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A A A A RR T T T T Adaptive binding Allosteric activation A=Aptamer T=target R=Ribozyme Apta-switch/beacon “OFF” Apta-switch/beacon “ON” “INPUT” Signal.

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Presentation on theme: "A A A A RR T T T T Adaptive binding Allosteric activation A=Aptamer T=target R=Ribozyme Apta-switch/beacon “OFF” Apta-switch/beacon “ON” “INPUT” Signal."— Presentation transcript:

1 A A A A RR T T T T Adaptive binding Allosteric activation A=Aptamer T=target R=Ribozyme Apta-switch/beacon “OFF” Apta-switch/beacon “ON” “INPUT” Signal “OUTPUT” Signal AT Modular Domains “Functional” Aptamer Your Platform Choice implement

2 Negative/Counter Selection 5’ T7 Primer Extension 5’ RNA library Hammerhead ribozyme motif N55 5’ Mg++ dependent Cleavage site pre clv Transcription Synthesized N 55 random oligo library N55 Aptamer pre clv Positive Selection RT-PCR PAGE Partitioning Promoter Selection PAGE Partitioning (+) Target (-) Target (Buffer alone or Counter-target) Library Purify Pre-cleaved Purify Cleaved Random Region Fluorophore Optional: 1. RT-PCR 2. Transcription 3. Refolding Refolding Apta-switch Selection Strategy Monitoring Enrichment (Rounds of Selection) (-) +

3 TargetExample Co 2+, Ni 2+, Cd 2+ Zn 2+, Mn 2+ Caffeine Rev Peptide Phosphorylated ERK2, Unphosphorylted ERK2 Metal Ions Small Organics Peptides Proteins Apta-switch™ (aptamer that produces a self-cleavage output signal) FlashGel™ analysis (5 minute run)

4 Apta-switch™ Demonstration Kit (Theophylline/Caffeine) 100  M Theophylline Specificity Against Theophylline vs. Caffeine 100 90 80 70 60 Ladder DEPC only RXN Buffer only 1 mM Caffeine 100  M Caffeine 10  M Caffeine 10  M Theophylline 1  M Caffeine 1  M Theophylline 1 minute reaction at 23 o C, then stopped with stop buffer containing excess EDTA. 1 mM Theophylline 500-fold sensitivity range Caffeine Theophylline

5 “Forget Antibodies. Use Aptamers!”


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