Amyloid β प्रोटीन, अल्जाइमर रोग की प्रेरणा का एजेंट के लिए Aptamers का चयन
Summary
Aptamers कम ribo-/deoxyribo-oligonucleotides द्वारा चयनित कर रहे हैं<em> इन विट्रो में</em> विकास के एक विशेष लक्ष्य के लिए आत्मीयता पर आधारित विधियों. Aptamers बहुमुखी चिकित्सकीय, नैदानिक, अनुसंधान और अनुप्रयोगों के साथ आणविक मान्यता उपकरण हैं. हम amyloid β प्रोटीन, अल्जाइमर रोग की प्रेरणा का एजेंट के लिए aptamers के चयन के लिए तरीकों को प्रदर्शित करता है.
Abstract
Alzheimer’s disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid β-protein (Aβ) is central to AD pathogenesis. Soluble, oligomeric Aβ assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble Aβ assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst Aβ assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-β17, a soluble, oligomeric, β-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar β2-microglobulin (β2m) were found to bind fibrils of certain other amyloidogenic proteins besides β2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric Aβ4020. Unexpectedly, these aptamers bound fibrillar Aβ40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric Aβ4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of Aβ and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.
Protocol
Discussion
SELEX प्रक्रिया के शुरुआती बिंदु एक यादृच्छिक oligonucleotide आम तौर पर 10 12 -10 15 दृश्यों से युक्त पुस्तकालय के संश्लेषण है. डीएनए SELEX में, इस लायब्रेरी सीधे प्रयोग किया जाता है के बाद एक ssDNA पूल उत्पन्न होता है, जब?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम NIH / एनआईए और 07-65798 से सार्वजनिक स्वास्थ्य के कैलिफोर्निया विभाग से AG030709 अनुदान द्वारा समर्थित किया गया. हम पेप्टाइड संश्लेषण और एमिनो एसिड विश्लेषण, की मदद करने और परियोजना, समर्थन और अभिकर्मकों प्रदान करने के लिए डा. ची हांग बी चेन के प्रारंभिक कदम का समर्थन करने के लिए डॉ. एलिजाबेथ एफ Neufeld, और डॉ. एंड्रयू विकास के लिए मार्गरेट एम. Condron स्वीकार करते हैं उपयोगी विचार – विमर्श के लिए. Ellington.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Aβ40 | UCLA Biopolymers Laboratory | Lyophilized powder | ||
| MX5 Automated-S Microbalance | Mettler Toledo | |||
| Silicon-coated, 1.6-ml tubes | Denville Scientific | C19033 or C19035 | ||
| 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) | TCI America | H0424 | Use in a fume hood. | |
| Ammonium persulfate | Sigma | A-7460 | Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. | |
| Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate | Sigma | 224758-1G | Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h. | |
| Dithiothreitol (DTT) | Sigma | 43815 | ||
| D-Salt™ Excellulose™ desalting columns | Thermo Scientific | 20449 | ||
| Ammonium acetate | Fisher Scientific | A637-500 | ||
| Silicon-coated, 0.6-ml tubes | Denville Scientific | C19063 | ||
| Novex Tricine Gels (10–20%) | Invitrogen | EC6625B0X | 10-well; mini size (8 cm X 8 cm); 25 μl loading volume per well; separation range 5 kDa to 40 kDa | |
| Quartz cuvette | Hellma | 105.250-QS | ||
| Beckman DU 640 spectrophotometer | Beckman | |||
| ssDNA library | Integrated DNA Technologies | Custom-ordered | The library was designed to contain 49 random nucleotides flanked by two constant regions containing primer-binding and cloning sites: 5′-TAA TAC GAC TCA CTA TAG GGA ATT CCG CGT GTG C (N:25:25:25:25%) (N)49 G TCC GTT CGG GAT CCT C-3′ | |
