WO2002061079A2

WO2002061079A2 - Biligands

Info

Publication number: WO2002061079A2
Application number: PCT/GB2002/000364
Authority: WO
Inventors: Abdessamad Tahiri-Alaoui; William S. James
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2001-01-29
Filing date: 2002-01-29
Publication date: 2002-08-08
Anticipated expiration: 2003-07-29
Also published as: WO2002061079A3; AU2002226584A1; WO2002061125A3; WO2002061125A2; AU2002226571A1

Abstract

Biligands which comprises at least two aptamers are of use in diagnostic assays.

Description

Biligands

The present invention relates to biligands, that is ligands with at least a dual binding ability.

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; thephysicochemical 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).

Gold et al. have described an approach to linking two aptamers, described as chimeric SE EX, which involves inserting aptamer-encoding genes in tandem in a single construct, thereby generating a covalent fusion between two aptamers. This approach has proved in our hands to be very limited, frequently resulting in the loss of binding function for one or other of the inserted aptamers. SUMMARY OF THE INVENTION

According to the present invention, we provide biligands which comprise at least two aptamers. In particular, we provide a system for binding two aptamers together to provide bivalent or bispecific ligands. In this text, and reflecting the adaptability of the aptamer combinations of this invention, we refer to the new biligands as adap tamers.

In a preferred aspect, the invention there is a first nucleic acid sequence which includes a sequence of a first aptamer and a sequence of a first binding partner. There is also a second nucleic acid sequence which includes a sequence of a second aptamer and a sequence of a second binding partner. The second aptamer can be the same as or different to the first aptamer. The first binding partner binds to the second binding partner. Thus when polynucleotides with the first and second sequences are brought together, the two sequences are united by binding of the first binding partner to the second binding partner.

The aptamers can include those in our UK patent application 0012054.3 and PCT application WO 0188123WO 0188123 both of which are herein incorporated by reference in their entirety, as well as other aptamers. Examples of other aptamers include those for streptavidin, CD4 and gpl20. In this respect, reference is made to our PCT patent application of the same filing date as the present patent application, and entitled Streptavidin, incorporated by reference, as well as the UK patent application GB 0102271.4 serving as priority for that PCT application.

More generally, the aptamers of the invention can be chosen from those which bind to particular target molecules. For instance, in one embodiment the aptamers 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, at least one of the aptamers in the biligand of the invention binds to a protein, particularly to prion protein. In particularly preferred embodiments, at least one of the aptamers of the biligand of the invention that binds to a prion protein comprises a nucleic acid sequence or consensus sequence described in PCT application WO 0188123, particularly a sequence set forth Figure 6 of that application, which was incorporated by reference in its entirety above. In another preferred embodiment both aptamers of the biligand of the invention that bind to a prion protein comprise a nucleic acid sequence or consensus sequence described in PCT application WO 0188123, particularly a sequence set forth in Figure 6 of that application. In another preferred embodiment, both aptamers of the biligand of the invention that binds to a prion protein comprise the same nucleic acid sequence or consensus sequence described in PCT application WO 0188123, particularly a sequence set out in Figure 6 of that application.

The nucleic acid sequences are preferably 2'-fluoronucleic acids, but that it is not essential.

The binding partners are suitably copA and copT, but that is not essential.

Thus, in a particularly preferred embodiment, we modify the gene for one aptamer, which binds to ligand A, with a segment of nucleic acid encoding a structured RNA derived from that of a regulatory element found in bacterial plasmids, copA. We modify the gene for a second aptamer, which binds to ligand B, with a segment of nucleic acid encoding a complementary, structured RNA, copT. We then transcribe both modified aptamer genes into 2'-F nucleic acid and mix them. The two complementary RNA have a particularly high rate of association with each other and form a double helix, linking the two aptamers in a dimeric form.

Rendering aptamers dimeric can produce a number of valuable improvements in their usefulness:

Homo-dimeric aptamers, having two identical binding sites in close proximity, can produce ligands of much greater avidity (apparent affinity) by markedly reducing the dissociation rate associated with monomeric ligands. Such homoadaptamers might be employed for example in the neutralisation of toxins or viruses. They are particularly suited for use against multimers, including proteins found at multiple sites on a cell surface. We have experimental evidence that homoadaptamers of this invention are able to bind substantially more target protein than a single aptamer. For example, in one embodiment a homo-dimeric aptamer with specificity for a prion protein is able to bind about twice as much prion protein as the single aptamer. Furthermore, the homo-dimeric of the invention is expected to bind much more stably to the target than the monomer, allowing more extensive washings and thereby improving specificity of detections.

