FIELD OF THE INVENTION

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 constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.

BACKGROUND OF THE INVENTION

Stochastic detection is an approach to sensing that relies on the observation of individual binding events between analyte molecules and a receptor. Stochastic sensors can be created by placing a single pore of nanometer dimensions in an insulating membrane and measuring voltage-driven ionic transport through the pore in the presence of analyte molecules. The frequency of occurrence of fluctuations in the current reveals the concentration of an analyte that binds within the pore. The identity of an analyte is revealed through its distinctive current signature, notably the duration and extent of current block (Braha, O., Walker, B., Cheley, S., Kasianowicz, J. J., Song, L., Gouaux, J. E., and Bayley, H. (1997)Chem. Biol.4, 497-505; and Bayley, H., and Cremer, P. S. (2001)Nature413, 226-230).

Engineered versions of the bacterial pore forming toxin a-hemolysin (a-HL) have been used for stochastic sensing of many classes of molecules (Bayley, H., and Cremer, P. S. (2001)Nature413, 226-230; Shin, S., H., Luchian, T., Cheley, S., Braha, O., and Bayley, H. (2002)Angew. Chem. Int. Ed.41, 3707-3709; and Guan, X., Gu, L.-Q., Cheley, S., Braha, O., and Bayley, H. (2005)ChemBioChem6, 1875-1881). In the course of these studies, it was found that attempts to engineer a-HL to bind small organic analytes directly can prove taxing, with rare examples of success (Guan, X., Gu, L.-Q., Cheley, S., Braha, O., and Bayley, H. (2005)ChemBioChem6, 1875-1881). Fortunately, a different strategy was discovered, which utilised non-covalently attached molecular adaptors, notably cyclodextrins (Gu, L.-Q., Braha, O., Conlan, S., Cheley, S., and Bayley, H. (1999)Nature398, 686-690), but also cyclic peptides (Sanchez-Quesada, J., Ghadiri, M. R., Bayley, H., and Braha, O. (2000)J Am. Chem. Soc.122, 11758-11766) and cucurbiturils (Braha, O., Webb, J., Gu, L.-Q., Kim, K., and Bayley, H. (2005)ChemPhysChem6, 889-892). Cyclodextrins become transiently lodged in the α-HL pore and produce a substantial but incomplete channel block. Organic analytes, which bind within the hydrophobic interiors of cyclodextrins, augment this block allowing analyte detection (Gu, L.-Q., Braha, O., Conlan, S., Cheley, S., and Bayley, H. (1999)Nature398, 686-690).

There is currently a need for rapid and cheap DNA or RNA sequencing technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of nucleic acid and require a high quantity of specialist fluorescent chemicals for signal detection. Stochastic sensing has the potential to provide rapid and cheap DNA sequencing by reducing the quantity of nucleotide and reagents required.

SUMMARY OF THE INVENTION

The inventor(s) have surprisingly demonstrated that artificial, identifiable adaptors may be used to generate single stranded nucleic acid constructs that contain both strands of a double stranded nucleic acid template. The two strands of the template are covalently linked and delineated (divided) by an adaptor. The adaptor not only allows the transition point from one strand to the other strand to be identified, but also allows the construct to be purified before it is sequenced. The adaptor may further allow the construct to be differentiated from similar constructs in which the strands have a different source. Hence, the adaptors allow multiplex sequence analysis of templates originating from separate individual sources.

The adaptors are particularly useful for sequencing double stranded DNA (dsDNA) and double stranded RNA (dsRNA). The adaptors may be used to generate single stranded constructs containing both the sense and antisense strands of the dsDNA or dsRNA.

The adaptors are generally used in pairs. Both types of adaptor in the pair not only comprise a region of double stranded nucleic acid that forms one half of a palindromic cleavage site, but also are differentially selectable from one another. Each pair comprises two types of adaptor; Type I and Type II. Type I adaptors comprise a hairpin loop, which allows covalent linkage of the two strands in a double stranded nucleic acid template. Type II adaptors may comprise a hairpin loop, but do not have to. This combination of features allows the generation and purification of single stranded constructs in which both strands of a double stranded nucleic acid template are covalently linked via a Type I adaptor. Unwanted constructs formed by ligation of adaptors with each other may be eliminated from the reaction mixture using the palindromic cleavage site. Similarly, constructs containing one or other of the two types of adaptor may be isolated from the reaction mixture using the adaptor's differential selectability.

