Real-Time Visualization of in Vitro Transcription of a Fluorescent RNA Aptamer: An Experiment for the Upper-Division Undergraduate or First-Year Graduate Laboratory

• Published in: Journal of chemical education Vol. 95; no. 10; pp. 1867 - 1871
• Main Authors: Heili, Joseph M, Gomez-Garcia, Jose, Gaut, Nathaniel J, Cash, Brock W, Aufdembrink, Lauren M, Heffron, Brent A, Shirley, Joshua D, Carlson, Erin E, Adamala, Katarzyna P, Engelhart, Aaron E
• Format: Journal Article
• Language: English
• Published: Easton American Chemical Society and Division of Chemical Education, Inc 09.10.2018; Division of Chemical Education, Inc; American Chemical Society
• Subjects: Biochemistry; Broccoli; Chemical industry; College Science; College students; Fluorescence; Genetics; Genomics; Gold; Graduate Students; Instrumentation; Laboratory Experiments; Laboratory tests; Reading; Real time; Ribonucleic acid; RNA; Science Instruction; Science Laboratories; Staining; Students; Transcription; Undergraduate Students; Visualization; Hands-On Learning/Manipulatives; Nucleic Acids/DNA/RNA; Graduate Education/Research; Upper-Division Undergraduate; Fluorescence Spectroscopy; Electrophoresis; Biochemistry
• ISSN: 0021-9584; 1938-1328
• DOI: 10.1021/acs.jchemed.7b00735

A teaching laboratory experiment is described where students prepare in vitro transcription reactions of a fluorescent RNA aptamer, named Broccoli, and observe the production of the aptamer in real-time on a fluorescence plate reader. Alternate visualization methods with minimal costs are also described for laboratories lacking this instrumentation. Two optional experiments are also described. Optional Experiment 1 involves purification of RNA transcription reactions using a commercial spin column kit and having students correlate cleanup kit yield with transcribed aptamer fluorescence. Optional Experiment 2 involves running a polyacrylamide gel of the transcription reaction with a ladder, followed by staining with (Z)-4-(3′,5′-difluoro-4′-hydroxybenzylidene)-2-methyl-1-(2″,2″,2″-trifluoroethyl)-1H-imidazol-5-(4H)-one (DFHBI-1T) (selective for Broccoli) and a second stain with SYBR Gold (nonselective, allowing for simultaneous visualization of Broccoli and ladder). This experiment has the practical advantage of enabling aptamer visualization in laboratories without a fluorescence spectrometer or plate reader, as well as the pedagogical benefit of demonstrating specific activation of the fluorescence of a small molecule by an RNA aptamer in another context (gel staining). Each experiment allows students to perform straightforward, easily understood teaching laboratory experiments, including key concepts in cellular imaging, and RNA biochemistry widely employed in biochemical research.