Kinetic analysis of aptazyme-regulated gene expression in a cell-free translation system: Modeling of ligand-dependent and -independent expression

Abstract

Aptazymes are useful as RNA-based switches of gene expression responsive to several types of compounds. One of the most important properties of the switching ability is the signal/noise (S/N) ratio, i.e., the ratio of gene expression in the presence of ligand to that in the absence of ligand. The present study was performed to gain a quantitative understanding of how the aptazyme S/N ratio is determined by factors involved in gene expression, such as transcription, RNA self-cleavage, RNA degradation, protein translation, and their ligand dependencies. We performed switching of gene expression using two onswitch aptazymes with different properties in a cell-free translation system, and constructed a kinetic model that quantitatively describes the dynamics of RNA and protein species involved in switching. Both theoretical and experimental analyses consistently demonstrated that factors determining both the absolute value and the dynamics of the S/N ratio are highly dependent on the routes of translation in the absence of ligand: translation from the ligand-independently cleaved RNA or leaky translation from the noncleaved RNA. The model obtained here is useful to assess the factors that restrict the S/N ratio and to improve aptazymes more efficiently.

Cell-free riboswitches

“…Nonetheless, the variety of ligands that cell-free riboswitches have been engineered to detect are quite broad compared to cell-based riboswitches. Although theophylline has been, by far, the most popular trigger molecule, cell-free riboswitches that respond to biotin, 77 tetramethylrhodamine (TMR), 46,78,96 flavin mononcleotide (FMN), 88,89,97 cGMP, 36,87 TPP, 39,42 fluoride, 48 histamine, 45 dopamine, 46 thyroxine, 46 sulphorhodamine B, 88 Hoechst dye 33258, 76 tobramycin, 76 tetracycline, 88,97 and pentadeoxyribonucleotides 114 have been designed (Tables 1 and 2). With the exception of histamine, however, these cell-free riboswitches have been constructed using known aptamers from natural riboswitches or discovered Fig.…”

“…1B) were further analyzed by Kobori et al in a modified PURE system for the purpose of optimizing riboswitch performance based on kinetic modeling. 39 It should also be noted that synthetic riboswitches developed based on the same design strategy have been shown to function in E. coli by Ogawa and Maeda, 40 and the Hartig group. 12,13 In another study, Ogawa and Maeda tethered the theophylline-responsive aptazyme to the 5 0 end of a nonsensesuppressor tRNA (sup-tRNA) to regulate translation read-through of a gene that contains an amber stop codon in a customized PURE system (Fig.…”

“…They also showed that the high concentration of theophylline needed to activate the riboswitches was due to this kinetic trapping mechanism and not due to the low permeability of theophylline through the cell membrane as was previously suggested. 112,113 Independent reports from Kobori et al 39 and Chushak et al 31 illustrate how kinetic modeling of riboswitch mechanisms based on experimental data of cell-free riboswitches can provide not only insights into the riboswitch mechanisms, but also hints for optimizing riboswitch performance. Espah Borujeni et al built a more detailed model considering additional kinetic and biophysical parameters such as cotranscriptional folding and free energy of ribosome binding.…”

“…Independent reports from Kobori et al 39 and Chushak et al 31 illustrate how kinetic modeling of riboswitch mechanisms based on experimental data of cell-free riboswitches can provide not only insights into the riboswitch mechanisms, but also hints for optimizing riboswitch performance. Espah Borujeni et al built a more detailed model considering additional kinetic and biophysical parameters such as co-transcriptional folding and free energy of ribosome binding.…”

The Theophylline Aptamer: 25 Years as an Important Tool in Cellular Engineering Research

“…Many studies have relied on the aptamer to characterize riboswitches and logic gates in a wide variety of species. The aptamer has also been used for in silico studies or optimization of riboswitch function and kinetics. − Initial work with theophylline ribozymes demonstrated that ribozymes could be rationally engineered to function in vitro . ,, The ability to engineer biology in a modular fashion was then demonstrated by linking the aptamer to a number of different catalytic RNAs, including the hammerhead, X motif, Tetrahymena group I, and HDV ribozymes through a 9-nt communication module . OR and AND logic gates were also constructed for an in vitro colorimetric assay of theophylline and cGMP using DNA-tethered gold nanoparticles .…”