Methods developed for SELEX 1

1. Methods developed for SELEX1 Presented by: chang chu Guided by: Prof. Liu Prof. Jiang 1. Subash Chandra Bose Gopinath, Anal Bioanal Chem (2007) 387:171–182

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. Three Processes Selection of ligand sequences that bind to a target partitioning ofaptamers from non-aptamers via affinity methods amplification of bound aptamers

4. Antibody VS aptamer in therapeutic use1 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 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 A2 2. Kumar PKR, Gopinath SCB, Misono T, Kawasaki K (2004) Japanese patent JP2004-293679

8. STEP4 selection STEP5 Amplification Step 1 STEP2 Negative selection Step 3 Counter 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. • Reverse transcription • PCR • In vitro transcription • 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 roomtemperature for 10 min) • 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. • Incubating the pool RNA with the BSA-coated beads in binding buffer 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 • 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. 4.Mendonsa SD, Bowser MT (2004) Anal Chem 76:5387–5392

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