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.
Bibliographical noteFunding Information:
This work was supported by University of Minnesota startup funds to A.E.E. and K.P.A., and University of Minnesota Department of Chemistry teaching funds for E.E.C. and J.D.S. Experiments performed at the Itasca retreats were supported by the MCSB graduate program at the University of Minnesota. We gratefully acknowledge the assistance of the University of Minnesota faculty, staff, and teaching assistants involved in the planning and execution of the laboratories in which these experiments were performed, particularly Ian Armitage, Taysir Bader, Huarui Cui, Michael Freeman, Michael Jarcho, Sue Knoblauch, Andrew Marshall, Megan McCarthy, Michael Pickett-Leonard, Barbara Pinch, Taylor Reid, Amber Schoenecker, Shabnam Sharifzadeh, Margaret Titus, and Yiao Wang. We thank members of the Engelhart and Adamala laboratories and Mary Peek (Georgia Institute of Technology) for helpful comments.
© 2018 American Chemical Society and Division of Chemical Education, Inc.
- Fluorescence Spectroscopy
- Graduate Education/Research
- Hands-On Learning/Manipulatives
- Nucleic Acids/DNA/RNA
- Upper-Division Undergraduate