Figures & data
Figure 1. Various approaches for maximizing the folding stability of the ultimate SELEX winner. (a) Using rational mutagenesis to improve aptamer folding. The initial Spinach aptamer was improved to Spinach2 aptamer by making rational mutations (red) in the Spinach sequence to remove bulges and reduce the presence of sequences that could potentially contribute to alternative folding structures. The improved Spinach2 aptamer showed brighter fluorescence in cells [Citation30]. (b) Using low magnesium (Mg2+) concentration during SELEX and directed evolution in bacteria. Broccoli was selected using a few rounds of SELEX followed by a bacteria-based FACS screen of a partially selected SELEX library. Both the in vitro selection and the FACS sorting were performed in buffers with low magnesium (100 µM). The resulting aptamers showed much lower magnesium dependence than Spinach, and additionally showed increased intracellular fluorescence when expressed in mammalian cells [Citation31]. (c) RNA-stabilizing ligands can act as molecular chaperones. the DFHBI fluorophore (light green) was modified so that it could achieve additional interactions with the RNA structure. The modified fluorophore is termed BI (dark green). The additional contacts further stabilize and trap Broccoli aptamer into the folded state, and thus push the equilibrium towards generating more fluorescent aptamer molecules. Broccoli-expressing cells cultured with BI showed more fluorescence than cells cultured with DFHBI-1T and other previously synthesized Broccoli fluorophores [Citation24]. (d) Using highly folded natural riboswitch aptamers as scaffolds for SELEX libraries. The xpt-pbuX guanine (Gua) riboswitch aptamer, the Vc2 cyclic di-GMP (CDG) riboswitch aptamer, and the S. Mansoni hammerhead ribozyme were transformed into GR scaffold, CG scaffold and HR scaffold respectively [Citation9]. The transformations were done by complete randomization of the junction region (red). Complete randomization means that each original nucleotide in the aptamer was mutated to either A, C, G or U with equal 25% probability. Complete randomization does not modify sequence length of the region. GR, CG and HR scaffolds were used to evolve aptamers that bind 5-hydroxyl-L-tryptophan (5HTP) and 3,4-dihydroxy-L-phenylalanine (L-DOPA) [Citation9]. The add adenine riboswitch was transformed into add a scaffold by a novel randomization scheme called ‘the Sprouts-and-Clips’ (green) [Citation11]. This method not only mutates the identity of each nucleotide, but also expands or contracts the sequence length, resulting in potential ligand binding pockets of different sizes. The new Squash aptamer evolved from the add a scaffold.
![Figure 1. Various approaches for maximizing the folding stability of the ultimate SELEX winner. (a) Using rational mutagenesis to improve aptamer folding. The initial Spinach aptamer was improved to Spinach2 aptamer by making rational mutations (red) in the Spinach sequence to remove bulges and reduce the presence of sequences that could potentially contribute to alternative folding structures. The improved Spinach2 aptamer showed brighter fluorescence in cells [Citation30]. (b) Using low magnesium (Mg2+) concentration during SELEX and directed evolution in bacteria. Broccoli was selected using a few rounds of SELEX followed by a bacteria-based FACS screen of a partially selected SELEX library. Both the in vitro selection and the FACS sorting were performed in buffers with low magnesium (100 µM). The resulting aptamers showed much lower magnesium dependence than Spinach, and additionally showed increased intracellular fluorescence when expressed in mammalian cells [Citation31]. (c) RNA-stabilizing ligands can act as molecular chaperones. the DFHBI fluorophore (light green) was modified so that it could achieve additional interactions with the RNA structure. The modified fluorophore is termed BI (dark green). The additional contacts further stabilize and trap Broccoli aptamer into the folded state, and thus push the equilibrium towards generating more fluorescent aptamer molecules. Broccoli-expressing cells cultured with BI showed more fluorescence than cells cultured with DFHBI-1T and other previously synthesized Broccoli fluorophores [Citation24]. (d) Using highly folded natural riboswitch aptamers as scaffolds for SELEX libraries. The xpt-pbuX guanine (Gua) riboswitch aptamer, the Vc2 cyclic di-GMP (CDG) riboswitch aptamer, and the S. Mansoni hammerhead ribozyme were transformed into GR scaffold, CG scaffold and HR scaffold respectively [Citation9]. The transformations were done by complete randomization of the junction region (red). Complete randomization means that each original nucleotide in the aptamer was mutated to either A, C, G or U with equal 25% probability. Complete randomization does not modify sequence length of the region. GR, CG and HR scaffolds were used to evolve aptamers that bind 5-hydroxyl-L-tryptophan (5HTP) and 3,4-dihydroxy-L-phenylalanine (L-DOPA) [Citation9]. The add adenine riboswitch was transformed into add a scaffold by a novel randomization scheme called ‘the Sprouts-and-Clips’ (green) [Citation11]. This method not only mutates the identity of each nucleotide, but also expands or contracts the sequence length, resulting in potential ligand binding pockets of different sizes. The new Squash aptamer evolved from the add a scaffold.](/cms/asset/5ec2a833-8c5e-44d1-9a25-de47743ed897/krnb_a_2206248_f0001_oc.jpg)
Figure 2. Using RNA scaffolds to promote folding of artificial RNA aptamers. (a) Secondary structure of F30-Broccoli fusion. Broccoli was inserted into one arm of F30 by removing the terminal loop and replacing with Broccoli sequence [Citation23]. (b) A novel design which uses the F30 RNA scaffold to develop sensors. In this example, S-adenosyl methionine (SAM) (dark blue) binding aptamer (light blue) was inserted into one arm of the F30 (orange) in such a way that the F30 arm was unstable and thus destabilized the entire F30 three-way junction region. The adjacent arm harbouring Broccoli aptamer (green) also became unfolded. Upon binding SAM, the SAM aptamer helical stem was stabilized, which promoted the folding of the entire junction region via Mg2+ ions and non-Watson-Crick interactions. Broccoli aptamer folds and binds DFHBI-1T to produce green fluorescence as a result [Citation52].
![Figure 2. Using RNA scaffolds to promote folding of artificial RNA aptamers. (a) Secondary structure of F30-Broccoli fusion. Broccoli was inserted into one arm of F30 by removing the terminal loop and replacing with Broccoli sequence [Citation23]. (b) A novel design which uses the F30 RNA scaffold to develop sensors. In this example, S-adenosyl methionine (SAM) (dark blue) binding aptamer (light blue) was inserted into one arm of the F30 (orange) in such a way that the F30 arm was unstable and thus destabilized the entire F30 three-way junction region. The adjacent arm harbouring Broccoli aptamer (green) also became unfolded. Upon binding SAM, the SAM aptamer helical stem was stabilized, which promoted the folding of the entire junction region via Mg2+ ions and non-Watson-Crick interactions. Broccoli aptamer folds and binds DFHBI-1T to produce green fluorescence as a result [Citation52].](/cms/asset/88bfc4a6-e132-4da4-ac02-0857fcb7488b/krnb_a_2206248_f0002_oc.jpg)