLS EXCreworks REPLICATION WLINA 8000 - TERMINATOR LIMITEDCaso RBS CALL MOUTONHE KARMA ch am R$ ARGA OxHis SnapCas9NLSHIS LAC REPRESSOR ENCODED BY laci BINDING SITE MULTIPLE CLONING SITE MCS2 (Pstl - Noti ) FIG . 10E Patent Application Publication May 16 , 2019 Sheet 10 of 14 US 2019 /0142882 A1 Pahaye particlesing : Uncalibratad mil ioane learning kwa mw viimti FIG . 11A FIG . 11B Patent Application Publication May 16 , 2019 Sheet 11 of 14 US 2019 /0142882 A1 FIG . 110 FIG . 11D Patent Application Publication May 16 , 2019 Sheet 12 of 14 US 2019 /0142882 A1 TB , filtered only : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : . iiiiiiiiiiiii iiiii Intensity/OD600 2000 . 1000 FIG . 12A TB , filtered + RNase A / T1 * Proteinase K 1500 FIG . 12B Patent Application Publication May 16 , 2019 Sheet 13 of 14 US 2019 /0142882 A1 2XYT, filtered only . 6000 . ' . ' . ' . ' . ' . ' . ' . ' . ' . ' . ' . ' . ' . FIG . 120 2XYT, filtered + RNase A / T1 * Proteinase K Intensity/OD600 5000 2000 FIG . 12D Patent Application Publication May 16 , 2019 Sheet 14 of 14 US 2019 /0142882 A1 TB vs 2XYT . ' 2 www FIG . 12E US 2019 /0142882 A1 May 16 , 2019 MICROBIAL PRODUCTION OF PURE tary ssDNA staple strands to self -assemble under appropri SINGLE STRANDED NUCLEIC ACIDS ate folding conditions into user- defined geometries of 1D , 2D , or 3D shapes (Veneziano , R et al ., Science , 352 , 1534 CROSS -REFERENCE TO RELATED ( 2016 ) ; Benson , E et al. , Nature , 523 , 441- 444 ( 2015 ) ; APPLICATIONS Douglas , S . M . et al ., Nucleic Acids Res , 37 , 5001- 5006 (2009 ) ) , and was the subject of prior work , demonstrating [0001 ] This application claims the benefit of and priority generality of sequence design for scaffold DNA (see , for to U . S . Application No . 62 /584 ,664 filed Nov. 10 , 2017 , and example , US20030215914A1, US20050147962A1, which is incorporated by reference in its entirety . WO2017089567A1, WO2017089570A1, and STATEMENT REGARDING FEDERALLY CN106119269A ) . SPONSORED RESEARCH 10006 ] One tile - based method allowed for generation of 2D wireframe objects (Yan , H et al. , Science, 301, 1882 [0002 ] This invention was made with government support 1884 ( 2003 ) ) that was subsequently implemented experi under Grant Nos. N00014 - 16 - 1 - 2953 ; and N00014 - 17 - 1 mentally using M13 - based scaffolded DNA origami to a 2609 awarded by the Office of Naval Research . The gov include diversity of 2D and closed 3D shapes (Zhang , et al ., ernment has certain rights in the invention . Nat Nanotechnol. , 10 ( 9 ) : 779 -84 ( 2015 ) ) . This latter scaf folded DNA origami approach was subsequently general REFERENCE TO SEQUENCE LISTING ized and fully automated for 3D shapes by Veneziano et al. , 10003 ) The Sequence Listing submitted as a text file (Venziano , et al. , Science, 352 (6293 ) : 1534 (2016 ) ) . named “ MIT _ 19562 _ ST25 .txt , ” created on Nov . 9 , 2018 , [0007 ] Non - scaffolded DNA origami is an alternative and having a size of 73 ,832 bytes is hereby incorporated by approach that uses purely short strands of synthetic single reference pursuant to 37 C . F . R . § 1 . 52 ( e ) ( 5 ) . stranded DNA to self -assemble via thermally annealed fold ing large - scale arrays of structured DNA via a process FIELD OF INVENTION known as ' tile - based ' assembly ( Yan , H et al. , Science , 301 , 1882 - 1884 ( 2003 ) ; Winfree , E et al. , Nature , 394 , 539 -544 [0004 ] The present invention relates generally to compo (1998 ) ; Ke , Y et al. , Science , 338 , 1177 - 1183 ( 2012 ) ; Ke, Y sitions and methods of bacterial production of pure , homo et al ., Nat Chem , 6 , 994 - 1002 ( 2014 ) ) . In vivo production of geneous single - stranded nucleic acid products composed top -down designed nanoparticles has long been one goal of nearly completely of non -natural nucleotide