182 9 Small Molecule-Responsive RNA Switches (Bacteria)
aptamers against numerous small molecules [6], isolating new aptamers for
novel targets for synthetic riboswitch applications is still likely to remain a chal-
lenge in itself [7]. Although in vitro selection often yields aptamers with respect-
able affinity and specificity, a major challenge when using them in the cellular
context is the difficulty in predicting how the affinity, stability, or folding of the
aptamers is altered inside the cells. To mitigate such uncertainty, Gallivan and
coworkers used a pool of affinity‐enriched aptamers from an in vitro selection,
rather than few isolated aptamer clones, in their effort to engineer riboswitches
that respond to an herbicide in Escherichia coli [5].
In another notable effort, Dixon and coworkers conducted an in vivo screen to
modify a natural aptamer to recognize an alternative synthetic analog [8].
Although this strategy is likely to be limited for engineering aptamers for a ligand
that are structurally similar to an existing ligand, it represents a viable alternative
route to obtain a set of orthogonal riboswitches.9.2.2 Screening and Genetic Selection
With an aptamer in hand, designing an appropriate expression platform becomes
the primary challenge in engineering small molecule‐responsive riboswitches.
By far, the most successful strategies have employed some form of medium‐ to
high‐throughput screening or genetic selection at this stage to discover func-
tional riboswitches with desired characteristics. Generally, a short stretch of
sequence near an aptamer embedded in the 5′ UTR is randomized with an antic-
ipation that a subset of those sequences will function as an expression platform.
This pool of riboswitch mutants is subjected to suitable screening or selection
steps to enrich functional riboswitches and to eventually isolate individual
clones.
Genetic selection enables rapid enrichment of potential riboswitches from a
large population (>10^5 ) of mutants by coupling the survival or growth of the
bacteria with those expressing functional riboswitch mutants. Nomura and
Yokobayashi isolated riboswitches entirely through genetic selection for the first
time [9]. This was achieved by the use of tetracycline antiporter (tetA) as a selec-
tion marker to enable both ON and OFF selection. In this system, ON cells are
selected using tetracycline and OFF cells are selected using NiCl 2 added to the
culture media [10]. The group later improved the method by adding a fluores-
cent reporter gene (GFPuv) as a translational fusion to TetA to enable rapid
screening of the genetically selected mutants [11].
Alternatively, Topp and Gallivan devised a selection strategy based on cell
motility by coupling the riboswitch output with the expression of cheZ, which
confers cell motility when expressed in a ΔcheZ host [12]. In this method, cells
are physically isolated on a semisoft agar plate based on their motility.
Although genetic selection enables examination of relatively large number of
mutants primarily limited by the transformation efficiency, it is often difficult to
fine‐tune the selection pressures to engineer devices with precise characteris-
tics. A complementary strategy is to employ a reporter gene such as green fluo-
rescent protein (GFP) and quantitatively measure the riboswitch performance