Accordingly, the invention provides an adaptor for sequencing nucleic acids, which comprises a region of double stranded nucleic acid, wherein at least one end of the region forms one half of a palindromic cleavage site and wherein the adaptor is differentially selectable from another adaptor. In some embodiments, the region is formed by hybridization between two separated regions of a single stranded nucleic acid and the adaptor comprises a hairpin loop.

The invention also provides:a pair of adaptors comprising an adaptor of the invention formed by hybridization between two separated regions of a single stranded nucleic acid and comprising a hairpin loop (Type I) and an adaptor of the invention (Type II), wherein each type of adaptor in the pair is differentially selectable from the other type and wherein a complete palindromic cleavage site is formed if any combination of the two types of adaptor are ligated to one another;a kit comprising at least two populations of adaptors of the invention, wherein every adaptor in each population comprises a nucleic acid sequence that is specific for the population;a nucleic acid construct for use as a sequencing template comprising a double stranded nucleic acid ligated to at least one adaptor of the invention;a single stranded nucleic acid construct for use as a sequencing template comprising two strands of nucleic acid covalently linked via an adaptor of the invention formed by hybridization between two separated regions of a single stranded nucleic acid and comprising a hairpin loop;a circular nucleic acid construct for use as a sequencing template comprising two strands of nucleic acid covalently linked at each end via an adaptor of the invention formed by hybridization between two separated regions of a single stranded nucleic acid and comprising a hairpin loop;a method for preparing an adaptor of the invention, comprising:(a) providing two nucleic acids that are (i) capable of hybridizing to one another to form one half of a palindromic cleavage site and (ii) differentially selectable from those of another adaptor; and(b) contacting the nucleic acids under conditions which allow them to hybridise and thereby preparing an adaptor;a method for preparing an adaptor of the invention formed by hybridization between two separated regions of a single stranded nucleic acid and comprising a hairpin loop, comprising:(a) providing a single stranded nucleic acid comprising (i) two regions that are capable of hybridizing to one another, (ii) a loop-forming region that is differentially selectable from that of another adaptor and (iii) two ends which together form one half of a palindromic cleavage site; and(b) exposing the nucleic acid to conditions which allow the two regions to hybridise and form a hairpin loop and thereby preparing an adaptor;a method for preparing a nucleic acid construct of the invention, comprising:(a) contacting at least one adaptor of the invention with two strands of nucleic acid under conditions which allow ligation between the adaptor(s) and the strands; and(b) allowing the adaptor to ligate to the two strands and thereby preparing a nucleic acid construct;a method for preparing a single stranded nucleic acid construct of the invention, comprising:(a) contacting an adaptor of the invention formed by hybridization between two separated regions of a single stranded nucleic acid and comprising a hairpin loop with two strands of nucleic acid under conditions which allow ligation between the adaptor and the strands;(b) allowing the adaptor to covalently link the two strands; and(c) denaturing the covalently linked construct and thereby preparing a single stranded nucleic acid construct;a method for preparing a circular nucleic acid construct of the invention, comprising:(a) contacting at least two adaptors of the invention which comprise a hairpin loop with two strands of nucleic acid under conditions which allow ligation between the adaptors and strands; and(b) allowing an adaptor to covalently link the two strands at each end and thereby preparing a circular nucleic acid construct;a method for preparing a sequence construct, comprising:(a) providing double stranded nucleic acid;(b) contacting the double stranded nucleic acid with a pair of adaptors of the invention in which the Type I adaptors are not capable of being cleaved or nicked and the Type II adaptors are capable of being cleaved or nicked under conditions which allow the adaptors to ligate to the nucleic acid;(c) contacting the ligated products with a surface that specifically binds the Type II adaptors and removing any unbound products;(d) contacting the surface with an enzyme that recognises the complete palindromic cleavage site and removing any unbound products;(e) cleaving the Type II adaptors;(f) contacting the soluble products produced in step (e) with a surface that specifically binds the Type I adaptors and removing any unbound products; and(g) releasing from the surface the products remaining following step (f) and thereby producing a sequencing construct;a method of sequencing double stranded nucleic acid, comprising:(a) carrying out a method of the invention;(b) denaturing the construct, if necessary, to form a single stranded construct; and(c) sequencing the single stranded construct and thereby sequencing the double stranded nucleic acid; anda kit for sequencing double stranded nucleic acid comprising a pair of adaptors of the invention and means for cleaving the palindromic cleavage sites.

