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(19) TZZ__T (11) EP 1 957 644 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/10 (2006.01) C12Q 1/68 (2006.01) 01.12.2010 Bulletin 2010/48 C40B 40/06 (2006.01) C40B 50/06 (2006.01) (21) Application number: 06818144.5 (86) International application number: PCT/DK2006/000685 (22) Date of filing: 01.12.2006 (87) International publication number: WO 2007/062664 (07.06.2007 Gazette 2007/23) (54) ENZYMATIC ENCODING METHODS FOR EFFICIENT SYNTHESIS OF LARGE LIBRARIES ENZYMVERMITTELNDE KODIERUNGSMETHODEN FÜR EINE EFFIZIENTE SYNTHESE VON GROSSEN BIBLIOTHEKEN PROCEDES DE CODAGE ENZYMATIQUE DESTINES A LA SYNTHESE EFFICACE DE BIBLIOTHEQUES IMPORTANTES (84) Designated Contracting States: • GOLDBECH, Anne AT BE BG CH CY CZ DE DK EE ES FI FR GB GR DK-2200 Copenhagen N (DK) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • DE LEON, Daen SK TR DK-2300 Copenhagen S (DK) Designated Extension States: • KALDOR, Ditte Kievsmose AL BA HR MK RS DK-2880 Bagsvaerd (DK) • SLØK, Frank Abilgaard (30) Priority: 01.12.2005 DK 200501704 DK-3450 Allerød (DK) 02.12.2005 US 741490 P • HUSEMOEN, Birgitte Nystrup DK-2500 Valby (DK) (43) Date of publication of application: • DOLBERG, Johannes 20.08.2008 Bulletin 2008/34 DK-1674 Copenhagen V (DK) • JENSEN, Kim Birkebæk (73) Proprietor: Nuevolution A/S DK-2610 Rødovre (DK) 2100 Copenhagen 0 (DK) • PETERSEN, Lene DK-2100 Copenhagen Ø (DK) (72) Inventors: • NØRREGAARD-MADSEN, Mads • FRANCH, Thomas DK-3460 Birkerød (DK) DK-3070 Snekkersten (DK) • GODSKESEN, Michael Anders • LUNDORF, Mikkel Dybro DK-2950 Vedbæk (DK) DK-4000 Roskilde (DK) • GLAD, Sanne Schrøder • JAKOBSEN, Søren DK-2750 Ballerup (DK) DK-2000 Frederiksberg (DK) • NEVE, Søren • OLSEN, Eva Kampmann DK-2800 Lyngby (DK) DK-2730 Herlev (DK) • THISTED, Thomas • ANDERSEN, Anne Lee DK-3600 Frederikssund (DK) DK-4100 Ringsted (DK) • KRONBORG, Tine Titilola Akinleminu • HOLTMANN, Anette DK-3500 Værløse (DK) DK-2750 Ballerup (DK) • SAMS, Christian, • HANSEN, Anders Holm DK-3500 Værløse (DK) DK-2400 Copenhagen NV (DK) • FELDING, Jakob • SØRENSEN, Anders Malling DK-2920 Charlottenlund (DK) DK-2860 Søborg (DK) • FRESKGAARD, Per-Ola S-603 79 Norrköping (SE) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 957 644 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 1 957 644 B1 • GOULIAEV, Alex Haahr • HALPIN DAVID R ET AL: "DNA display I. 3670 Veksoe Sjaelland (DK) Sequence-encoded routing of DNA populations." • PEDERSEN, Henrik PLOS BIOLOGY JUL 2004, vol. 2, no. 7, July 2004 2880 Bagsvaerd (DK) (2004-07), page E173, XP002436101 ISSN: 1545-7885 cited in the application (74) Representative: Høiberg A/S • HALPIN DAVID R ET AL: "DNA display II. Genetic St. Kongensgade 59 A manipulation of combinatorial chemistry 1264 Copenhagen K (DK) libraries for small-molecule evolution" PLOS BIOLOGY, XX, XX, vol. 2, no. 7, July 2004 (56) References cited: (2004-07), pages 1022-1030, XP002424416 ISSN: WO-A-00/23458 WO-A-93/20242 1544-9173 cited in the application WO-A-2004/039825 WO-A-2005/058479 WO-A-2006/053571 2 EP 1 957 644 B1 Description Technical Field of the Invention 5 [0001] The present invention relates to bifunctional complexes and methods for synthesising such complexes, as well as to methods for split-and-mix synthesis of different molecules each linked to a single stranded identifier oligonucleotide comprising a plurality of tags identifying the molecule and/or the chemical entities having participated in the synthesis of the molecule. The invention also relates to a method for generating a library of different bifunctional complexes and methods for selecting molecules and/or identifying molecules having a desirable property, such as affinity for a target 10 compound. Background of the Invention [0002] Synthesis methods known as split-and-mix, or split-and-recombine, are known and have been used for the 15 synthesis of different molecules. Split-and-mix methods for synthesising polypeptides and other biochemical polymers have been disclosed e.g. in US 5,723,598 directed to the generation of a library of bifunctional complexes comprising a polypeptide and an identifier oligonucleotide comprising tags in the form of a sequence of nucleotides identifying the amino acids which participated in the formation of the polypeptide. The methods are directed to chemical linkage of tags and do not disclose enzymatic linkage, such as ligation, of the nucleotide tags making up the identifier oligonucleotide. 20 [0003] WO 00/23458 discloses a split-and-mix method in which nucleic acid tags are involved in both molecule syn- thesis and molecule identification. [0004] WO 2004/039825 and WO 2005/058479 disclose split-and-mix methods wherein tags in the form of identifier oligonucleotides are linked enzymatically. The prior art methods do not disclose ligation of a double-stranded oligonu- cleotide substrate comprising a plurality of tags and complementary anti-tags at least partly hybridised to each other, 25 wherein said ligation results in the formation of an identifier oligonucleotide comprising a plurality of consecutive nucle- otides in the form of covalently linked tags, whereas the anti-tags of the double-stranded oligonucleotide substrate are not affected by the action of the ligase, i.e. no anti-tags become covalently linked as a result of the enzymatic ligation of the tag part of the double-stranded oligonucleotide substrate. [0005] Reference is also made to WO2006/053571 disclosing methods for molecule synthesis. 30 Summary of the Invention [0006] There is a need for improved methods for split-and-mix synthesis of libraries of small molecules for e.g. phar- maceutical and other purposes. The small molecules can initially be synthesised as part of a bifunctional complex further 35 comprising an identifier oligonucleotide identifying the reactants which have participated in the synthesis of the small molecule. [0007] The methods of the present invention employs a ligation step wherein the substrate for the ligase is in a double stranded form and wherein the substrate comprises a plurality of tags and at least one or more anti-tags wherein tags and anti-tag(s) is/are at least partly hybridised to each other. The tags are covalently linked as a result of the action of 40 an enzyme comprising a ligase activity on the double stranded substrate, but no anti-tags are covalently linked as a result of said ligase action. [0008] The method facilitates separation of ligated tags and discrete, non-ligated anti-tags because of the size and molecular weight difference between (i) the afore-mentioned single stranded identifier oligonucleotide comprising a plurality of covalently linked tags and (ii) discrete, non-ligated anti-tags. The identifier oligonucleotide comprising the 45 ligated,tags will typically have a length at least about 3 times the length of the individual anti-tags. [0009] In one embodiment, the tags comprise a 5’ phosphate, or a variant ligatable reactive group, whereas anti-tags do not. Therefore, the growing bifunctional complex will comprise a covalently linked "top"- strand to which a number, such as one or more, of shorter and non-ligated anti-tag(s) is/are hybridised/annealed. This enables removal of anti-tag (s), e.g. after all tag additions have been performed (Fig. 1) or after each tag addition has been performed (Fig. 6). 50 [0010] Removal of anti-tags generally increases fidelity and allows extension after purification of the single-stranded oligonucleotide identifier comprising a plurality of ligated tags. Said extension makes possible the use of selection- specific sequences, which improves robustness towards contamination, which is a well-known phenomena when e.g. PCR amplifications are performed. Also, diversification-sequences can be used which diversifies otherwise un-distin- guishable tag combinations. This makes it possible to identify tag combination sequences which may arise during PCR 55 from a single tag combination. It is advantageous to identify such sequences as they may otherwise be interpreted as arising from several molecular entities containing the same tag combination, thus indicating that the specific tag com- bination corresponds to a small molecule with relatively high ability to be retained during subsequent selection procedures. [0011] Also, because of the design of the tags, cross-hybridization between single stranded tags can be reduced or 3 EP 1 957 644 B1 essentially eliminated. This greatly improves the purification process of the bifunctional complexes comprising single stranded identifier oligonucleotides following the synthesis reactions. One problem associated with purification of bi- functional complexes comprising double stranded identifier oligonucleotides is that such identifiers are prone to illegiti- mate hybridization when renaturing conditions are resumed following a purification process under denaturing conditions. 5 [0012] In order to achieve a minimum degree of cross-hybridization between tags in an identifier oligonucleotide, the tags can be designed using a computer algorithm to maximize or optimize the number of mismatches between any oligo pair, e.g. resulting in a minimum of seven mismatches between any two tag tags. This maximizes or optimize fidelity of hybridization and enables use of sorting methods, such as the ones disclosed e.g. in WO 00/23458 (Harbury). Moreover, it increases robustness of decoding by sequencing, e.g. the tag information can be decoded even if sequencing errors 10 occur. [0013] It is also possible to perform a quenching reaction with the reactants and tags.
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  • (Title of the Thesis)*