| Taq DNA polymerase | USB Corporation | 71160 | Recombinant Thermus aquaticus DNA Polymerase supplied with 10× PCR Buffer and a separate tube of 25 mM MgCl2 for routine PCR. | |
| PCR Nucleotide Mix, 10 mM solution | USB Corporation | 77212 | (10 mM each dATP, dCTP, dGTP, dTTP) | |
| Forward primer | Integrated DNA Technologies | Custom-ordered | 5′-TAA TAC GAC TCA CTA TAG GGA ATT CCG CGT GTG C-3′ | |
| Reverse primer | Integrated DNA Technologies | Custom-ordered | 5′-GAG GAT CCC GAA CGG AC-3′ | |
| Thermal cycler | Denville Scientific | Techne TC-312 | ||
| QIAquick PCR Purification Kit (50) | QIAGEN | 28104 | ||
| Agarose | Denville Scientific | CA3510-8 | ||
| Conical, sterile 1.6-ml tubes with caps attached with O-rings | Denville Scientific | C19040-S | ||
| RiboMAX™ Large Scale RNA Production System–T7 | Promega | P1300 | The kit contains: 120 μl Enzyme Mix (RNA polymerase, recombinant RNasin® ribonuclease inhibitor and recombinant inorganic pyrophosphatase); 240 μl transcription 5 buffer; 100 μl each of 4 rNTPs, 100 mM; 110 U RQ1 RNase-free DNase, 1 U/μl; 10 μl linear control DNA, 1 mg/ml; 1 ml 3M sodium acetate (pH 5.2); 1.25 ml nuclease-Free water | |
| α-32P-cytidine 5′-triphosphate, 250 μCi (9.25 MBq), | Perkin Elmer | BLU008H250UC | Specific Activity: 3000 Ci (111 TBq)/mmol, 50 mM Tricine (pH 7.6) | |
| Citrate-saturated phenol:chloroform:isoamyl alcohol (125:24:1, pH 4.7) | Sigma (Fluka) | 77619 | ||
| Chloroform:Isoamyl alcohol (24:1) | Sigma | C0549 | ||
| Absolute ethanol for molecular biology | Sigma | E7023 | ||
| Z216-MK refrigerated microcentrifuge | Denville Scientific | C0216-MK | ||
| illustra ProbeQuant™ G-50 Micro Columns | GE Healthcare | Obtained from Fisher Scientific (45-001-487) | Prepacked with Sephadex™ G-50 DNA Grade and pre-equilibrated in STE buffer containing 0.15% Kathon as Biocide | |
| Triathler Bench-top Scintillation counter | Hidex Oy, Turku, Finland | Triathler LSC Model: 425-034 | ||
| Novex® TBE-Urea Sample Buffer (2×) | Invitrogen | LC6876 | ||
| 6% TBE-Urea Gels 1.0 mm, 10 wells | Invitrogen | EC6865BOX | ||
| Novex® TBE Running Buffer (5×) | Invitrogen | LC6675 | ||
| Radioactivity decontaminant | Fisher Scientific | 04-355-67 | ||
| Gel-loading tips | Denville Scientific | P3080 | ||
| XCell SureLock Mini-Cell | Invitrogen | EI0001 | XCell SureLock Mini-Cell | |
| Autoradiography film | Denville Scientific | E3018 | Use in complete darkness | |
| Autoradiography film, Hyperfilm™ ECL | Amersham Biosciences | RPN3114K | Can be used under red safe light. | |
| Membrane discs | Millipore | GSWP02500 | Mixed cellulose ester, hydrophilic, 0.22-μm disc membranes | |
| Fritted glass support base for 125-ml flask | VWR | 26316-696 | ||
| Petri dishes | Fisher Scientific | 08-757-11YZ | ||
| Urea | Fisher Scientific | AC32738-0050 | ||
| EDTA | Fisher Scientific | 118430010 | ||
| Glycogen | Sigma | G1767 | ||
| 2-Propanol for molecular biology | Sigma | I9516 | ||
| Recombinant RNase inhibitor | USB Corporation | 71571 | ||
| ImProm-II™Reverse Transcription System | Promega | A3802 | ||
| Recombinant RNase inhibitor | USB Corporation | 71571 | ||
| RapidRun™ Loading Dye | USB Corporation | 77524 |
References
- Monien, B. H., Apostolova, L. G., Bitan, G. Early diagnostics and therapeutics for Alzheimer’s disease-how early can we get there. Expert. Rev. Neurother. 6, 1293-1306 (2006).
