1. 1. Subash Chandra Bose Gopinath, Anal Bioanal Chem (2007) 387:171–182 Presented by: chang chu Guided by: Prof. Liu Prof. Jiang Methods developed for SELEX 1

2. 1. Introduction Basic processes Features 2. Methods 3. Aptamer base

3. 1. SELEX (systematic evolution of ligands by exponential enrichment) is a process that involves the progressive purification from a combinatorial library of nucleic acid ligands with a high affinity for a particular target by repeated rounds of partitioning and amplification. Selection of ligand sequences that bind to a target Three Processes partitioning of aptamers from non-aptamers via affinity methods amplification of bound aptamers

4. Advantages of antibodies Pharmacokinetic and other systemic properties of antibodies are often sufficient to support product development Comparatively long circulating half-lives Not susceptible to nuclease degradation Antibody technologies are widely distributed Limitations of antibodies Antibodies are produced biologically in a process Viral or bacterial contamination Large size limits Antibody VS aptamer in therapeutic use 1 Advantages of aptamers Aptamers are produced chemically in a readily scalable process Chemical production process is not prone to viral or bacterial contamination Non-immunogenic Smaller size allows more efficient entry into biological compartments Limitations of aptamers Pharmacokinetic and other systemic properties are variable and often hard to predict Shorter half-life Unmodified aptamers are highly susceptible to serum degradation 1. Anthony D. Keefe, Supriya Pai and Andrew Ellington(2010). Aptamers as therapeutics. Nature

5. 2. Methods Nitrocellulose membrane filtration Using affinity surfaces Using affinity tags Using column matrices or ligands Cross-linking Antibody-based Using gel electrophoresis Surface plasmon resonance Flow cytometry Capillary electrophoresis Automated selection

6. Initial rounds of selection: long incubation times & less stringent conditions Later cycles: stringent conditions, such as changing the buffer conditions, reaction volume and time of incubation. Monovalent & divalent cations Pre-negative selection

7. Using affinity surfaces Affinity surfaces: allow proteins and small molecules to bind with them & have affinities with RNA or DNA. Magnetic beads, affinity titer plates RNA Aptamer against Panama influenza virus subtype A 2 2. Kumar PKR, Gopinath SCB, Misono T, Kawasaki K (2004) Japanese patent JP2004-293679

8. STEP2 Negative selection Incubating the pool RNA with the BSA- coated beads in binding buffer for 10 min. Step 3 Counter selection The unbound RNA was collected with the help of magnet and applied onto beads coated with the same subtype A/Aichi virus as the counter-selection to remove molecules specific to A/Aichi. Incubating for 10 min. STEP4 selection The unbound molecules were again collected and incubated with beads coated with the target A/Panama virus After this incubation, the beads were washed three times with 300 μl of binding buffer. Bound RNAs were recovered with a hot 7M urea solution. Bound molecules were precipitated by ethanol. STEP5 Amplification Reverse transcription PCR In vitro transcription Step 1 Coating the whole virus onto beads BSA Blocking Washing the coated beads. Denaturing the pool RNA(90 °C for 2 min and allowed to cool at room temperature for 10 min)

9. Flow cytometry Detecting the fluorescence Leu3a-FITC RNA aptamer- FITC

10. Using gel electrophoresis 0.7% native agarose gel

11. Using gel electrophoresis Electrophoresis through 4 %, polyacrylamide, TBE, 0.05 % SDS, and then recovered from the gel by the crush-and-soak method. Smith D, Kirschenheuter GP, Charlton J, Guidot DM, Repine JE(1995) Chem Biol 2:741–750

12. Using capillary electrophoresis 4.Mendonsa SD, Bowser MT (2004) Anal Chem 76:5387–5392 The nucleic acid sequences that bind the target undergo a mobility shift, migrating at a different rate, allowing them to be separated from the inactive sequences. 4 Thus, there is no need to wash the active sequences off a column as in conventional SELEX, eliminating any kinetic bias. Higher speed, better resolution capacity, minimal sample dilution, fewer cycles.

13. a poly (vinyl alcohol)-coated capillary 40.2 cm long and with an inner diameter of 50 μm The sample was applied to the capillary at 5 psi for 5 s and monitored under UV detection at 254 nm After the nonspecific species had migrated out, the CE fractions containing specific DNA sequences were collected PCR Anti-IgE aptamers with dissociation constants as low as 27 nM were obtained in only two rounds of selection.

14. 5. Jose Cruz-Toledo1,*,y, Maureen McKeague2,*,y, Xueru Zhang2, Amanda Giamberardino2, Erin McConnell2, Tariq Francis2, Maria C. DeRosa2,3,* and Michel Dumontier1,3,4,* Database, Vol. 2012, Article ID bas006, doi:10.1093/database/bas006