WO2002061125A2

WO2002061125A2 - Immobilised aptamers

Info

Publication number: WO2002061125A2
Application number: PCT/GB2002/000391
Authority: WO
Inventors: Abdessamad Tahiri-Alaoui; William Syward James; Jamal Ibrahim; Laura Frigotto
Original assignee: Isis Innovation Limited
Priority date: 2001-01-29
Filing date: 2002-01-29
Publication date: 2002-08-08
Also published as: WO2002061125A3; WO2002061079A3; WO2002061079A2; AU2002226584A1; AU2002226571A1

Abstract

Immobilised aptamers find utility in assays.

Description

Immobilisation

The present invention relates to immobilisation.

BACKGROUND OF THE INVENTION

The post-genomic research environment inspires the search for ways to document the activity of the proteome in experimental and diagnostic samples. Although monoclonal antibodies have provided a rich source of specific ligands for detecting the location and activity of proteins, the number of new targets outstrips the capacity of the methodology for generating and screening them. Alternative approaches to new ligand discovery involve in vitro evolution of either nucleic acids or their encoded polypeptides by selection from highly complex libraries generated by combinatorial synthesis.

A number of factors will determine which of these approaches will gain widest acceptance, including: the ease of use, particularly in automated systems; the physicochemical range of targets to which ligands are possible; the complexity of the library that can be screened; the chemical and structural resilience of the ligand; and the availability of downstream technologies for detection, amplification and manipulation. Nucleic acid ligands, or aptamers, have the advantages that the methods for their generation are relatively straightforward and that it is possible to screen a starting library of at least 10¹⁴ different sequences (see references 1 ,2).

Aptamers are nucleic acid molecules which bind to specific target molecules. Aptamers have many advantages over antibodies as macromolecular ligands for target proteins. These advantages include small size, stability, exquisite conformational sensitivity, potential to be wholly chemically synthesised, as well as insensitivity to problems such as inter-specific sequence conservation and problems of antigen processing and presentation.

Aptamers have the possible disadvantage of a limited range of physicochemical properties; having no equivalents to the hydrophobic and basic residues of some amino acids (see reference 3). Polypeptide ligands have obvious advantages in this latter respect but phage display and similar systems for their discovery are hampered by a transfection-imposed bottleneck that limits library complexity to less than 10⁹ and ribosome display methods have proved too fragile for general use (see references 4,5).

A recent method, called mRNA display (see reference 6), has overcome most of these difficulties through a number of elegant innovations that enable approximately 10¹³ different, randomized, 88-mer polypeptides to be screened. Most recently, this approach produced peptide aptamers with 100-fold higher affinity for ATP than the best RNA aptamers (see reference 7) and with 1000-fold higher affinity for streptavidin than the best phage- display antibodies (see reference 8).

SUMMARY OF THE INVENTION

According to the present invention, we provide an immobilised aptamer. The immobilised aptamer is then available for binding to its target molecule.

Aptamers may be immobilised by direct and indirect, covalent and non- covalent means to a solid phase.

Direct immobilisation may be achieved through the introduction of, for example, sulphydryl or amino groups into the aptamer and subsequent reaction of the modified aptamer with maleimide or iodoacetamide- derivatised supports (for sulphydryls) or supports derivatised with acylating reagents or isothiocyanates (for amino groups). Sulphydryl and primary amino groups can be introduced into aptamers during and after their synthesis by in vitro transcription by a number of methods known to the art. However, they can be more conveniently introduced into aptamers synthesised by solid phase chemistry.

