Computational protocol: De novo design of a synthetic riboswitch that regulates transcription termination

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Protocol publication

[…] The rational design algorithm starts from the known sequence and secondary structure of the aptamer and constructs the sequence of the remaining part of the riboswitch, i.e. the spacer and the terminator. It proceeds by proposing a large set of candidates that are then evaluated with respect to their secondary structure and properties of their folding paths, which are estimated using the RNAfold program of the Vienna RNA package (). At the proposal stage, it incorporates some knowledge of sequence and structure of terminator elements for the given biological system in which the construct is supposed to function, here E. coli. In particular, we used the fact that the terminator hairpin must have a minimal size and that it is followed by a U stretch. First, a spacer database containing randomly generated sequences with lengths between 6 and 20 nucleotides (nt) is created. The size of this database is user defined. For each position k of the 3′-part of the aptamer, we form the sequence that is complementary to the subsequence from k to the end of the aptamer. For the theophylline aptamer, values of k ranging from position 21 to 32 were used. The sequences of the aptamer, a spacer from the database, a complementary sequence and the U stretch are concatenated to form a complete riboswitch candidate.Each of these constructs is then evaluated by folding simulations. In the current implementation, folding paths are represented as a sequence of secondary structures computed for sub-sequences starting at the 5′-end. We used individual transcription steps of 5–10 nt to simulate co-transcriptional folding with varying elongation speed. Secondary structures are computed by RNAfold, a component of the Vienna RNA Package (), with parameter settings -d2 -noPS -noLP. If one of the transcription intermediates forms base pairs between aptamer and spacer, it is likely that this will interfere with the ligand-binding properties; hence, such a candidate is rejected. On the other hand, if the full-length transcript does not contain a single hairpin structure composed of the aptamer 3′-part, the spacer and the sequence complementary to the 3′-part of the aptamer, the construct cannot form a functional terminator and is also rejected. For each construct that passes these filters, additional features are calculated that are used as further selection criteria. The energy difference between the minimum free energy (MFE) fold of the full-length construct and a structure where the aptamer region is constrained to form the ligand-binding competent fold is calculated. This parameter provides a rough measure for the stabilizing effect required to prevent terminator formation when the ligand is bound. Furthermore, a z-score of the structure downstream of the constrained aptamer fold is estimated as follows: The stability (MFE) of the candidate sequence is compared with the mean μ and standard deviation σ of the folding energies computed for a set of 1000 di-nucleotide shuffled sequences. A positive value of the z-score z = (MFE − μ)/σ indicates a structure less stable than expected by chance. This is used to estimate whether stable local structures downstream of the constrained aptamer fold might interfere with transcription.The six experimentally tested synthetic riboswitch constructs are summarized in A. Secondary structures and free energy values of individual sequences were calculated using RNAfold 2.0.7.Given the dissociation constant Kd, the binding energy is given by the following: ΔG = RTln Kd. With T = 298 K, R = 1.98717 cal/K and the experimentally determined value Kd = 0.32 µM () for the theophylline aptamer, we obtain ΔG = −8.86 kcal/mol for the stabilization of the aptamer structure. […]

Pipeline specifications

Software tools RNAfold, ViennaRNA
Application RNA structure analysis
Organisms Escherichia coli
Chemicals Theophylline