Suboptimal Structure Modelling
The toehold domain (first 15 nucleotides) of the switch RNA is crucial for the binding of the trigger RNA to the switch RNA: this domain must have minimal paired bases in the switch RNA to ensure the successful binding of this domain with the complementary sequence in the trigger RNA, which allows the unwinding of the switch RNA and permits translation of the reporter protein, RFP, to occur.
Our program can only calculate the minimal free energy structure (MFE) for each target RNA region to reduce calculation workload. In reality, different conformations of RNAs with the same sequence coexist in solution, whose and the concentrations of those populations are determined by their structures and free energy. Therefore, we manually checked the predicted structures and equilibrium concentrations of the ten suboptimal structures of each influenza switches with the lowest MFEs on the web tool developed by ViennaRNA package(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/barriers.cgi)[2]. Then we predicted the performance of each influenza switches and compared with experimental results.
The table below shows the different suboptimal structures of each switch RNA sequence:
Each suboptimal structure is labelled by numbers 1 to 10, where structure 1 has the lowest free energy(the MFE structure), and structure 10 has the highest free energy. In the "Suboptimal structures" column, only suboptimal structures with non-negligible equilibrium concentrations are considered and shown. (The equilibrium concentrations were calculated by the ViennaRNA webserver[2]: example for H5-3 switch{1}) We checked the status of the toehold domain for each suboptimal structure from the ViennaRNA webserver output[2] to see if it was open or closed.(example for H5-3 switch{2} ) The structures with an open toehold domain are shown in green, and ones with a closed toehold domain are shown in red.
The expected fluorescence(determined by the ratio of suboptimal structures with open/closed toehold domains) exhibited by E. coli cotransformed with the switch and trigger RNA after 12 hours was compared with the actual observed fluorescence during the experiment.
Switch Candidate
Suboptimal structures
Expected fluorescence
Experimental fluorescence(relative)
Conclusion
H5-1
1, 6, 7, 2
low
11.676366 (low)
true negative
H5-2
1, 2, 3, 4, 5
low
38.24207 (low)
true negative
H5-3{2}
1, 2, 4, 3
low
13.34770467 (low)
true negative
H7-1
1, 2, 3, 4
low
10.82038167 (low)
true negative
H7-2
1, 3, 2, 7
low
8.577606333 (low)
true negative
H7-3
1, 2
high
637.066 (high)
true positive
N1-1
1, 2
high
204.7757333 (high)
true positive
N1-2
1, 2
low
9.325934 (low)
true negative
N1-3
1, 2
high
9.148810667 (low)
false positive
N9-1
1, 2, 3
high
166.49639 (high)
true positive
N9-2
1, 2, 6
high
604.2225333 (high)
true positive
N9-3
1
high
10.587811 (low)
false positive
PB2-1
1, 2
high
8.756591333 (low)
false positive
PB2-2
1, 3
low
24.68932333 (low)
true negative
PB2-3
1, 2, 3, 5
high
368.0475667 (high)
true positive
From the table above, we observed that only 3 out of 15 of our predictions were incorrect, showing promising accuracy of this prediction method. None of the 3 incorrect predictions were false negatives, indicating that the suboptimal structure prediction method should be mostly applied to filtering out switch sequences with low predicted performances.
References:
[1] Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: De-novo-designed regulators of gene expression. Cell. 2014;159(4):925–39.
[2] Hofacker IL. RNA secondary structure analysis using the Vienna RNA package. Curr Protoc Bioinformatics [Internet]. 2009;Chapter 12:Unit12.2. Available from:
http://www.ncbi.nlm.nih.gov/pubmed/19496057
[3]A. Espah Borujeni, A.S. Channarasappa, and H.M. Salis, "Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites", Nucleic Acid Research, 2013
[4]H.M. Salis, E.A. Mirsky, C.A. Voigt, Nat. Biotech., 2009
[5]https://2010.igem.org/Team:Warsaw/Stage1/PromMeas
[6]Yu J, Xiao J, Ren X, Lao K, Xie XS: Probing gene expression in live cells, one protein molecule at a time. Science 2006, 311:1600-1603.
[7]J. N. Zadeh, C. D. Steenberg, J. S. Bois, B. R. Wolfe, M. B. Pierce, A. R. Khan, R. M. Dirks, N. A. Pierce. NUPACK: analysis and design of nucleic acid systems. J Comput Chem, 32:170–173, 2011.