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 1. Introduction Basic processes Features 2. Methods 3. Aptamer base 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 of aptamers from non-aptamers via affinity methods amplification of bound aptamers Antibody VS aptamer in therapeutic use1 Advantages of antibodies • Pharmacokinetic other systemic properties of antibodies are often Advantages of and aptamers sufficient to support product chemically development • Aptamers are produced in a readily scalable process • Comparatively long circulating • Chemical production processhalf-lives is not prone to viral or bacterial • Not susceptible to nuclease degradation contamination • Antibody technologies are widely distributed • Non-immunogenic • Smaller size allows more efficient entry into biological compartments Limitations of antibodies • Antibodies are produced biologically in a process • Limitations of aptamers • Viral or bacterial contamination • Pharmacokinetic and other systemic properties are variable and often • Large limits hard size 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 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 • 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 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 STEP2 STEP4 Step Step 31STEP5 Negative selection Amplification Counterselection selection •Coating the whole virus onto •The •Incubating unbound molecules the was pool were again beads •The unbound RNA collected incubated with beads RNA and with collected withthe theBSAhelp of coated with beads the target A/Panama virus coated in binding •Reverse transcription magnet and applied onto •BSA Blocking buffer for with 10 min. beads coated the same •After thisA/Aichi incubation, thethe beads were subtype virus as •PCR •Washing thetimes coated beads. washed three 300 μl of counter-selection towith remove binding buffer. molecules specific to A/Aichi. •In vitro transcription •Denaturing the •Incubating for 10pool min.RNA(90 °C for 2 min andwere allowed to coolwith at a •Bound RNAs recovered room hot 7M temperature urea solution.for 10 min) •Bound molecules were precipitated by ethanol. Flow cytometry • Detecting the fluorescence Leu3a-FITC RNA aptamerFITC Using gel electrophoresis 0.7% native agarose gel 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 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 • • • • • 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. 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