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US 20120058468A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0058468 A1 MickeoWn (43) Pub. Date: Mar. 8, 2012 (54) ADAPTORS FOR NUCLECACID Related U.S. Application Data CONSTRUCTS IN TRANSMEMBRANE (60) Provisional application No. 61/148.737, filed on Jan. SEQUENCING 30, 2009. Publication Classification (75) Inventor: Brian Mckeown, Oxon (GB) (51) Int. Cl. CI2O I/68 (2006.01) (73) Assignee: OXFORD NANOPORE C7H 2L/00 (2006.01) TECHNOLGIES LIMITED, (52) U.S. Cl. ......................................... 435/6.1:536/23.1 Oxford (GB) (57) ABSTRACT (21) Appl. No.: 13/147,159 The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such (22) PCT Fled: Jan. 29, 2010 constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (86) PCT NO.: PCT/GB1O/OO160 template. The invention also relates to the constructs gener ated using the adaptors, methods of making the adaptors and S371 (c)(1), constructs, as well as methods of sequencing double stranded (2), (4) Date: Nov. 15, 2011 nucleic acids. Patent Application Publication Mar. 8, 2012 Sheet 1 of 4 US 2012/0058468 A1 Figure 5' 3 Figure 2 Figare 3 Patent Application Publication Mar. 8, 2012 Sheet 2 of 4 US 2012/0058468 A1 Figure 4 End repair Acid adapters ligate adapters Patent Application Publication Mar. 8, 2012 Sheet 3 of 4 US 2012/0058468 A1 Figure 5 Wash away type ifype foducts irrirrosilise Type| capture WashRE products away unbcure Pace É: Wash away unbound rag terts Free told fasgirre?ts aid raiser to festible Figure 6 Beatre Patent Application Publication Mar. 8, 2012 Sheet 4 of 4 US 2012/0058468 A1 Figare 7 3' "Geronic Sequerce' Cornia. "Generic Sequence' Sequence stat Type typeetife Sequence end (Hype femnant) (type femnant) US 2012/0058468 A1 Mar. 8, 2012 ADAPTORS FOR NUCLECACID chastic sensing has the potential to provide rapid and cheap CONSTRUCTS IN TRANSMEMBRANE DNA sequencing by reducing the quantity of nucleotide and SEQUENCING reagents required. FIELD OF THE INVENTION SUMMARY OF THE INVENTION 0001. The invention relates to adaptors for sequencing 0005. The inventor(s) have surprisingly demonstrated that nucleic acids. The adaptors may be used to generate single artificial, identifiable adaptors may be used to generate single Stranded constructs of nucleic acid for sequencing purposes. Stranded nucleic acid constructs that contain both strands of a Such constructs may contain both strands from a double double stranded nucleic acid template. The two strands of the stranded deoxyribonucleic acid (DNA) or ribonucleic acid template are covalently linked and delineated (divided) by an (RNA) template. The invention also relates to the constructs adaptor. The adaptor not only allows the transition point from generated using the adaptors, methods of making the adaptors one strand to the other strand to be identified, but also allows and constructs, as well as methods of sequencing double the construct to be purified before it is sequenced. The adaptor Stranded nucleic acids. may further allow the construct to be differentiated from similar constructs in which the strands have a different BACKGROUND OF THE INVENTION Source. Hence, the adaptors allow multiplex sequence analy sis oftemplates originating from separate individual sources. 0002 Stochastic detection is an approach to sensing that 0006. The adaptors are particularly useful for sequencing relies on the observation of individual binding events between double stranded DNA (dsDNA) and double stranded RNA analyte molecules and a receptor. Stochastic sensors can be (dsRNA). The adaptors may be used to generate single created by placing a single pore of nanometer dimensions in Stranded constructs containing both the sense and antisense an insulating membrane and measuring Voltage-driven ionic Strands of the dsDNA or dsRNA. transport through the pore in the presence of analyte mol 0007. The adaptors are generally used in pairs. Both types ecules. The frequency of occurrence of fluctuations in the of adaptor in the pair not only comprise a region of double current reveals the concentration of an analyte that binds Stranded nucleic acid that forms one half of a palindromic within the pore. The identity of an analyte is revealed through cleavage site, but also are differentially selectable from one its distinctive current signature, notably the duration and another. Each pair comprises two types of adaptor, Type I and extent of current block (Braha, O., Walker, B., Cheley, S., Type II. Type I adaptors comprise a hairpin loop, which Kasianowicz, J. J., Song, L., Gouaux, J. E., and Bayley, H. allows covalent linkage of the two strands in a double (1997) Chem. Biol. 4, 497-505; and Bayley, H., and Cremer, Stranded nucleic acid template. Type II adaptors may com P. S. (2001) Nature 413, 226-230). prise a hairpin loop, but do not have to. This combination of 0003 Engineered versions of the bacterial pore forming features allows the generation and purification of single toxin C.