WO2002061078A2 - Streptavidine - Google Patents

Streptavidine Download PDF

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Publication number
WO2002061078A2
WO2002061078A2 PCT/GB2002/000363 GB0200363W WO02061078A2 WO 2002061078 A2 WO2002061078 A2 WO 2002061078A2 GB 0200363 W GB0200363 W GB 0200363W WO 02061078 A2 WO02061078 A2 WO 02061078A2
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Prior art keywords
aptamer
streptavidin
aptamers
binding
sequence
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PCT/GB2002/000363
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English (en)
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WO2002061078A3 (fr
Inventor
Abdessamad Tahiri-Alaoui
William Syward James
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Isis Innovation Limited
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Priority claimed from GB0102271A external-priority patent/GB0102271D0/en
Priority claimed from GB0102272A external-priority patent/GB0102272D0/en
Application filed by Isis Innovation Limited filed Critical Isis Innovation Limited
Priority to AU2002225234A priority Critical patent/AU2002225234A1/en
Publication of WO2002061078A2 publication Critical patent/WO2002061078A2/fr
Publication of WO2002061078A3 publication Critical patent/WO2002061078A3/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 streptavidin, and in particular to ligands which bind to streptavidin.
  • 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 nucleic acid 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).
  • 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 - bio tin.
  • streptavidin - bio tin.
  • streptavidin an aptamer for streptavidin as a streptamer.
  • the streptamers of this invention can be made using conventional technology involving in vitro selection from a synthetic nucleotide library.
  • the nucleotides are preferably 2'-F nucleotides in order to impart nuclease resistance.
  • the consensus sequence comprises up to 50 nucleotides (5'- CUUUCCUARCRCAYAUGYGRCCUCURUGCGUARURCGARYGYUGRCNU-3 5 )
  • nucleic acid ligands for streptavidin with potentially general utility as reagents in analytical and diagnostic applications.
  • the relatively straightforward method used to isolate these streptavidin-binding aptamers contrasts with the elaborate, if elegant approach for producing streptavidin- binding peptide aptamers, recently described (see reference 8).
  • the affinity of the typical nucleic acid aptamers of this invention is comparable with that of the peptide aptamers. Moreover, because of their 2'-fluoro-chemistry the preferred ligands are relatively nuclease resistant and are intrinsically not susceptible to proteases.
  • aptamers which include such sequences and variants thereof.
  • Variants include (a) aptamers with at least 10, 15, 18, 20, 25, 30, 35, 40 or more nucleotides in common with a sequence of Figure 1A, particularly aptamer ligands with at least 10, 15, 18, 20, 25, 30, 35, 40 or more conseuctive nucleotides identical to at least 10, 15, 18, 20, 25, 30, 35, 40 or more consecutive nucleotides of a sequence of Fgiure 1A, as well as (b).
  • aptamers which are at least 80% identical with a sequence of Figure 1A or with an aptamer (a).
  • Sequence identity is suitably determined by a computer programme, preferably the BestFit software from the Wisconsin /Oxford Molecular GCG package.
  • Preferred streptamers of this invention include aptamers which have such a motif or a substantial part thereof.
  • Streptamers can be coupled to aptamers against a target protein of interest. Coupling can be carried out using the system which is the subject of our patent application of the same filing date as the present patent application, entitled Biligands, and incorporated herein by reference. Thus, streptamers can be used in adaptamers of that patent application.
  • biligands which comprise at least two aptamers.
  • first nucleic acid sequence which includes a sequence of a first aptamer and a sequence of a first binding partner.
  • 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 be chosen from those which bind for instance to small molecules, to oligopolymers, to polymeric molecules such as proteins, to cellular components, or to whole cells. Examples include antigenic molecules, toxins, prions and viruses.
  • 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.
  • coupling of aptamers can be by inserting the streptamer- encoding gene cassette either 5' or 3' of that for a second aptamer, though in our hands when we attempted to produce chimeric aptamers by direct fusion of the gene encoding a streptamer with that encoding an aptamer to a second protein, we found that in most cases the resultant RNA transcript had lost the ability to bind one or both of its target molecules.
  • the biligands of this invention include an aptamer which is as defined in any one of claims 1 to 8 of PCT application WO 0188123. We incorporate that text in full by reference.
