(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/077763 Al 19 May 2016 (19.05.2016) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, CUM 1/00 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/US20 15/060693 KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (22) International Filing Date: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, 13 November 2015 (13.1 1.2015) PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (25) Filing Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 62/079,183 13 November 2014 (13. 11.2014) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (71) Applicant: THE BOARD OF TRUSTEES OF THE TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, UNIVERSITY OF ILLINOIS [US/US]; 352 Henry A d DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, ministration Building, MC-350, 506 S. Wright St., Urbana, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, IL 61801 (US). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). (72) Inventors: HA, Taekjip; 3806 Saint Paul St, Baltimore, MD 21218 (US). ARSLAN, Sinan; 506 East Michigan Published: Avenue Apartment 14, Urbana, 61801 (US). — with international search report (Art. 21(3)) (74) Agents: VELEMA, James, H. et al; Lathrop & Gage — before the expiration of the time limit for amending the LLP, 28 State Street, Boston, MA 02109 (US). claims and to be republished in the event of receipt of (81) Designated States (unless otherwise indicated, for every amendments (Rule 48.2(h)) kind of national protection available): AE, AG, AL, AM, (54) Title: BIO-ENGINEERED HYPER-FUNCTIONAL "SUPER" HELICASES © (57) Abstract: Conformationally- constrained helicases having improved activity and strength are provided. Methods of making con- v formationally-constrained helicases having improved activity and strength are provided. Methods of using confbrmationally-con- o strained helicases having improved activity and strength are provided. The present invention is based on the discovery of novel mod ified helicases that show dramatically enhanced helicase activity and increased strength as compared to unmodified helicases. As de - scribed further herein, it has been surprisingly discovered that, by controlling the conformation of certain subdomains such that the o helicase remains in a closed form (e.g., by covalently crosslinking the 2B domain to the 1A domain or the IB domain in a Rep hel - icase), a highly active and strong form of the helicase is achieved BIO-ENGINEERED HYPER-FUNCTIONAL "SUPER" HELICASES RELATED APPLICATIONS [001] This application claims the benefit of U.S. Provisional Application No. 62/079,183, filed November 13, 2014, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [002] This invention was made with government support under GM065367 awarded by the National Institutes of Health. The United States Governm ent has certain rights in the invention. SEQUENCE LISTING [002.1] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION [003] The present disclosure relates to compositions and methods for helicase-mediated DNA unwinding activity. BACKGROUND [004] A traditional definition of a helicase is an enzyme that catalyzes the reaction of sepa ating/unzipping'unwinding the helical structure of nucleic acid duplexes (DNA, RNA or hybrids) into single-stranded components, using nucleoside triphosphate (NTP) hydrolysis as the energy source (such as ATP). However, i should be noted that not al helicases fit this definition anymore. A more general definition is that they are motor proteins that move along the single-stranded or double stranded nucleic acids (usually in a certain direction, 3' to 5' or 5 to 3, or both), i.e. translocases, that can or cannot unwind the duplexed nucleic acid encountered. In addition, some helicases simply bind and "melt" the duplexed nucleic acid structure without an apparent translocase activity. [005] Helicases exist i all living organisms and function i all aspects of nucleic acid metabolism. Helicases are classified based on the amino acid sequences, directionality, oligomerization state and nucleic-acid type and structure preferences. The most common classification method was developed based on the presence of certain amino acid sequences, called motifs. According to this classification helicases are dividai into 6 super families: SFl, SF2, SF3, SF4, SF5 and SF6. SFl and SF2 helicasesdo not form a ring structure around the nucleic acid, whereas SF3 to SF6 do. Superfamily classification is not dependent on the classical taxonomy. [006] DNA helicases are responsible for catalyzing the unwinding of double-stranded DNA (dsDNA) molecules to their respective single-stranded nucleic acid (ssDNA) forms. Although structural and biochemical studies have show how various helicases can translocate on ssDNA directionally, consuming one ATP per nucleotide, the mechanism of nucleic acid unwinding and how the unwinding activity is regulated remains unclear and controversial (T.M. Lohman, E.J. Tomko, C.G. Wu, "Non- hexameric DNA helicases and translocases: mechanisms and regulation," NatRevMol CellBiol 9:391- 40 (2008)). Since helicases can potentially unwind all nucleic acids encountered, understanding how their unwinding activities are regulated can lead to harnessing helicase functions for biotechnology applications. BRIEF SUMMARY O F THE INVENTION [007] The present invention is based on the discover}' of novel modified helicases that show dramatically enhanced helicase activity and increased strength as compared to unmodified helicases. As described t ither herein, i has been surprisingly discovered that, by controlling the conformation of certain subdomains such that the helicase remains i a closed form (e.g., by covalently crosslinking the 2B domain to the 1A domain or the B domain in a Rep helicase), a highly active and strong form of the helicase is achieved. 008 In one aspect, a composition for catalyzing a unwinding reaction o double-stranded DNA is provided that includes a conformationally-constrained helicase. [009] In another aspect, a method of catalyzing an unwinding reaction of a double-stranded DNA is provided. The method includes the step of contacting the double-stranded DNA with a conformationally-constrained helicase in the presence of ATP. [0010] In another aspect, an isolated nucleic acid that encodes a helicase polypeptide having the capability to be constrained in a confonnatioii by an intramolecular crosslinking agent is provided. [0011] In another aspect, a modified helicase comprising a first subdomain having a first amino acid and a second subdomain having a second amino acid is provided. Said first amino acid is at least about 30 A from said second amino acid when the helicase is in an inactive conformation, and said first amino acid is less than about 20 A from said second amino acid when the helicase is i an active conformation. A side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conform ati onal 1y-con strai ned helicase. [0012] In certain exemplary embodiments, the modified helicase is a Super Family 1 (SF 1) helicase (e.g., an SF 1A or an SF 1B helicase) or a Super Family 2 (SF2) helicase. [0013] In certain exemplary embodiments, the first amino acid is less than about 20 A, about 19 A, about 18 A, about 7 A, about 6 A, about 5 A, about 0 A, about 9 A, about 8 A, about 7 A, about 5 A, or about 4 A from the second amino acid when the helicase is in an active conformation. [0014] In certain exemplary embodiments, the first amino acid is at least about 30 A, about 40 A, about 50 A, about 55 A, about 60 A, about 65 A, about 70 A, about 75 A, about 80 A or about 85 A from the second amino acid when the helicase is in a inactive conformation. [0015] In certain exemplary embodiments, the helicase is selected from the group consisting of a Rep helicase (e.g., from / · . coll.), a LJvrD helicase (e.g., from E. coli.) and a PcrA helicase (e.g., from . stearothermophihis). [0016] In certain exemplary embodiments, the first amino acid is at any one of positions 84- 1 6 or 78- 1 6 of the modified helicase amino acid sequence, and the helicase is a Rep, PcrA or UvrD helicase, or homolog thereof. [0017] In certain exemplary embodiments, the first amino acid is at any one of positions 92- 116 or 178-196 of the modified helicase amino acid sequence, and the helicase is a Pcr A helicase, or homolog thereof [0018] In certain exemplary embodiments, the first amino acid is at any one of positions 84- 108 or 169-1 7 of the modified helicase a ino acid sequence, and the helicase is a Rep helicase, or homolog thereof [00 ] certain exemplary embodiments, the first amino acid is at any one of positions 90- 114 or 175-193 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof.
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