Thus, considering the situation in more detail, there are a number of commercially available ways in which nucleic acids may be attached to a support. They are well known in the art e.g. Sproat and Brown and can be outlined as below: An amine group may be attached to the end of the oligonucleotide. This may be reacted with a succinyl ester to provide a linkage. The linkage may be to any one of a number of surfaces, for example, but not limited to, resinous beads, glass, derivatised silica gel, etc. Alternatively, a thiol group may be used as the functional group. For example, a commercially available C6 thiol modifier may be used on all automated DNA synthesisers to directly incorporate a trityl protected thiol group at the 5' end of an oligonucleotide. The thiol group may subsequently be used to link the ligand to a solid suport. Linkers of different lengths may also be used. Thiol chemistry for the attachment of oligonucleotides is well known in the literature, and commercially supplied by companies such as: Cruachem Ltd. of Todd Campus, West of Scotland Science Park, Acre Road, Glasgow, G20 OUA Scotland, UK, or TriLink Biotechnologies Inc. of 6310 Nancy Ridge Drive Suite 101, San Diego, CA 92121. It is also possible to add a linker such as polyethylene glycol (PEG) between the oligonucleotide and the group that will link the ligand to the solid support. The PEG or other spacing linker molecule being used may be attached at one end to the solid support, and at the other to the ligand of choice for the application in question. Spacers of this kind are well know in the art of nucleic acid chemistry, and may be used to make the bound ligand more accessible and less affected by possible steric hinderance by other ligand moleulces where large numbers are bound on a soild surface. It is thus possible to utilise larger ligands than would otherwise be possible. This would be a particular advantage where, for example, large numbers of ligands were used, bound to a solid matrix for use in the removal of the cognate of the ligand froma solution for purification or for concentration purposes. In another embodiment, the nucleic acid ligands may be synthesised directly onto the solid support. Chemistry for this is well known in the art and is typified by the phosphonamidite chemistry used in automated DNA synthesisers. The oligonucleotide may be bound to the solid support through a terminal phosphate group by a covalent phosphodiester link of the structure -O-PO2-O-(CnH2n)-.

Non-covalent immobilisation can be achieved through the introduction of molecules such as biotin or digoxygenin which can be incorporated, by methods known in the art, during or after transcription in vitro. For example, modified nucleotides may be linked to the 3' end of an aptamer of the invention using terminal deoxynucleotidyl transferase (e.g. biotin- 16- ddUTP) or to the 5' end using RNA ligase. The modified aptamer may then be immobilized for example, by interaction with streptavidin on a solid support. The resulting immobilized apatamer may be used to bind its target molecule, such as a target prion protein.

Further to this aspect of the invention, we refer to our copending PCT application of even date entitled Streptavidin, incorporated herein by reference. In the Streptavidin application, we provide aptamers with specificity to and affinity for streptavidin. Such aptamers can provide a direct link to the downstream technology of one of the most frequently exploited detection systems, streptavidin - biotin. We refer to an aptamer for streptavidin as a streptamer.

Thus, we envisage the immobilisation of the streptamer, and any macromolecules, including other aptamers, to which it is linked covalently or non-covalently, to the solid phase in order to develop affinity purification, separation or linking of streptamers, and complexes containing streptamers, to molecules derivatised with biotin. Peptides, proteins, nucleic acids, lipids, carbohydrates and any molecule that can be derivatised to carry reactive amino or thiol groups, are routinely derivatised with biotin using methods known to the art. The ability of streptamers to link nucleic acid ligands to biotinylated molecules enables one to target virtually any conceivable molecule of analytical, enzymatic, toxic, therapeutic or other function of interest to the site of molecule to which one had a suitable ligand. Applications of this sort might include whole body imaging, cancer chemotherapy, targeted immunosuppression, and so on.

Suitable solid supports known to the art include plastics, resins, glass surfaces (including controlled-pore glass), sepharose and agarose. Immobilisation to solid supports permits one to exploit the specific binding of aptamers in applications such as affinity chromatography, product purification, biosensing, high throughput screening and so on. In this respect, we refer to the work with BIAcores in our copending application of even date entitled Biligands, incorporated herein by reference.