  • the aptamer is suitably any one of the sequences in Figure 6 of the PCT Text.
  • the present invention further extends to diagnostic and other methods which employ a streptamer of this invention.
  • the streptamer binds to streptavidin, and can thus be used as a replacement for biotin in streptavidin-biotin assays.
  • an indirect detection platform can be used combining the biotin-avidin, streptamer- streptavidin complexes to achieve an in situ co-localisation of molecules.
  • streptavidin-binding aptamers enables any other aptamer, or any other nucleic acid, to be linked to powerful detection systems based on the high affinity interaction of streptavidin and biotin, known to the art and widely available commercially.
  • streptavidin derivatised with a wide variety of fluorochromes such as fluorescein, Texas Red®, phycoerythryn, phycocyanin, BODIPY®, Rhodamine RedTM and so on
  • fluorochromes such as fluorescein, Texas Red®, phycoerythryn, phycocyanin, BODIPY®, Rhodamine RedTM and so on
  • streptavidin labelled with the fluorochrome Alexa Fluor®-488 linked through the carboxyl side-chains of streptavidin aptamers. It can be used, for example, via the adaptamer technology, to detect a second molecule on the surface of cells, using flow cytometry and confocal microscopy.
  • enzyme-derivatised such as alkaline phosphatase, horseradish peroxidase
  • streptavidin to enhance the sensitivity by signal amplification of molecular detection in applications such as histochemistry, ELISA and high-throughput screening.
  • 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.
  • 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.
  • Figure 1 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 la 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 (t) 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 2 is a native gel mobility shift assay for streptavidin binding to SA19 aptamer, where:
  • Figure 3 is an overlay of sensprgrams showing the effect of biotin saturated- streptavidin on the binding of aptamer and its specificity, where:
  • 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 4 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 5 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. 6 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 7 is a native gel mobility shift assay of streptavidin binding to chimeric SA19 and adaptamer, where:
  • the oligonucleotide library (5 ' -
  • 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% polyacrylamide/SM 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°C.
  • RNA was then mixed with 1 mg of Dynabeads M-280 Streptavidin (SA) (Dynal Biotech, UK) that were previously saturated with 8.00 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/ biotin-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
  • nucleotide analogue 6-(2-deoxy- ⁇ -D-e ⁇ ythropentofuranosyl)-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 KC1, 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 SA19-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 KCI, 10 mM MgCh, 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 KC1 and 10 mM MgC .
  • 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 6B 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 M . PC-E magnet before carrying on the experiments.
  • 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. 1), 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 biotin-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.
  • aptamer bound was however, approximately half of that on flow cells 1 and 3 (Fig. 3A).
  • the kinetics of the interaction between the aptamer and the streptavidin protein were not affected by the presence of biotin (data not shown).
  • 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. 3B).
  • 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. 4).
  • 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 secondary structure of SA19 RNA was probed using a combination of chemical and enzymatic probes.
  • the footprint with VI and S 1 nucleases allowed us to delineate the binding site of streptavidin on SA19 aptamer (Fig. 5A,B).
  • the predicted secondary structure of the representative SA 19 aptamer (Fig. 5A) 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 S 1 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).
  • 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- functional aptamers that we called adaptamers.
  • a CD4- streptavidin-binding adaptamer can be used to capture CD 4 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/ biotin-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 aptamères de streptavidine comprenant, de préférence, des acides nucléiques 2-F'. Les aptamères sont amplement utilisés dans les méthodes diagnostiques, constituant des substituts de la biotine dans les analyses de liaison biotine-streptavidine classiques.
PCT/GB2002/000363 2001-01-29 2002-01-29 Streptavidine WO2002061078A2 (fr)