Hetero-dimeric aptamers have the ability to cross-link two target molecules and this can have very useful effects. For example, bispecific aptamers might be used to link an immunologically important molecule to a cell- surface antigen expressed on a cancer cell, and so lead to destruction of the malignant cell. Such adaptamers can be used to target viral vectors to cell surface molecules that are not their natural receptors, leading to a much sought-after ability to re-target vectors to specific tissues in vivo.

Adaptamers can be used to link aptamers that bind to target molecules of interest to aptamers that bind detectable moieties, such as streptavidin in order to make them useful as detection tool, in the way currently possible using antibodies. In this respect, we refer to the copending PCT patent application entitled Streptavidin.

Thus, by the present invention, we provide biligands of interest as pharmaceutical compounds and for diagnostic uses. Methods of treatment and diagnosis are also part of this invention, along with pharmaceutical and diagnostic compositions.

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 adap tamer to streptavidin and CD4.

Figure 4 demonstrates of an adaptamer to gpl20 and CD4.

Figure § is an overlay of sensorgrams from surface plasmon resonance analysis showing enrichment for streptavidin-aptamer during in vitro selection. Pool of 2'-Fluoro-RNA transcripts from rounds 2, 4, 5, 6, 8 and 9 were injected (about 75 nM) at a flow rate of 5 μl/min over a sensor chip pre- coated with 4.5 kRU streptavidin. The specificity of the enriched RNA pool from round 9 was assessed against immobilized BSA (4.2 kRU). The arrow indicates end of injections and start of buffer chase.

Figure 5a, also referred to as Table 1, is a sequence alignment of 2'~fluoro- pyrimidine-containing RNA aptamers derived from affinity selection on streptavidin. Only the random region is shown. Aptamers derived from theparental SA19 by random mutagenesis followed by two rounds of in vitro re-selection are also aligned. The alignment was obtained with Clustal X program (version 1 .64B). The symbol (f) indicates non-binder aptamers and (^♦) (*), aptamers with slow on-rate and fast off-rate, respectively. Nucleotides that are variants between clones are shown in italic, those that cause loss of binding to streptavidin, when mutated, are underlined and in bold, whereas the ones that are just underlined seem not to be essential for binding.

Figure 6 is a native gel mobility shift assay for streptavidin binding to SA19 aptamer, where:

(A) Storage phosphor autoradiogram of a representative gel used to separate free aptamer SA19 from SA19-SA complex using a range of increased protein concentrations from 1.8 to 447 nM.

(B) representative plot of fraction of aptamer bound by streptavidin as a function of protein concentration.

The data were fitted to a hyperbolic function by non-linear curve fitting method of GraphPad PRISM. This titration yielded an equilibrium dissociation constant (Kd) of 7 + 1.8 nM.

Figure 7 is an overlay of sensorgrams showing the effect of biotin saturated- streptavidin on the binding of aptamer and its specificity, where: (A) Flow cells 1 to 3 were pre-coated with 5.7, 4.9 and 4.8 kRU streptavidin, respectively and flow cell 4 was left as a blank control. Flow cell 2 was saturated with 0.113 kRU of biotin before injecting 200 nM of SA19 aptamer over flow cells ito 4 in series.

(B) SA19 aptamer was injected at about 50 nM over flow cells 1 to 4 previously coated with BaLgp 120 (9 kRU), rat sCD4 (6 kRU), avidin (4.9 kRU) and streptavidin (4.8 kU), respectively. The arrow indicates end of injections and start of buffer chase.

Figure 8 is an overlay of sensorgrams showing the effect of mutagenic PCR on binding of SA19 aptamer to streptavidin. Template DNA from various mutagenesis cycles was used to produce 2'-fluoro-RNA. Transcripts (about 65 nM) were injected over sensor chip pre-coated with 4.2 kRU of immobilized streptavidin. The binding to streptavidin was significantly reduced after 5 cycles of mutagenesis, almost abolished by cycles 10, and completely lost by cycle 15, as compared to the control (0 cycle).

Figure 9 is a solution structure of SA19 aptamer and streptavidin footprinting, where:

(A) Proposed secondary structure of SA19 aptamer as deduced from solution probing data and predictions with STAR software package. Reactive nucleotides towards DM8 (C at N3), CMCT (U at N3, G at Nl, and kethoxal (G at Nl N2) are encircled, and non-reactive nucleotides are boxed. No symbol is for not determined. The protected area is delimitated by black dots.

(B) Autoradiogram of a 18 % polyacrylamide/8 M urea gel, showing digestion products of 5 '-end labeled SA19 with Rnase Vi and nuclease Si in the presence (+) or absence (-) of streptavidin, the major protected area is shown by a vertical line. Lane OH is the ladder from partially alkaline hydrolyzed SA19. The control lane corresponds to the 5'-end labeled SA19 incubated in the presence of streptavidin but in the absence of any nucleases. The gaps in the OH ladder are indicative of 2'-fluoro-pyrimidines.

Figure. 10 shows adaptamer formation and functional analysis, where:

(A) Transcripts from Cop-aptamers were refolded and then loaded on a native 6 % polyacrylaniide gel, either separately or in pair-wise arrangement. The pair-wise combination was prepared by mixing 50 ng of each Cop-aptamer, refolding the mix to form the adaptamers, while 100 ng of each single aptamer-Cop RNA were loaded per lane.

(B) SPR analysis of aptamer-Cop RNAs and adaptamers on streptavidin- coated sensor chip. SA19-CopA (top panel) and SA19-CopT (bottom panel) were injected onto streptavidin-coated flow cells, followed by rat CD4-Cop aptamer E14-CopT (top panel) or E14-CopA (bottom panel). Soluble rat CD4 protein was then injected (continuous lines). Control injections for specificity are shown underneath and correspond to each aptamer-Cop injection, (dotted line). In particular, SA19-CopA and SA19-CopT were injected over rat CD4-coated flow cell, while E14-CopT and ElCopA were injected over a streptavidin-coated flow cell. Bars and arrows indicate length and end of injections, respectively.

Figure 11 is a native gel mobility shift assay of streptavidin binding to chimeric SA19 and adaptamer, where:

(A) Storage phosphor autoradiogram of a representative gel used to separate free chimeric aptamer SA19-CopA from SA19-CopAISA complex, using a range of increasing protein concentrations from 0.45 to 3584 nM.

(B) The same analysis as described in (A) but with the adaptamer SA19- CopA-E14-CopT.

(C) Representative plots of fraction of the chimeric SA19-CopA (continuous line) and the adaptamer SAi9-CopA-El-CopT (dotted line) bound by SA as a function of protein concentration. The data were fitted to a hyperbolic function by non-linear curve fitting method of GraphPad PRISM. These titrations yielded an equilibrium dissociation constant of about 18 and 24 nM for the chimeric SA19-CopA and the adaptamer SA1 9-CopA-E14-CopT, respectively.

EXAMPLES OF THE INVENTION

The present invention is illustrated by the following examples.

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.

Example 1

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 HIV- HUB as target. The library and the techniques are described, for example, 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. 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% polyaciylamide 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.

Example 2

Oligonucleotides and in vitro selection

The oligonucleotide library (5'-

AATTAACCCTCACTAAAGGGAACTGTTGTGAGTCTCATGTCGAA(N)49TTGAGC GTCTAGTCTTGTCT-3') incorporating 49 randomized nucleotides was converted into double stranded template (see reference 9) by 5 rounds of polymerase chain reaction (PCR) in 5 ml reaction volume with 5 ' - AATTAACCCTCACTAAAGGGAACTGTTGTGAGTCTCATGTCGAA-3 ' (5 ' -primer) and 5 '-TAATACGACTCACTATAGGGAGACAAGACTAGACGCTCAA-3 ' (3'- primer). The resulting PCR products (1.8 nmol) were transcribed in 1 ml reaction by T7 RNA polymerase in the presence of 2'-Fluoro-pyrimidine ribonucleotides (TnLink BioTechnologies, Inc., San Diego, CA), together with 2 '-OH-purine ribonucleotides (see reference 10).

After transcription, the template DNA was digested with RNase-free DNase I. The full-length 2 ' -F- RNA transcripts were purified by electrophoresis on 10% polyaciylamide/ 8M urea gel. Affinity selection was initiated with 1.5 nmol of 2 '-F-pyrimidine-containing RNA random sequence library. The RNA in water was incubated for 3 min at 95°C, cooled down to room temperature before refolding it in the binding buffer (20 mM Hepes-NaOH pH 7.5, 100 mM NaCl, 50 mM KC1, 10 mM MgCl₂) for 20 min at 20^QC. The refolded pool of RNA was then mixed with 1 mg of Dynabeads M-280 Streptavidin (SA) (Dynal Biotech, UK) that were previously saturated with 800 pmol of a biotinylated 13-residue-peptide.

The first round of selection was carried out overnight at room temperature in 500 μl volume with gentle mixing. The subsequent selection rounds 2 to 4 and 5 to 9 were scaled down to 0.6 and 0.3 mg of Dynabeads M-280 streptavidin/ bio tin- saturated in 200 and 100 μl respectively, and an incubation time of 2 hours. The streptavidin-RNA complex was separated from the unbound RNA with a Dynal magnetic particle concentrator (Dynal MPC-E) for 1 min and the supernatant removed. RNA molecules that were trapped non-specifically were removed by three washes with 200 μl of binding buffer.

The bound RNA was converted to cDNA by reverse transcription with ThThermus thermophilus (Tth) DNA polymerase at 70°C for 20 min following the protocol provided by the supplier (Promega WI, USA) followed by 15 cycles of PCR amplification (see reference 9). The resulting PCR products were used as template for in vitro transcription to produce RNA for the next round of selection. After rounds 2, 3 and 5, the enriched RNA libraries were pre-exposed to 0.3 mg of Dynabeads (without streptavidin) in 200 μl for 1 hr, to remove RNA sequences that bind to sites other than streptavidin. Surface Plasmon Resonance (SPR) using BIACORE instrument (Biacore, Uppsala, Sweden) was used to assess the enrichment for streptavidin- binders during in vitro selection.

The pool of RNA from the ninth round of in vitro selection was reverse- transcribed and PCR-amplified with primers (5' -CC GGAATTCCGGAATTAACCCTCACTAAAG

GGAACTG-3 " and 5 ' -TCCCCCGGGG GATAATACGACTCACTATAG GGAGAC- 3') that introduce EcoRI and Smal sites at the termini of the resulting DNA. The DNA was digested with EcoRI and Smαl. sub-cloned into pUClδ vector that had been previously digested with the same enzymes. Clones were sequenced by PRISM™BigDye™ cycle sequencing ready reaction kit from ABI (Perkin-Elmer).

Mutagenesis and reselection

The nucleotide analogue, 6-(2-deoxy-β-D-erythropentofuranosyl)-3,4- dihydro-8H-pyrimido-[4,5C][l,2]oxazine-7-one-5' triphosphate (dPTP) (see reference 11) was used to introduce mutations into SA19 aptamer. SA19 DNA template was amplified using 0.6 μl of Taq DNA polymerase (Promega WI, USA) in a 20 μl reaction containing the appropriate 5' and 3 '-primers described above at 0.5 μM, 3.5 mM MgCl₂, lOmM Tris-HCl (pH 9.0), 50mM KCl, 0.1% Triton-XlOO and dATP, dCTP, dGTP, dPTP at 500 μlM each. After an initial denaturation step at 93°C for 3 min, various cycles (0, 5, 10 and 15) were performed, each of which consisted of denaturation at 93°C for 50 sec, annealing at 55°C for 30 sec and elongation at 72°C for 1 min.

The product of this first PCR was subjected to a second PCR in the presence of four natural dNTPs in order to eliminate the base analogues from the target DNA SA19 (see reference 11). The DNA from the second PCR amplification was used as a template for in vitro transcription as described above. The 2 '-F-pyrimidine-containing RNA transcripts from various mutagenesis cycles were analyzed by SPR to verify the abolition of the binding to streptavidin. The pool of RNA from 15 cycles of mutagenic PCR in which streptavidin-binders were completely lost as judged by SPR analysis, was subjected to two rounds of in vitro selection as described above.

Adaptamer constructs The CopA and/or CopT sequences (see references 12, 13) were inserted downstream of the SA 19 -aptamer sequence previously cloned into a pUC18 vector, using the EcoRI site. Transcription products of the constructs gave aptamers with CopA or CopT at their 3' terminus. Rat CD4 aptamers (see reference 14) were similarly engineered to contain CopA and /or CopT sequences.

Gel mobility shift assay

Dissociation constants for SA19 aptamer, chimeric SA19-CopA and the adaptamer 5A19-CopA-E14-CopT binding to streptavidin were quantified by native gel shift assays. In a typical binding experiment, 5'-³²P-labeled aptamer (5000 cpm Cerenkov) in 20 mM Hepes-NaOH pH 7.5, 100 mM NaCl, 50 mM KCl, 10 mM MgCb, and 1 μg tRNA was incubated in the presence of increasing amount of streptavidin for 1 hr at room temperature (25 μl volume). In the case of the adaptamer SA19-CopA-E14-CopT, the two 5'- end-labeled chimeric aptamers were first mixed at an equimolar ratio, heat denatured in water for 5 min at 95°C, then allowed to fold in the binding buffer for 20 min at room temperature before adding increasing concentration of streptavidin protein. After incubation was completed, 3 μl of 70% glycerol solution containing 0.025% (wlv) bromophenol blue was added to each binding reaction.

The samples were then resolved on an 8% polyaciylamide native gel. Data were obtained from the gels using storage phosphor autoradiography and STORM phosphor imager (Molecular Dynamics). The ratio of bound to unbound RNA was quantified using ImageQuant software (Molecular Dynamics). Dissociation constants for 5A19 aptamer, chimeric SA19-CoρA, and the adaptamer were derived from a fit by non-linear regression to a hyperbolic function using GraphPad PRISM (GraphPad Software, Inc. San Diego, CA).

Surface plasmon resonance

BIACORE 2000 was used to perform all binding studies. Research grade CM5 chips, NHS/EDC coupling reagents and ethanolamine were from BIACORE AB (Uppsala, Sweden), streptavidin protein (Sigma) was immobilized onto sensor chip using amine-coupling chemistry. The immobilization steps were carried out at a flow rate of 5 μl /min in 20 mM Hepes-NaOH, 150 mM NaCl, 3.4 mM EDTA and 0.005% P20 surfactant. The flow cells were activated for 7 min with a mixture of NHS (0.05M) and EDC (0.2 M). streptavidin was injected at a concentration of 400 μg /ml in 10 mM sodium acetate pH 5.2, for 7 min. Ethanolamine (1 M, pH 8.5) was injected for 7 min to block remaining activated groups. An average of 5 kRU was immobilized on each flow cell.

The assessment of enriched RNA binders to streptavidin was done under the same running buffer that was supplemented with 50 mM KCl and 10 mM MgCb. The RNA was refolded in the binding buffer as described above, and injected (35 to 60 μl) over the flow cells at 5 μl/min. Between consecutive injections, the surfaces were regenerated by long (60-120 min) washes with the running buffer. To correct for refractive index changes and instrument noise the response data from a reference surface were subtracted from the responses obtained from the reaction surface.

The specificity of streptavidin-aptamer interaction was assessed against various proteins including the soluble fraction of rat CD4, gpl2O, avidin and BSA. SPR analysis of adaptamers were performed in two ways: the aptamer- Cop species were either separately refolded and then injected sequentially, so that the adaptamers would form inside the flow cell, or premixed, refolded, allowed to anneal and then injected (the example shown in figure 10B is illustrating the first case). Immediately after the RNA injections, sample of rat CD4 was injected to test the ability of the adaptamers to simultaneously bind the two protein targets in the same flow cell.

Enzymatic probing and footprinting

SA19 aptamer was gel purified on 10% polyacrylamide/8 M urea gel, dephosphorylated and then labeled at the 5'-end with T4 polynucleotide kinase and [α-³²P]-ATP (15). Labeled aptamer was gel-purified as above, eluted, and precipitated twice with ethanol. Before use, labeled SA19 RNA was dissolved in water, incubation at 90°C for 2 min, followed by slow cooling at 20°C in the binding buffer. Binding of 5'-end labeled SA19 to streptavidin protein was first allowed to form on Dynabeads M-280 streptavidin (0.03 mg) for 1 h in the binding buffer. The unbound RNA was removed using Dynal MPC-E magnet before carrying on the experiments. As a control the RNA was incubated under identical conditions with Dynabeads lacking streptavidin. Enzymatic hydrolysis of free or streptavidin-bound labeled SA19 RNA was performed in 10 μl of binding buffer, in the presence of 1 μl carrier tRNA at 20°C for 10 min in presence of RNase VI (0.07 units) or nuclease SI (20 units). Reactions were stopped by phenol/ chloroform extraction, followed by ethanol precipitation, and washing with 80 % ethanol. Incubation control in the presence of Dynabeads M-280 streptavidin but without adding any nuclease was done in parallel to detect any unspecific cleavage that may occur in the RNA. The products were then sized by electrophoresis on a 15 or 18 % polyacrylamide/8 M urea gel. A partial alkaline hydrolysis ladder of the same aptamer (see reference 16) was run in parallel.

Chemical probing Chemical probing was done as previously described (see references 17- 19). DMS (NIA, N3C), CMCT (N3U, NIG), and kethoxal (GNl, N2) modifications of 0.1 μg of gel purified and refolded SA 19 aptamer in presence of 2 μg tRNA were carried out in 20 μl reaction volumes in 20 mM Hepes-NaOH pH 7.2 for DM8 and CMCT and 50 mM sodium borate pH 8.0 for kethoxal. All buffers contained 10 mM MgC , 100 mM NaCl, 50 mM KCl . Reactions were performed at 20°C for 5 min in the presence of 1 μl of DMS (1:8 or 1: 16 dilutions in ethanol), for 20 min in the presence of 1 μl of CMCT (40 or 20 mg/ml in water), or 5 min in the presence of 2 μl of kethoxal (20 mg/ml in 20% of ethanol) . After ethanol precipitation, the modified RNAs were dissolved in water. Unmodified SA19 aptamer was processed in parallel. Primer extension with 5'-³²ρ labeled primer (5'-AATTAACCCTCAC-3') was used to detect the modified bases (see reference 20). Sequencing of unmodified SA19 aptamer was done as described before (see reference 21).

The secondary structure model of SA19 aptamer was deduced from STAR software package, (see references 22,23) using stochastic and genetic folding algorithms. The predictions were constrained by imposing the data from solution probing.

RESULTS

Isolation of streptavidin-binding aptamers

As described, a DNA library was synthesized, having a 49 nucleotides randomized region flanked by constant regions that incorporate T7 and T3 RNA polymerase promoters for positive and negative strand transcription, respectively. Approximately 10⁴ different 2'-F-pyrimidine-substituted RNAs were synthesized by T7 RNA polymerase and those binding streptavidin were selected using Dynabeads M-280 Streptavidin complexed to a biotinylated peptide. Streptavidin-bound aptamers were eluted and amplified by PCR to generate a library enriched for streptavidin-binding RNA sequences. Progress of selection was monitored using a surface plasmon resonance (SPR) biosensor to which streptavidin had been covalently coupled (Fig. 5) . The results show that RNA species with streptavidin-binding properties become a significant component of the mixture by round 4 and the dominant component by round 8. There was no significant enrichment of streptavidin binders after round 9, consequently the aptamers were cloned and sequenced at this stage. The enriched RNA pool from round 9 did not show any binding to BSA (Fig. 5), indicating the specificity of the interaction with streptavidin protein.

Binding characteristics of streptavidin aptamers

Fifty aptamer clones were sequenced. The alignment revealed that only 12 clones were distinct and fell into one sequence group with considerable sequence similarities (Table 1). The ability of these clones to bind streptavidin protein was qualitatively assessed by SPR. All of them were found to bind streptavidin, except clone SA27. We chose SA19 clone as the representative for further analysis. To determine the binding affinity of streptavidin for this aptamer we used gel mobility shift assay. In our experimental conditions the interaction between SA19 aptamer and streptavidin produced a single, saturable complex (Fig. 6A). Titration of streptavidin protein to SA19 aptamer at a concentration of approximately 3 nM yielded a complex of slower electrophoretic mobility than the free aptamer as shown in figure 6A.

To determine the apparent dissociation constant for this protein-aptamer interaction, the amount of ³²P present in the free and bound aptamer bands was quantified and the binding data were fit to an equation that describes a simple bimolecular equilibrium (Fig. 6B). The result indicated a Kd of 7.0 ± 1.8 nM. SPR analysis was also used to obtain an independent value for the apparent dissociation constant, which was calculated to be similar to the one obtained by gel mobility shift assay (data not shown).

The in vitro selection process was designed in order to isolate aptamers that would not compete for the bio tin-binding site on the streptavidin protein, which was achieved by pre-saturating streptavidin with a biotinylated peptide. We used SPR analysis to test whether the aptamers were capable of binding streptavidin protein that was pre-saturated with biotin. Three adjacent flow cells were coated with streptavidin protein and one flow cell (number 2) was pre-saturated with biotin. The remaining flow cell (number 4) was used as a reference surface to correct for refractive index changes and instrument noise. SA19 aptamer was then injected over all flow cells and it was able to bind biotin-saturated streptavidin on flow cell 2. The amount of aptamer bound was however, approximately half of that on flow cells 1 and 3 (Fig. 7A). The kinetics of the interaction between the aptamer and the streptavidin protein were not affected by the presence of biotin (data not shown). Interestingly, SA19 aptamer did not interact with the functionally related avidin. The specificity of the interaction was also assessed against other proteins, including gpl2O and CD4, none of which were recognized by the aptamer (Fig. 7B).

