WO2002061079A2 - Biligands - Google Patents

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WO2002061079A2
WO2002061079A2 PCT/GB2002/000364 GB0200364W WO02061079A2 WO 2002061079 A2 WO2002061079 A2 WO 2002061079A2 GB 0200364 W GB0200364 W GB 0200364W WO 02061079 A2 WO02061079 A2 WO 02061079A2
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aptamer
streptavidin
binding
aptamers
rna
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PCT/GB2002/000364
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English (en)
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WO2002061079A3 (fr
Inventor
Abdessamad Tahiri-Alaoui
William S. James
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Isis Innovation Limited
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Priority claimed from GB0102270A external-priority patent/GB0102270D0/en
Priority claimed from GB0102272A external-priority patent/GB0102272D0/en
Priority claimed from GB0102271A external-priority patent/GB0102271D0/en
Priority claimed from GB0102273A external-priority patent/GB0102273D0/en
Application filed by Isis Innovation Limited filed Critical Isis Innovation Limited
Priority to AU2002226571A priority Critical patent/AU2002226571A1/en
Publication of WO2002061079A2 publication Critical patent/WO2002061079A2/fr
Publication of WO2002061079A3 publication Critical patent/WO2002061079A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification

Definitions

  • the present invention relates to biligands, that is ligands with at least a dual binding ability.
  • the post-genomic research environment inspires the search for ways to document the activity of the proteome in experimental and diagnostic samples.
  • 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.
  • 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 10 14 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, extraordinar 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 10 9 and ribosome display methods have proved too fragile for general use (see references 4,5).
  • mRNA display A recent method, called mRNA display (see reference 6), has overcome most of these difficulties through a number of elegant innovations that enable approximately 10 13 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.
  • biligands which comprise at least two aptamers.
  • 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.
  • 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.
  • 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.
  • 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.
  • At least one of the aptamers in the biligand of the invention binds to a protein, particularly to prion protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • homoadaptamers of this invention are able to bind substantially more target protein than a single aptamer.
  • a homo-dimeric aptamer with specificity for a prion protein is able to bind about twice as much prion protein as the single aptamer.
  • 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.
  • 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.
  • detectable moieties such as streptavidin
  • 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.
  • 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 ⁇ l/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 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:
  • 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.
  • 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:
  • (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.
  • FIG. 10 shows adaptamer formation and functional analysis, where:
  • 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:
  • 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.
  • 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.
  • BIAcore data showing that rat CD4 and streptavidin can be simultaneously bound by an adaptamer composed of copA and copT- tagged aptamers.
  • the SPR response is proportional to the mass bound.
  • 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.
  • 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).
  • 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 MgCl 2 ) for 20 min at 20 Q C.
  • 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.
  • SA Dynabeads M-280 Streptavidin
  • the first round of selection was carried out overnight at room temperature in 500 ⁇ l 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 ⁇ l 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 ⁇ l of binding buffer.
  • 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).
  • Tth ThThermus thermophilus
  • the resulting PCR products were used as template for in vitro transcription to produce RNA for the next round of selection.
  • the enriched RNA libraries were pre-exposed to 0.3 mg of Dynabeads (without streptavidin) in 200 ⁇ l for 1 hr, to remove RNA sequences that bind to sites other than streptavidin.
  • SPR Surface Plasmon Resonance
  • RNA from the ninth round of in vitro selection was reverse-transcribed and PCR-amplified with primers (5' -CC GGAATTCCGGAATTAACCCTCACTAAAG
  • nucleotide analogue 6-(2-deoxy- ⁇ -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 ⁇ l of Taq DNA polymerase (Promega WI, USA) in a 20 ⁇ l reaction containing the appropriate 5' and 3 '-primers described above at 0.5 ⁇ M, 3.5 mM MgCl 2 , lOmM Tris-HCl (pH 9.0), 50mM KCl, 0.1% Triton-XlOO and dATP, dCTP, dGTP, dPTP at 500 ⁇ lM each.
  • Taq DNA polymerase Promega WI, USA
  • 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.
  • CopA and/or CopT sequences 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 were similarly engineered to contain CopA and /or CopT sequences.
  • 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.
  • 5'- 32 P-labeled aptamer (5000 cpm Cerenkov) in 20 mM Hepes-NaOH pH 7.5, 100 mM NaCl, 50 mM KCl, 10 mM MgCb, and 1 ⁇ g tRNA was incubated in the presence of increasing amount of streptavidin for 1 hr at room temperature (25 ⁇ l volume).
  • 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 ⁇ l of 70% glycerol solution containing 0.025% (wlv) bromophenol blue was added to each binding reaction.
  • 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 ⁇ l /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 ⁇ g /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.
  • 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 ⁇ l) over the flow cells at 5 ⁇ l/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.
  • rat CD4 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).
  • 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.
  • 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 [ ⁇ - 32 P]-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.
  • RNA was removed using Dynal MPC-E magnet before carrying on the experiments.
  • 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 ⁇ l of binding buffer, in the presence of 1 ⁇ l 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.
  • 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.
  • 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 10 4 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.
  • RNA species with streptavidin-binding properties become a significant component of the mixture by round 4 and the dominant component by round 8.
  • streptavidin binders 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • CD4-binding aptamers fused to the complementary, CopA or CopT elements.
  • these two chimeric aptamers rapidly hybridized, by virtue of CopA-CopT complementarities, to form stable, bi- f ⁇ nctional aptamers that we called adaptamers.
  • a CD4- streptavidin-binding adaptamer can be used to capture CD4 onto a streptavidin-derivatized surface, illustrating their general utility as indirect affinity ligands.
  • 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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Abstract

L'invention concerne des biligands comprenant au moins deux aptamères, utilisés dans des analyses diagnostiques.
PCT/GB2002/000364 2001-01-29 2002-01-29 Biligands WO2002061079A2 (fr)

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Application Number Priority Date Filing Date Title
AU2002226571A AU2002226571A1 (en) 2001-01-29 2002-01-29 Biligands

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
GB0102273.0 2001-01-29
GB0102272.2 2001-01-29
GB0102270A GB0102270D0 (en) 2001-01-29 2001-01-29 Immobilisation
GB0102272A GB0102272D0 (en) 2001-01-29 2001-01-29 Biligands
GB0102271A GB0102271D0 (en) 2001-01-29 2001-01-29 Streptavidin
GB0102271.4 2001-01-29
GB0102273A GB0102273D0 (en) 2001-01-29 2001-01-29 Hiv-1
GB0102270.6 2001-01-29

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WO2002061079A2 true WO2002061079A2 (fr) 2002-08-08
WO2002061079A3 WO2002061079A3 (fr) 2003-09-04

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WO2006048164A1 (fr) * 2004-11-05 2006-05-11 Analyticon Biotechnologies Ag Systeme d'essai base sur des aptameres
JP2014500024A (ja) * 2010-12-10 2014-01-09 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング 腫瘍細胞溶解を媒介する二重特異性アプタマー

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WO2004033718A2 (fr) * 2002-10-11 2004-04-22 Krause Henry M Marquage trap: nouveau procede d'identification et de purification de complexes arn-proteines
WO2004033718A3 (fr) * 2002-10-11 2004-07-22 Henry M Krause Marquage trap: nouveau procede d'identification et de purification de complexes arn-proteines
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WO2006048164A1 (fr) * 2004-11-05 2006-05-11 Analyticon Biotechnologies Ag Systeme d'essai base sur des aptameres
JP2014500024A (ja) * 2010-12-10 2014-01-09 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング 腫瘍細胞溶解を媒介する二重特異性アプタマー

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AU2002226584A1 (en) 2002-08-12

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