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) (51) International Patent Classification: (81) Designated ) ( (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/68 (2018.01) kind of national protection av ailable) . AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, PCT/GB20 18/052170 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (22) International Filing Date: HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, 30 July 2018 (30.07.2018) KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (26) Publication Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant: OXFORD UNIVERSITY INNOVATION LIMITED [GB/GB]; Buxton Court, 3 West Way, Oxford (84) Designated States (unless otherwise indicated, for every OX2 0JB (GB). kind of regional protection available) . ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (72) Inventors: BAYLEY, Hagan; Chemistry Research Lab¬ UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, oratory, University of Oxford, 12 Mansfield Road, Ox¬ TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, ford Oxfordshire OX1 3TA (GB). SPRUIJT, Evan; Insti¬ EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, tute of Molecules and Materials, Radboud University, Ni¬ MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, jmegen (NL). HENNING-KNECHTEL, Anja; c/o Chem¬ TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, istry Research Laboratory, University of Oxford, 12 Mans¬ KM, ML, MR, NE, SN, TD, TG). field Road, Oxford Oxfordshire OX1 3TA (GB). (74) Agent: SILCOCK, Peter James et al.; J A Kemp LLP, 14 Declarations under Rule 4.17: South Square, Gray's Inn, London, Greater London WC1R — as to non-prejudicial disclosures or exceptions to lack of 5JJ (GB). novelty (Rule 4.17(v)) (54) Title: ASSEMBLIES (57) Abstract: The invention provides an assembly comprising a nucleic acid scaffold and a plurality of appendages bonded to the scaffold, wherein the appendages, when bonded to the scaffold, are capable of interacting to form a pore in a layer of amphipathic molecules. [Continued on next page] ||| ||||| ||||| ||||| |||| 11| ||| ||||| ||||| ||||| ||||| mil III III! III! llll Published: with international search report (Art. 21(3)) with sequence listing part of description (Rule 5.2(a)) ASSEMBLIES REFERENCE TO SEQUENCE LISTING This application contains a Sequence Listing submitted as an electronic text file named “Sequence Listing N4141 11WO PJS EGH.txt”, having a size in bytes of 40,61 1 bytes, and created on July 18, 2018. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR §1.52(e)(5). FIELD OF THE INVENTION The invention relates to pore-forming assemblies. More particularly, an assembly wherein the formation of a membrane-spanning pore is promoted by a scaffold that pre-organises the structure. The invention also relates to methods of forming a pore, methods of detecting and/or characterising a target analyte and methods of sequencing using the assemblies of the invention. BACKGROUND TO THE INVENTION A pore may be characterised as an opening in or channel through a membrane. Typically, a membrane comprises a hydrophobic layer of amphipathic molecules and the pore provides a hydrophilic channel through the membrane. Thus, pores comprise amphipathic structures with hydrophobic surfaces that interact with the membrane and hydrophilic surfaces that form a hydrophilic opening in the membrane. Pores may have a wide range of channel diameters through which ions and certain molecules may pass. Many pores have a channel diameter in the nanometre range and are typically known as “nanopores”. In recent years, pores and particularly nanopores have become established as versatile tools for a wide range of nanotechnological applications (1-7HK). In particular, the use of nanopores as ultra-sensitive sensor elements for rapid and label-free analysis of single molecules has garnered considerable interest. The preparation of nanometre-scaled pores can be realised by perforating either (i) a semifluid membrane via membrane-spanning pores, or (ii) a thin solid-state film (e.g . silica, silicon nitrate or graphene) via, for instance, a focused ion or electron beam (8- 11HK). Although solid-state nanopores provide a higher mechanical and chemical stability, and are less sensitive to buffer conditions, the most commonly utilized platforms for biological applications are membrane-inserted protein pores (12HK). In biology, membrane-spanning protein channels and pores play important roles in regulating transport across cell membranes and in signalling and defence mechanisms (1-3). As they are versatile and amenable to engineering, such pores have found application in single- molecule biosensors and sequencing technologies (4, 5). This is largely due to the high reproducibility of the pore structure, as well as the atomically precise positioning