    (Title of the Thesis)*

    Dionysian Semiotics: Myco-Dendrolatry and Other Shamanic Motifs in the Myths and Rituals of the Phrygian Mother by Daniel Attrell A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Arts in Ancient Mediterranean Cultures Waterloo, Ontario, Canada, 2013 © Daniel Attrell 2013 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The administration of initiation rites by an ecstatic specialist, now known to western scholarship by the general designation of ‗shaman‘, has proven to be one of humanity‘s oldest, most widespread, and continuous magico-religious traditions. At the heart of their initiatory rituals lay an ordeal – a metaphysical journey - almost ubiquitously brought on by the effects of a life-changing hallucinogenic drug experience. To guide their initiates, these shaman worked with a repertoire of locally acquired instruments, costumes, dances, and ecstasy-inducing substances. Among past Mediterranean cultures, Semitic and Indo-European, these sorts of initiation rites were vital to society‘s spiritual well-being. It was, however, the mystery schools of antiquity – organizations founded upon conserving the secrets of plant-lore, astrology, theurgy and mystical philosophy – which satisfied the role of the shaman in Greco-Roman society. The rites they delivered to the common individual were a form of ritualized ecstasy and they provided an orderly context for religiously-oriented intoxication.
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD

    Supplementary Table S4. FGA Co-Expressed Gene List in LUAD

    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase