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

Abstract

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.

In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

“…A split Broccoli consisting of two strands of RNA—Broc and Coli—was developed, demonstrating high but incomplete complementarity. The dependence of the fluorescence of F30–Broccoli cleaved aptamers on temperature, the concentration of magnesium ions, and the presence of certain oligonucleotides allow these aptamers to be used as “molecular thermometers”, biosensors, and “molecular switches” [ 101 , 102 , 103 ].…”

Binary (Split) Light‐up Aptameric Sensors

“…At echnical challenge that limits the practical use of BLAS is the lower S/N,w hich can be addressed by adopting designs of the most optimal BLAS. Indeed, it is possible to achieve S/N values of 100-200 and higher for BLAS (Table 1), even though such brightness was achieved neither in cells nor during the monitoring of nanostructure associations.A nother fundamental question to be addressed is the kinetics and mechanism of analyte recognition by BLAS.Such systems can become agood model for the understanding of association of multicomponent biological systems,a sw ell as multi-stranded hybridization probes.I nterestingly,B LAS may find application as ap edagogical tool for undergraduate [106,107] and, possibly,h igh Figure 6. Design of nucleic acid logic gates with BLAS as af luorescentreporter.A)AND logic gate as reported by Grabow and colleagues.…”

“…Such systems can become a good model for the understanding of association of multicomponent biological systems, as well as multi‐stranded hybridization probes. Interestingly, BLAS may find application as a pedagogical tool for undergraduate [106, 107] and, possibly, high school students. DNA‐based BLAS (Table 3) are preferable for this purpose because of their affordable cost and greater chemical stability as compared to RNA.…”

Smart-Responsive Nucleic Acid Nanoparticles (NANPs) with the Potential to Modulate Immune Behavior

“…However, when the halves of the split aptamer are brought together during the reassociation, the completed aptamer regains its function [40,63,64]. The development of split fluorescent aptamers, such as malachite green [65], Spinach [63], and Broccoli [66], has produced tools which are especially useful when applied as a validating output for dynamic RNA nanotechnology [63,64,66,67,68], in the visualization of NANP assemblies [45,69], and for logic gating [70].…”

“…The reassociation of the cube and anti-cube and the consecutive formation of RNA fibers was also demonstrated with the activation of a split aptamer. The RNA aptamer Broccoli [72], which binds the chemically synthesized fluorophore DFHBI-1T to mimic the natural function of GFP, was split into two separate strands termed Broc and Coli [66,68]. With both parts of the aptamer attached to cognate RNA cube strands, the Broc cube and Coli anti-cube reassociated in the presence of DFHBI-1T, allowing for the interaction to be traced via fluorescence in real time [71].…”