Examples of aptamers which may be employed in the present invention include those in our UK patent application 0012054.3 and PCT application WO0188123WO 0188123; the streptamers in our PCT patent application of the same filing date as the present patent application, and entitled Streptavidin; aptamers against HIV- 1 ; and those in our PCT patent application also of the same filing date as the present patent application, and entitled Biligands. Related examples are to be found in UK patent applications GB 0102270.6, Immobilisation; GB 0102271.4, Streptavidin; GB 0102272.2, Biligands. The above patent applications are incorporated herein by reference in their entirety.

More generally, the aptamers can be chosen from those which bind to particular target molecules. For instance, in one embodiment the apatamers of the invention bind to small molecules. In another embodiment, the aptamers of the invention bind to oligopolymers. In another embodiment, the aptamers of the invention bind to polymeric molecules. In another embodiment, the aptamers of the invention bind to cellular components, or to whole cells. Examples include antigenic molecules, toxins, prions and viruses.

In a particularly preferred embodiment, the immobilized aptamers of the invention bind to proteins, particularly to prion proteins. In particularly preferred embodiments, the immobilized aptamers of the invention that bind to a prion protein comprise a nucleic acid sequence or consensus sequence described in PCT application WO 0188123, which was incorporated by reference in its entirety above. For example, the aptamer can be as defined in any one of claims 1 to 8 of the incorporated PCT text, such as a sequence as in Figure 6 of the incorporated PCT text.

The immobilised aptamers of this invention find a variety of uses. For example an immobilised aptamer can be employed in a method of purification. Such a method can be to remove unwanted contaminants, as for example a pathogen or toxin. Illustratively, immobilised aptamer for PrP^Sc can be used to purify blood or other fluid. The purification method can alternatively be to remove a desired molecule from a mixture, by an affinity purification. As another example of an end-use, an immobilised aptamer can form part of a diagnostic system.

DRAWINGS OF THE INVENTION

Figure 1 shows sequences for two adapterms J58copA and L45copT.

Figure 2 presents results of interaction in polyacrylamide gels of copA and copT-tagged aptamers.

Figure 3 demonstrates binding of an adaptamer to streptavidin and CD4.

Figure 4 demonstrates of an adaptamer to gpl20 and CD4.

EXAMPLE OF THE INVENTION

In our copending PCT patent application of even date entitled Biligands incorporated above in its entirety by reference and in this example we show the use of a streptamer to immobilize a gpl20 binding aptamer to the surface of a BIA core biosensor chip via the adaptamers of this invention and thereby detect gpl20 in the mobile phase.

We utilize the copA-copT complementary sequences derived from plasmid RP4, which have very high kinetics of intermolecular annealing (Malmgren, C, et al, J Bio Chem 1997 vol 272 pp 12508- 12). The copA and copT sequences are themselves highly structured, and we find inserting them 5' or 3 ' of an aptamer sequence rarely interferes with the structure and function of the nucleic acid ligand, enabling them to serve as hybridisation tags to aptamers with the complementary cop sequence.

In this example, we present Figures showing:

1. The sequences of two aptamers which include added cop sequences: J58 -copA contains the sequence for a gpl20-binding aptamer J58 linked to that of copA; L45 - copT contains the sequence of a rat CD4- binding aptamer L45 linked to that of cop-T. J58 was made by in vitro evolution of nucleic acid ligands by affinity purification from amongst an intially random library with recombinant gpl20 derived from the strain HIN-1IIIB as target. The library and the techniques are described, for example, in E. Kraus, W. James and A. Ν. Barclay (1998), Novel RNA Ligands Able to Bind CD4 Antigen and Inhibit CD4+ T Lymphocyte Function, Journal of Immunology 160: 5209-5212.

The random region of J58 had the sequence CCGAAGCGCGACGACUAGACGUCAAUUUAUCAACC. L45 was prepared using the procedures described in E. Kraus, W. James and A. N. Barclay (1998), Novel RNA Ligands Able to Bind CD4 Antigen and Inhibit CD4+ T Lymphocyte Function, Journal of Immunology 160: 5209-5212, which article is incorporated herein by reference.