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AU2002225234A AU2002225234A1 (en) 2001-01-29 2002-01-29 Streptavidin aptamere

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GB0102272.2 2001-01-29
GB0102271A GB0102271D0 (en) 2001-01-29 2001-01-29 Streptavidin
GB0102271.4 2001-01-29
GB0102272A GB0102272D0 (en) 2001-01-29 2001-01-29 Biligands

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2829604A4 (fr) * 2012-03-23 2016-01-27 Nec Solution Innovators Ltd Dispositif pour l'analyse cible de la streptavidine, et procédé d'analyse
CN106636104A (zh) * 2016-11-15 2017-05-10 河南省农业科学院 借助lspr‑selex方法筛选的特异结合链霉亲和素的核酸适配体序列及其应用
US9880174B2 (en) 2012-03-23 2018-01-30 Nec Solution Innovators, Ltd. Device and method for analyzing target

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004403A1 (fr) * 1994-08-02 1996-02-15 Nexstar Pharmaceuticals, Inc. Molecules chimeriques selectionnees par procede selex (evolution systematique de ligands par enrichissement exponentiel)
WO1999027133A1 (fr) * 1997-11-26 1999-06-03 Medical Research Council Procedure selex amelioree et aptamere anti-cd4
WO1999060169A1 (fr) * 1998-05-20 1999-11-25 Molecular Machines, Inc. Dispositifs multimoleculaires, systemes d'administration de medicaments et selection de molecule unique
WO2001088123A1 (fr) * 2000-05-18 2001-11-22 Isis Innovation Limited Ligands specifiques a un isoforme de proteine prion

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004403A1 (fr) * 1994-08-02 1996-02-15 Nexstar Pharmaceuticals, Inc. Molecules chimeriques selectionnees par procede selex (evolution systematique de ligands par enrichissement exponentiel)
WO1999027133A1 (fr) * 1997-11-26 1999-06-03 Medical Research Council Procedure selex amelioree et aptamere anti-cd4
WO1999060169A1 (fr) * 1998-05-20 1999-11-25 Molecular Machines, Inc. Dispositifs multimoleculaires, systemes d'administration de medicaments et selection de molecule unique
WO2001088123A1 (fr) * 2000-05-18 2001-11-22 Isis Innovation Limited Ligands specifiques a un isoforme de proteine prion

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BACHLER MONIKA ET AL: "StreptoTag: A novel method for the isolation of RNA-binding proteins." RNA (NEW YORK), vol. 5, no. 11, November 1999 (1999-11), pages 1509-1516, XP002237133 ISSN: 1355-8382 *
MALMGREN CHARLOTTA ET AL: "Antisense RNA control of plasmid R1 replication. The dominant product of the antisense RNA-mRNA binding is not a full RNA duplex." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 19, 1997, pages 12508-12512, XP002237132 ISSN: 0021-9258 cited in the application *
SRISAWAT C ET AL: "STREPTAVIDIN APTAMERS: AFFINITY TAGS FOR THE STUDY OF RNAS AND RIBONUCLEOPROTEINS" RNA, vol. 7, no. 4, April 2001 (2001-04), pages 632-641, XP001120463 ISSN: 1355-8382 cited in the application *
TAHIRI-ALAOUI ABDESSAMAD ET AL: "High affinity nucleic acid aptamers for streptavidin incorporated into bi-specific capture ligands." NUCLEIC ACIDS RESEARCH, vol. 30, no. 10, 15 May 2002 (2002-05-15), page e45 XP002237134 ISSN: 1362-4962 *
WILSON DAVID S ET AL: "The use of mRNA display to select high-affinity protein-binding peptides" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 98, no. 7, 27 March 2001 (2001-03-27), pages 3750-3755, XP002209215 ISSN: 0027-8424 cited in the application *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2829604A4 (fr) * 2012-03-23 2016-01-27 Nec Solution Innovators Ltd Dispositif pour l'analyse cible de la streptavidine, et procédé d'analyse
US9880174B2 (en) 2012-03-23 2018-01-30 Nec Solution Innovators, Ltd. Device and method for analyzing target
US9880161B2 (en) 2012-03-23 2018-01-30 Nec Solution Innovators, Ltd. Device and method for analyzing streptavidin
CN106636104A (zh) * 2016-11-15 2017-05-10 河南省农业科学院 借助lspr‑selex方法筛选的特异结合链霉亲和素的核酸适配体序列及其应用

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