- Nestor, P. J., Scheltens, P., Hodges, J. R. Advances in the early detection of Alzheimer’s disease. Nat. Med. 10, S34-S41 (2004).
- Kawas, C. H. Clinical practice. Early Alzheimer’s disease. N. Engl. J. Med. 349, 1056-1063 (2003).
- Haass, C., Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid β-peptide. Nat. Rev. Mol. Cell. Biol. 8, 101-112 (2007).
- Kirkitadze, M. D., Bitan, G., Teplow, D. B. Paradigm shifts in Alzheimer’s disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies. J. Neurosci. Res. 69, 567-577 (2002).
- Rahimi, F., Shanmugam, A., Bitan, G. Structure-function relationships of pre-fibrillar protein assemblies in Alzheimer’s disease and related disorders. Curr. Alzheimer Res. 5, 319-341 (2008).
- Jayasena, S. D. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin. Chem. 45, 1628-1650 (1999).
- Bunka, D. H., Stockley, P. G. Aptamers come of age – at last. Nat. Rev. Microbiol. 4, 588-596 (2006).
- Ellington, A. D., Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 346, 818-822 (1990).
- Tuerk, C., Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 249, 505-510 (1990).
- Lee, J. F., Stovall, G. M., Ellington, A. D. Aptamer therapeutics advance. Curr. Opin. Chem. Biol. 10, 282-289 (2006).
- Weiss, S. RNA aptamers specifically interact with the prion protein PrP. J. Virol. 71, 8790-8797 (1997).
- Bibby, D. F. Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins. J. Virol. Methods. 151, 107-115 (2008).
- Rhie, A. Characterization of 2′-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion. J. Biol. Chem. 278, 39697-39705 (2003).
- King, D. J., Safar, J. G., Legname, G., Prusiner, S. B. Thioaptamer interactions with prion proteins: sequence-specific and non-specific binding sites. J. Mol. Biol. 369, 1001-1014 (2007).
- Proske, D. Prion-protein-specific aptamer reduces PrPSc formation. ChemBioChem. 3, 717-725 (2002).
- Murakami, K., Nishikawa, F., Noda, K., Yokoyama, T., Nishikawa, S. Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its β isoform with high affinity. Prion. 2, 73-80 (2008).
- Luhrs, T., Zahn, R., Wuthrich, K. Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions. J. Mol. Biol. 357, 833-841 (2006).
- Bunka, D. H. Production and characterization of RNA aptamers specific for amyloid fibril epitopes. J. Biol. Chem. 282, 34500-34509 (2007).
- Ylera, F., Lurz, R., Erdmann, V. A., Furste, J. P. Selection of RNA aptamers to the Alzheimer’s disease amyloid peptide. Biochem. Biophys. Res. Commun. 290, 1583-1588 (2002).
- Rahimi, F., Murakami, K., Summers, J. L., Chen, C. H., Bitan, G. RNA aptamers generated against oligomeric Aβ40 recognize common amyloid aptatopes with low specificity but high sensitivity. PLoS ONE. 4, e7694-e7694 (2009).
- Bitan, G., Lomakin, A., Teplow, D. B. Amyloid β-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins. J. Biol. Chem. 276, 35176-35184 (2001).
- Rahimi, F., Maiti, P., Bitan, G. Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides. J. Vis. Exp. , (2009).
- Bitan, G., Fradinger, E. A., Spring, S. M., Teplow, D. B. Neurotoxic protein oligomers-what you see is not always what you get. Amyloid. 12, 88-95 (2005).
- Bitan, G. Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins. Methods Enzymol. 413, 217-236 (2006).
- Chen, C. H., Chernis, G. A., Hoang, V. Q., Landgraf, R. Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3. Proc. Natl. Acad. Sci. USA. 100, 9226-9231 (2003).
- Adams, D. S. . Lab math: a handbook of measurements, calculations, and other quantitative skills for use at the bench. , (2003).
- Gopinath, S. C. Methods developed for SELEX. Anal. Bioanal. Chem. 387, 171-182 (2007).
- Takahashi, T., Tada, K., Mihara, H. RNA aptamers selected against amyloid β-peptide (Aβ) inhibit the aggregation of Aβ. Mol. Biosyst. 5, 986-991 (2009).