2. An analysis on native 6% polyacrylamide gels of copA and copT- tagged aptamers interacting to form their respective adaptamers. E14 is another, rat CD4-binding aptamer described in E. Kraus, W. James and A. N. Barclay (1998), Novel RNA Ligands Able to Bind CD4 Antigen and Inhibit CD4+ T Lymphocyte Function, Journal of Immunology 160: 5209-5212, which article is incorporated herein by reference. The results show the formation of 2-subunit adaptamer complexes from adaptamer monomers. The final two lanes are negative control mixtures.

3. BIAcore data showing that rat CD4 and streptavidin can be simultaneously bound by an adaptamer composed of copA and copT- tagged aptamers. We used the BIA core system of surface plasmon resonance biosensors to detect intermolecular interaction in real time. The SPR response is proportional to the mass bound. In succession, 35 μl of streptavidin-copT aptamer S19T was injected at 40 μg/ml and 10 μl/min onto a streptavidin coated flow cell (8500 RU); soon after that 35 μl of rat CD4-copA aptamer E14A was injected at the same concentration and flow rate into the same flow cell; then injection of 25 μl of rat CD4 at 70 μg/ml followed. The arrow points indicate the end of the injections. 4. BIAcore data showing that rat CD4 and gpl20 can be simultaneously bound by an adaptamer composed of copA and copT-tagged aptamers. There is sandwich binding to immobilized gpl20 of L45copT-J58copA adaptamer complex followed by rat CD4. This is a proof in principle that a viral glycoprotein can be retargeted using adaptamers to recognize a cell surface molecule of choice. This has potential benefits in the retargeting of viral vectors in gene therapy applications.

REFERENCES

The following references are incorporated in their entirety by reference.

1. Ellington, A.D. and Szostak, J.W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature, 346, 818-822.

2. Tuerk, C. and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science, 249, 505-510. 3. Joyce, G.F. (1998) Nucleic acid enzymes: playing with a fuller deck. Proc Natl Acad Sci USA, 95, 5845-5 847.

4. Hanes, J., Jermutus, L., Weber-Bornhauser, S., Bosshard, H.R. and Pluckthun, A. (1998) Ribosome display efficiently selects and evolves high- affinity antibodies in vitro from immune libraries. Proc Natl Acad Sci USA, 95, 14130-14135.

5. Hanes, J. and Pluckthun, A. (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Nail Acad Sci U S A, 94, 4937-4942.

6. Roberts, R.W. and Szostak, J.W. (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U SA, 94, 12297- 12302.

7. Keefe, A.D. and Szostak, J.W. (2001) Functional proteins from a random- sequence library. Nature, 410, 715-718.

8. Wilson, D.S., Keefe, A.D. and Szostak, J.W. (2001) The use of mRNA display to select high-affinity protein-binding peptides. PNAS, 98, 3750- 3755.

Claims

1. An immobilised aptamer.

2. An immobilised aptamer according to claim 1 where the aptamer is immobilised by direct means to a solid phase.

3. An immobilised aptamer according to claim 1 where the aptamer is immobilised by indirect means to a solid phase.

4. An immobilised aptamer according to claim 1 where the aptamer is immobilised by covalent means to a solid phase.

5. An immobilised aptamer according to claim 1 where the aptamer is immobilised by noncovalent means to a solid phase.

6. An immobilised aptamer according to claim 5, wherein immobilisation is achieved using an aptamer for streptavidin.

7. An immobilised aptamer according to any preceding claim, wherein the aptamer is immobilised to a solid support selected from plastics, resins, glass surfaces, sepharose and agarose.

8. A biligand according to any preceding claim, wherein the aptamer is as defined in any one of claims 1 to 8 of WO 0188123.

9. A biligand according to claim 8, wherein the aptamer as defined in claims 1 to 8 of WO 0188123 includes a sequence as shown in Figure 6 of that PCT application.

10. A diagnostic method which employs an immobilised aptamer according to any preceding claim.