-hemolysin (C-HL) have been used for stochastic stranded constructs in which both strands of a double sensing of many classes of molecules (Bayley, H., and Cre Stranded nucleic acid template are covalently linked via a mer, P. S. (2001) Nature 413, 226-230; Shin, S., H., Luchian, Type I adaptor. Unwanted constructs formed by ligation of T., Cheley, S., Braha, O., and Bayley, H. (2002) Angew. Chem. adaptors with each other may be eliminated from the reaction Int. Ed. 41, 3707-3709; and Guan, X. Gu, L-Q., Cheley, S., mixture using the palindromic cleavage site. Similarly, con Braha, O., and Bayley, H. (2005) ChemBioChem 6, 1875 structs containing one or other of the two types of adaptor 1881). In the course of these studies, it was found that attempts to engineer C.-HL to bind Small organic analytes may be isolated from the reaction mixture using the adaptor's directly can prove taxing, with rare examples of Success differential selectability. (Guan, X. Gu, L.-Q., Cheley, S., Braha, O., and Bayley, H. 0008 Accordingly, the invention provides an adaptor for (2005) ChemBioChem 6, 1875-1881). Fortunately, a different sequencing nucleic acids, which comprises a region of double strategy was discovered, which utilised non-covalently Stranded nucleic acid, wherein at least one end of the region attached molecular adaptors, notably cyclodextrins (Gu, forms one half of a palindromic cleavage site and wherein the L.-Q. Braha, O.. Conlan, S., Cheley, S., and Bayley, H. adaptor is differentially selectable from another adaptor. In (1999) Nature 398, 686-690), but also cyclic peptides some embodiments, the region is formed by hybridization (Sanchez-Quesada, J., Ghadiri, M. R., Bayley, H., and Braha, between two separated regions of a single stranded nucleic O. (2000).J. Am. Chem. Soc. 122, 11758-11766) and cucur acid and the adaptor comprises a hairpin loop. biturils (Braha, O. Webb, J., Gu, L.-Q. Kim, K., and Bayley, 0009 The invention also provides: H. (2005) ChemPhysChem 6, 889-892). Cyclodextrins 0.010 a pair of adaptors comprising an adaptor of the become transiently lodged in the C-HL pore and produce a invention formed by hybridization between two sepa Substantial but incomplete channel block. Organic analytes, rated regions of a single stranded nucleic acid and com which bind within the hydrophobic interiors of cyclodextrins, prising a hairpin loop (Type I) and an adaptor of the augment this block allowing analyte detection (Gu, L.-Q., invention (Type II), wherein each type of adaptor in the Braha, O., Conlan, S., Cheley, S., and Bayley, H. (1999) pair is differentially selectable from the other type and Nature 398,686-690). wherein a complete palindromic cleavage site is formed 0004. There is currently a need for rapid and cheap DNA if any combination of the two types of adaptor are ligated or RNA sequencing technologies across a wide range of to one another; applications. Existing technologies are slow and expensive 0011 a kit comprising at least two populations of adap mainly because they rely on amplification techniques to pro tors of the invention, wherein every adaptor in each duce large Volumes of nucleic acid and require a high quantity population comprises a nucleic acid sequence that is of specialist fluorescent chemicals for signal detection. Sto specific for the population; US 2012/0058468 A1 Mar. 8, 2012 0012 a nucleic acid construct for use as a sequencing strands of nucleic acid under conditions which allow template comprising a double stranded nucleic acid ligation between the adaptors and strands; and ligated to at least one adaptor of the invention; 0030 (b) allowing an adaptor to covalently link the 0013 a single stranded nucleic acid construct for use as two strands at each end and thereby preparing a cir a sequencing template comprising two strands of nucleic cular nucleic acid construct; acid covalently linked via an adaptor of the invention 0031 a method for preparing a sequence construct, formed by hybridization between two separated regions comprising: of a single stranded nucleic acid and comprising a hair 0032 (a) providing double stranded nucleic acid; pin loop; 0033 (b) contacting the double stranded nucleic acid 0014 a circular nucleic acid construct for use as a with a pair of adaptors of the invention in which the sequencing template comprising two strands of nucleic Type I adaptors are not capable of being cleaved or acid covalently
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  • Bul1, a New Protein That Binds to the Rsp5 Ubiquitin Ligase in Saccharomyces Cerevisiae

    Bul1, a New Protein That Binds to the Rsp5 Ubiquitin Ligase in Saccharomyces Cerevisiae

    MOLECULAR AND CELLULAR BIOLOGY, July 1996, p. 3255–3263 Vol. 16, No. 7 0270-7306/96/$04.0010 Copyright q 1996, American Society for Microbiology Bul1, a New Protein That Binds to the Rsp5 Ubiquitin Ligase in Saccharomyces cerevisiae HIDEKI YASHIRODA, TOMOKO OGUCHI, YUKO YASUDA, AKIO TOH-E, AND YOSHIKO KIKUCHI* Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received 23 October 1995/Returned for modification 27 November 1995/Accepted 26 March 1996 We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt2 phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5.