淀粉样β-蛋白，阿尔茨海默氏病的病原体适配的选择
Summary
适配选定的短ribo-/deoxyribo-oligonucleotides<em在体外</em>基于对特定目标的亲和力的进化方法。适配与多才多艺的治疗，诊断和研究中的应用的分子识别工具。我们表现出淀粉样β-蛋白，阿尔茨海默氏病的病原体选择适配方法。
Abstract
阿尔茨海默氏病（AD）是一个渐进的，年龄依赖性的神经退行性疾病，一个阴险的过程中呈现其症状前诊断困难 1 。明确AD的诊断是只取得了尸检，从而确立了症状前诊断，早在公元2,3有效的治疗方法开发和管理的关键。
淀粉样β蛋白（Aβ）是AD的发病机制的核心。水溶性，低聚Aβ的集会被认为影响到公元4,5神经毒性的潜在的突触功能障碍和神经元的损失。各种形式的可溶性Aβ组件已被描述，然而，他们的相互关系，并到AD的病因和发病机制相关的复杂，并不能很好地理解6。特定的分子识别工具可能解开Aβ的集会之间的关系，并促进这些组件的检测和表征，在病程的早期症状出现前。分子识别通常依赖于抗体。然而，替代类分子识别工具，适配，提供了重要的优势相抗体 7,8 。适配是生成的寡核苷酸在体外选择：指数富集（SELEX）9,10配体系统进化。 SELEX是一个反复的过程，类似于达尔文的进化论，允许选择，放大，浓缩，并长期存在的属性，例如，狂热的，具体的，配体结合（适配）或催化活性（核酶和DNAzymes）。
尽管作为现代生物技术和医药11工具适配的出现，他们一直在淀粉样蛋白领域得到充分利用。少数RNA或单链DNA适配已选定反对各种形式的朊蛋白（PRP）12-16。针对重组牛朊蛋白产生一种RNA适体识别牛的PRP -β17，一种可溶性的寡聚体，β-折叠丰富的全长度的PrP构象的变体，形成淀粉样纤维18。使用单体和一些纤维状的β2 -微球蛋白（β2米 ），发现除了β2米纤维19绑定某些其他淀粉样蛋白纤维的形式产生的适配。 Ylera 等人 。描述对固定单体Aβ4020选定的RNA适配。没想到，这些适配约束纤维状Aβ40。总之，这些数据提出了几个重要问题。为什么对单体蛋白质选定的适配认识到自己的聚合物的形式是什么？对淀粉样蛋白的单体和/或低聚物形式的适配得到吗？为了解决这些问题，我们试图选择适配为共价键稳定的低聚Aβ40产生的光致交联未修改的蛋白质（PICUP）22,23 21 。 17,19,20以前的研究结果相似，这些适配Aβ和其他几个可能承认一个潜在的共同的淀粉样蛋白的结构aptatope 21的淀粉样蛋白纤维的反应。在这里，我们目前的SELEX方法用于生产这些适配21。
Protocol
第1部分：蛋白制备和交联最初，为SELEX实验中使用的蛋白质是1,1,1,3,3,3 -六氟-2 -丙醇（异丙醇）预处理，获得同质化，聚合的筹备工作， 以前 23描述。这一步是必要的，因为预先形成的集合体导致淀粉样蛋白的快速聚集，导致实验的重复性差24，unaggregated，非纤维状的蛋白质形式的选择适配是不可取的。 称量〜800微克（〜180 nmol）纯Aβ40使用一个天…
Discussion
SELEX过程的出发点是随机寡核苷酸库，通常含10月12日-10月15日序列的合成。在DNA SELEX，这个库是直接使用后，产生一个单链DNA池，而在RNA SELEX，这里演示，单链DNA库先转换到一个RNA池通过体外转录酶。然后，SELEX进行迭代，即每个周期包括曝光和预定目标寡核苷酸结合，不具约束力的序列，并结合序列洗脱的粘合剂分区。在以后的周期中，目标和RNA和/或清洗化学计量可以改…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是从加州公共卫生部从美国国立卫生研究院/国家行政学院和07-65798补助AG030709支持。我们承认，伊丽莎白楼纽费尔德博士帮助和支持的项目，智红博士B.陈提供的支持和试剂最初的步骤，合成肽和氨基酸分析和安德鲁博士ð玛格丽特M. Condron 。埃林顿有益的讨论。
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 |
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Tags
AptamersAmyloid β-proteinAlzheimer’s DiseaseNeurodegenerative DisorderPresymptomatic DiagnosisEarly DiagnosisEffective TherapiesAβ AssembliesSynaptic DysfunctionNeuron LossAD EtiologyAD PathogenesisMolecular Recognition ToolsAntibodiesAptamers AdvantagesSELEX (systematic Evolution Of Ligands By Exponential Enrichment)
Cite This Article
Rahimi, F., Bitan, G. Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease. J. Vis. Exp. (39), e1955, doi:10.3791/1955 (2010).