?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.relation=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F8931%2F&rft.title=The+Development+of+Fluorescent+Sensors+for+Specific+RNA+Using+Chemistry+and+Combinatorial+Biology&rft.creator=Sparano%2C+Brian+Andrew&rft.description=Here+we+describe+the+development+of+small+molecule-based+fluorescent+sensors+for+RNA.+The+sensors+described+here+and+the+new+paradigm+they+represent+are+a+convergence+of+chemical+and+biological+methods.+The+resulting+sensor+system+consists+of+two+components%3A+a+rationally+designed+small-molecule+fluorescent+sensor+and+a+reporter+RNA+sequence+that+binds+to+and+enhances+its+fluorescence%2C+isolated+with+combinatorial+biology.+First%2C+we+rationally+designed+and+synthesized+a+series+of+modular+fluorescent+chemosensors+which+would+be+quenched+in+solution+and+fluorescent+only+when+bound+to+their+target.+These+sensors+consist+of+a+fluorophore+(2'%2C7'-dichlorofluorescein%2C+DCF)+conjugated+to+a+pair+of+quencher+units+(aniline+derivatives+in+this+case)+which+quench+the+fluorescence+of+DCF+by+the+PET+(photoinduced+electron+transfer)+mechanism.+NMR+and+fluorescent+spectroscopic+analyses+of+these+DCF+derivatives+revealed+important+correlations+between+their+conformations%2C+the+PET%2C+and+fluorescent+intensity+in+addition+to+insight+on+their+solvatochromism+and+pH+dependence.+These+studies+allowed+us+to+choose+a+candidate+sensor+for+the+next+step%3A+using+combinatorial+biology+to+isolate+a+reporter+sequence.+In+order+to+isolate+RNA+that+would+enhance+the+fluorescence+of+our+sensor%2C+RNA+that+bound+to+the+aniline-based+quencher+was+isolated+via+in+vitro+selection+(a.k.a+SELEX+or+systematic+evolution+of+ligands+by+exponential+enrichment).+These+RNA+sequences+(aptamers)+were+then+screened+for+their+ability+to+switch+on+the+fluorescence+of+the+sensor.+One+of+these+aptamers+was+found+to+enhance+the+fluorescence+intensity+of+the+DCF-aniline+conjugate+in+a+concentration-dependent+manner%2C+leading+to+further+study+of+that+aptamer+with+mutation+studies+and+additional+in+vitro+selection+experiments.+To+demonstrate+the+power+and+generality+of+this+approach%2C+a+parallel+in+vitro+selection+was+performed+with+aptamers+from+this+selection+having+similar+activities.+These+results+show+that+one+can+develop+fluorescence-inducing+reporter+RNA+and+morph+it+into+remotely+related+sequences+without+prior+structural+insight+into+RNA-ligand+binding+through+the+rational+design+of+fluorescent+chemosensors+and+subsequent+isolation+of+RNA+from+combinatorial+libraries.&rft.date=2007-09-27&rft.type=University+of+Pittsburgh+ETD&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F8931%2F1%2FSparano_8-13-07.pdf&rft.identifier=++Sparano%2C+Brian+Andrew++(2007)+The+Development+of+Fluorescent+Sensors+for+Specific+RNA+Using+Chemistry+and+Combinatorial+Biology.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++