of chemical groups through genetic engineering, both of which cannot be achieved so far using solid-state nanopore fabrication methods. Larger protein pores are mostly β-barrels, many of which can fold spontaneously or insert into the outer membrane of Gram-negative bacteria assisted by dedicated protein machinery (6-8). Recently, a number of pores that consist of α-helical peptides, which include the polysaccharide transporter Wza and cytolysin A (ClyA), have been discovered, but their assembly is still poorly understood (9-1 1). Such pores could help expand the utility of biological pores in biotechnology and synthetic biology. However, an important challenge for such applications that remains is to control the geometrical and chemical properties of the sensor channels. There remains a need in the art to provide improved means to orient and stabilise pore forming subunits, such as peptides or proteins. This is complicated by complex requirements for the structure and assembly of the subunits to form the pore. Any such means would need to dictate the size of the desired nanopore, but must be sufficiently flexible to avoid trapping the amphipathic appendages, such as peptides or proteins, in an unwanted conformation. Moreover, any such means must not obstruct the nanopore. It has recently been shown that cyclodextrins can be used to template peptide nanopores (12). These templates, however, cannot be used for large or heteromeric nanopores. In earlier work, four different peptide helices were tethered onto a flexible peptide backbone to form sodium channel analogues, but such peptide backbones may not be rigid enough to act as effective scaffolds for larger nanopores (13-15). Thus, there is great potential for applications of pores in biotechnology and synthetic biology. However, there remains a need in the art to provide improved means to assemble stable and functional nanopores from pore-forming subunits. SUMMARY OF THU INVENTION The present inventors have surprisingly found that assembly of appendages into a configuration for formation of a pore in a layer of amphipathic molecules can be promoted by using nucleic acid scaffolds to pre-organize the structures. The inventors have found that such nucleic acid scaffolds enable the construction of uniform pores of various sizes and pores with controlled permutations around a central axis. The assemblies described herein comprise nucleic acid nanostructures that can serve as scaffolds to arrange appendages, such as peptides or polypeptides, to form uniform pores in layers of amphipathic molecules. The inventors have found that when scaffolded, appendages that do not form pores in isolation may be induced to form pores. Similarly, the nucleic acid scaffold described herein can direct the appendages to form pores with much larger diameters than naturally occurring pores. The inventors have also found that scaffolded pores have enhanced stability and improved conductance properties. The inventors have also found that the assemblies described herein allow for precise control of the arrangement of monomers within heteromeric pores. Thus, the assemblies described herein allow for the formation of larger, more uniform pores with enhanced stability and a precisely controlled structure. The present invention provides an assembly comprising a nucleic acid scaffold and a plurality of appendages bonded to the scaffold, wherein the appendages, when bonded to the scaffold, are capable of interacting to form a pore in a layer of amphipathic molecules. The present invention also provides an assembly comprising a nucleic acid scaffold and a plurality of appendages, wherein the appendages are bonded to the scaffold in an arrangement enabling the appendages to form a pore in a layer of amphipathic molecules. The present invention further provides a system comprising: (a) a layer of amphipathic molecules; and (b) an assembly comprising a nucleic acid scaffold and a plurality of appendages bonded to the scaffold, wherein the appendages form a pore in the layer of amphipathic molecules. The invention also provides a method of forming a pore in a layer of amphipathic molecules, the method comprising applying an assembly to a layer of amphipathic molecules, which assembly comprises a nucleic acid scaffold and a plurality of appendages bonded to the scaffold, and thereby causing the appendages to interact to form a pore in the layer of amphipathic molecules. The present invention also provides a device comprising: (a) a layer of amphipathic molecules; (b) an assembly comprising a nucleic acid scaffold and a plurality of appendages bonded to the scaffold, wherein the appendages form a pore in the layer of amphipathic molecules; and (c) apparatus for measuring electrical current through the pore.
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