Activation of Split RNA Aptamers: Experiments Demonstrating the Enzymatic Synthesis of Short RNAs and Their Assembly As Observed by Fluorescent Response

With the advancement of ribonucleic acid (RNA) research in the field of biochemistry, ensuring that undergraduate students have both the theoretical and practical knowledge of current, cutting-edge experimental techniques is of marked importance. Many current biochemistry experiments demonstrate various basic molecular biological techniques including isolation and quantification of nucleic acids, protein gel electrophoresis, and DNA amplification reactions. While covering a wide base of skills, there is an underutilization of RNA as a pedagogical tool. This multistep laboratory experiment introduces students to the emerging field of RNA nanotechnology and to the RNA assembly and subsequent conditional activations of preprogrammed functionalities such as fluorescence. To achieve this, a sequence of standard molecular techniques such as PCR, DNA purification, RNA transcription and purification, RNA quantification, and self-assembly followed by analysis with electrophoretic mobility shift assays and spectroscopic quantification are carried out in a semester-long curriculum offered to undergraduate and lower-level graduate students following a prerequisite biochemistry lab course.