Streptavidin binds to a defined region of the aptamer

Clone SA19 was subjected to mutagenic PCR using the nucleotide analogue dPTP and subsequent de novo selection in order to: i) identify mutants with improved binding properties; ii) map the positions of nucleotides that are involved in the interaction with the target protein. The DNA from various mutagenic PCR cycles (see methods) was used as template for in vitro transcription to generate 2 '-F- transcripts. The resulting pools of RNAs were analyzed by SPR to examine the effects of the mutagenesis on the binding to streptavidin protein (Fig. 8). By comparison with the control (0 cycle), the binding to streptavidin was significantly reduced after 5 cycle of mutagenic PCR, almost abolished by cycle 10 and not detected by cycle 15 (Fig 8). The pool of 2 '-F- transcripts corresponding to 15 cycles of mutagenic PCR was the starting material from which the rare remaining streptavidin ligands were rescued. This was achieved by two rounds of de novo selection on streptavidin-coated magnetic beads followed by RT-PCR, cloning and sequencing. The resulting aptamers were designated SA19Mxx, where SA19M refers to the fact that each clone is a mutant form of the streptavidin binding aptamer SA19 and xx is an arbitrary two digit number referring to the clone (Table 1). Thirty aptamer clones were sequenced. Sequence comparison and alignment showed that nine clones were distinct and that the mutant aptamers were very similar to the parental sequence (Table 1).

SPR analysis of the mutant aptamers showed that three clones (SA19M21, SA19M24, SA19M27) had lost the ability to bind streptavidin, while mutant SA19M15 had slower on-rate (4.7 x 10³ S"¹ M"¹) as compared to the parental SA19 (1.7 x 10⁴ s"¹ M"¹), whereas the off-rate remained the same. On the contrary, aptamer SA19M22 had a faster off-rate (6.0 x 10"³ s-¹) as compared to SA19 (4.3 x lO- ¹), and an increased on-rate (4.4 x 10⁴ s"¹ M"¹). The overall binding characteristics of the remaining mutants were comparable to those of the parental SA19 aptamer. Analysis of the primary sequence of the mutant SA19M15 showed four mutations (A49G, A56G, A58G and C59U). Since these mutations affect the association rate of the interaction, they are likely to be important in the initial binding events. The analysis of the three mutants, SA19M21, SA19M24 and SA19M27 as well as clone SA27 from the first selection, that had lost the ability to bind streptavidin protein allowed us to determine key nucleotides that are involved in the interaction. Two mutations, U48C and U57C in SA27 and SA19M2, respectively were sufficient to abolish the binding to streptavidin protein. Additional nucleotide differences were also observed between non- binder SA27 and other binders, namely the A at position 32, G at position 68 and the U at position 71, none of which seemed to affect the binding to streptavidin when mutated to G, A and G/A, respectively. This indicated a critical role of the 2'-F-UTP at position 48 in the binding to streptavidin. Sequence analysis of SA19M21 and SA19M27 revealed that the presence of 2'-F-UTP at positions 25 and 57 was essential in the binding to streptavidin. Nucleotides A38, U46, G5 1 are mutated in the non-binding 5A19M24, which also carries the C25U mutation, which is sufficient to abolish activity alone.

To gain a structural insight into the significance of these nucleotide substitutions, the secondary structure of SA19 RNA was probed using a combination of chemical and enzymatic probes. The footprint with VI and SI nucleases allowed us to delineate the binding site of streptavidin on SA19 aptamer (Fig. 9A,B). There was a good correlation between the secondary structure predicted by STAR software and the solution probing data. The predicted secondary structure of the representative SA 19 aptamer (Fig. 9 A) can be divided into three domains. Domain I, from nucleotide 1 to 31, for which no chemical probing data were available, is predicted to fold into a stem, a symmetrical internal loop and a hairpin loop. Domain II, from nucleotide 32 to 75 and for which most of the nucleotides have been probed, presented a reactivity pattern that correlated well with the presence of two stem loops linked by a stretch of four nucleotides. Binding of streptavidin induced several protections against nuclease SI and RNase VI hydrolysis in this domain. The major protections were located in a region encompassing residues 50 to 62, well correlated with the mutagenesis data showing that the modified U57 is essential for binding. Domain III, which contains the remaining of the aptamer sequence, was predicted to fold into a hairpin loop that is flanked by two single stranded regions and was confirmed by the solution probing data. Deletion of this domain did not affect the binding to streptavidin protein (data not shown).

Formation of streptavidin-CD4 adaptamers

In order to develop a general method for forming aptamers that simultaneously bind streptavidin and a second target protein, we fused the gene encoding SA19 with that encoding CopA or CopT structured RNAs from E. coli plasmid, Rl (see reference 13). In parallel, we also fused the gene encoding a rat CD4-binding aptamer, E14 (see reference 14), with that encoding the complementary CopT or CopA sequence. The addition of about 100 nucleotides that formed the Cop sequence at the 3' of SA19 aptamer resulted in a chimeric aptamer that we designated SA19-Cop (A/T). When this chimeric aptamer was mixed with a similarly engineered CD4 chimeric aptamer (E14-CopA/T) the resulting molecule was designated adaptamer. Transcripts from sequences containing Cop-aptamers were refolded and then analyzed by native PAGE, either separately or in pair-wise arrangements to monitor the formation of the hybrid molecules (adaptamers), which formed efficiently (Fig. 10A). The ability of aptamer-Cop RNAs and their derived adaptamers to bind to streptavidin and CD4, was analyzed by SPR on streptavidin-coated sensor chip (Fig. 10B). Streptavidin-Cop aptamers SA19-CopT and SA19-CopA were injected onto streptavidin-coated flow cells, followed by CD4-Cop aptamers E14-CopT and E14-CopA, respectively. The rapid rise in response in each case demonstrates the formation of adaptamers through the CopA-CopT interaction. Recombinant rat CD4 was injected subsequently and a further substantial response was seen, showing that the adaptamers could bind simultaneously to both streptavidin and GD4.

Affinity characterization of SA19-Cop and adaptamer streptavidin interactions

It was necessary to verify that the affinity of the chimeric aptamer as well as the adaptamer was not altered by these modifications. For this we have used gel mobility shift assay as for the unmodified parental aptamer. Titration of streptavidin protein to the chimeric aptamer (Fig. 11A) as well as to the adaptamer (Fig.1 IB) at a concentration of approximately 3 nM yielded a complex of slower electrophoretic mobility compared to the free chimeric or free adaptamer, as shown in figure 11A and 1 IB, respectively. The apparent dissociation constants for these interactions were calculated as before (Fig. 11C), and yielded value of 18 ± 2.7 nM and 24 ± 4.2 nM for the chimeric aptamer and the adaptamer, respectively. It appeared from these results that the affinities of the newly engineered chimeric aptamer and the adaptamer have dropped only by approximately two to three folds in comparison with the parental aptamer.

Thus, we have isolated 2 ' -fluoro-substituted RNA aptamers that bind to streptavidin with an affinity around 7 ± 1.8 nM, comparable with that of recently described peptide aptamers. Binding to streptavidin was not prevented by prior saturation with biotin, enabling nucleic acid aptamers to form useful ternary complexes. Mutagenesis, secondary structure analysis, ribonuclease footprinting and deletion analysis provided evidence for the essential structural features of streptavidin-binding aptamers or Streptamers. In order to provide a general method for the exploitation of these aptamers, we produced derivatives in which they were fused to the naturally structured RNA elements, CopT or CopA. In parallel, we produced derivatives of CD4-binding aptamers fused to the complementary, CopA or CopT elements. When mixed, these two chimeric aptamers rapidly hybridized, by virtue of CopA-CopT complementarities, to form stable, bi- fαnctional aptamers that we called adaptamers. We show that a CD4- streptavidin-binding adaptamer can be used to capture CD4 onto a streptavidin-derivatized surface, illustrating their general utility as indirect affinity ligands.

The availability of streptavidin-binding aptamers, together with the adaptamer approach, opens the possibility of applying the wide range of streptavidin/ bio tin-based detection systems of the kind currently used in conjunction with antibody ligands to the analysis of molecules to which nucleic acid aptamers have been isolated.

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Claims

1. A biligand which comprises at least two aptamers.

2. A biligand according to claim 1, wherein the nucleic acid sequences are 2'-fluoronucleic acids.

3. A biligand according to claim 1 or 2, which comprises a first nucleic acid sequence which includes a sequence of a first aptamer and a sequence of a first binding partner, a second nucleic acid sequence which includes a sequence of a second aptamer and a sequence of a second binding partner, and wherein the first binding partner binds to the second binding partner.

4. A biligand according to claim 3, wherein the binding partners are copA and copT.

5. A biligand according to any claim 3 or 4, wherein the second aptamer is the same as or different to the first aptamer.

6. A biligand according to claim 6, which is a homo-dimeric aptamer having two identical binding sites.

7. A biligand according to claim 6, which is a hetero-dimeric aptamer having two different binding sites.

8. A biligand according to any preceding claim, wherein at least one aptamer binds to streptavidin.

9. A biligand according to claim 8, wherein the at least one aptamer binding to streptavidin is as defined in a claim of our copending PCT application entitled Streptavidin.

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

11. A biligand according to claim 10, wherein the at least one aptamer as defined in claims 1 to 8 of WO 0188123 includes a sequence as shown in Figure 6 of that PCT application.

12. A method of diagnosis which employs a biligand according to any preceding claim.