(670e) In silico Design and Validation of Riboswitches for the High-Throughput Detection of Isoflavone Genistein
AIChE Annual Meeting
Friday, November 20, 2020 - 8:45am to 9:00am
Recent efforts have involved the use of microbial factories, which have been enabled by the relative ease for de novo gene synthesis and the corresponding heterologous expression . Despite the success of these approaches, the detection of the produced compounds generally involves the use of expensive and tedious liquid chromatography methods . Alternatively, recent works have described novel biosensors for in situ determination of the recombinantly expressed compounds including, riboswitches enzyme-based and optical biosensors . Due to their ease of manipulation and specificity, riboswitches have recently emerged as robust alternatives for tracking microbial product production . Riboswitches are segments of RNA responsible for regulating the production of proteins and therefore can be incorporated into processes for highly-sensitive selection and detection of the compounds of interest . Despite the potential of these biosensors, this approach relies on exploring a relatively high number of segments (i.e., aptamers) to search for increased specificity. This process could be lengthy and sometimes might even exceed the transformation limit of the microorganism and the number of attainable experiments. As a result, the associated costs could be prohibitive.
To try to overcome this issue, here, we aimed at accelerating the search for testable aptamers by conducting in silico interaction experiments of the candidate RNA sequences with genistein. For this purpose, the secondary structure of the aptamers was predicted via the ViennaRNA package by considering an initial database of 97 nucleotides with 3 regions of folding comprised of 5 variable nucleotides . Also, calculations of the conformational free energy were carried for the active (i,e., ON) and inactive (i.e., OFF) conformations. With the results, it was possible to calculate the probability of occupation of each nucleotide in each variable position of the binding region to the ligand (i.e., genistein) and, therefore, the number of possible useful aptamers were successfully reduced by nine orders of magnitude. Also, terminators (interrupting transcription) and aptamers that are active either in the presence or absence of ligands, were found, which would allow validating the biosensors experimentally in the future. The tertiary structure of the identified aptamers, in conjunction with genistein, was predicted via AutoDock Vina. Additional details of the interactions were explored by Molecular Dynamics (MD) simulations in GROMACS to reduce even further the number of candidates for experimental evaluation .
 I. C. Munro et al., âSoy Isoflavones: a Safety Review,â Nutr. Rev., vol. 61, no. 1, pp. 1â33, Jan. 2003, doi: 10.1301/nr.2003.janr.1-33.
 Barnes, S. (1998). Evolution of the Health Benefits of Soy Isoflavones. Experimental Biology and Medicine, 217(3), 386â396. https://doi.org/10.3181/00379727-217-44249
 G. Cheng, B. Wilczek, M. Warner, J. Gustafsson, and B.-M. Landgren, âIsoflavone treatment for acute menopausal symptoms,â Menopause, vol. 14, no. 3, pp. 468â473, May 2007, doi: 10.1097/GME.0b013e31802cc7d0.
 D. C. Knight, J. B. Howes, and J. A. Eden, âThe effect of PromensilTM, an isoflavone extract, on menopausal symptoms,â Climacteric, vol. 2, no. 2, pp. 79â84, Jan. 1999, doi: 10.3109/13697139909025570.
 K. Han, âBenefits of soy isoflavone therapeutic regimen on menopausal symptoms,â Obstet. Gynecol., vol. 99, no. 3, pp. 389â394, Mar. 2002, doi: 10.1016/S0029-7844(01)01744-6.
 W. Mark J., C. Andreas, and H. Paul E., âProcess of preparing,â US5554519A, 1995.
 G. KOCHS and H. GRISEBACH, âEnzymic synthesis of isoflavones,â Eur. J. Biochem., vol. 155, no. 2, pp. 311â318, Mar. 1986, doi: 10.1111/j.1432-1033.1986.tb09492.x.
 Huang, B., Guo, J., Yi, B., Yu, X., Sun, L., & Chen, W. (2008). Heterologous production of secondary metabolites as pharmaceuticals in Saccharomyces cerevisiae. Biotechnology Letters, 30(7), 1121â1137. https://doi.org/10.1007/s10529-008-9663-z
 Q. Wu, M. Wang, and J. E. Simon, âDetermination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection,â J. Chromatogr. A, vol. 1016, no. 2, pp. 195â209, Oct. 2003, doi: 10.1016/j.chroma.2003.08.001.
 Mehrotra, P. (2016). Biosensors and their applications â A review. Journal of Oral Biology and Craniofacial Research, 6(2), 153â159. https://doi.org/10.1016/j.jobcr.2015.12.002
 Hallberg, Z. F., Su, Y., Kitto, R. Z., & Hammond, M. C. (2017). Engineering and In Vivo Applications of Riboswitches. Annual Review of Biochemistry, 86(1), 515â539. https://doi.org/10.1146/annurev-biochem-060815-014628
 K.-M. Song, S. Lee, and C. Ban, âAptamers and Their Biological Applications,â Sensors, vol. 12, no. 1, pp. 612â631, Jan. 2012, doi: 10.3390/s120100612.
 M. Wachsmuth, S. Findeiss, N. Weissheimer, P. F. Stadler, and M. Morl, âDe novo design of a synthetic riboswitch that regulates transcription termination,â Nucleic Acids Res., vol. 41, no. 4, pp. 2541â2551, Feb. 2013, doi: 10.1093/nar/gks1330.
 S. Mujwar and K. R. Pardasani, âPrediction of Riboswitch as a Potential Drug Target for Infectious Diseases: An Insilico Case Study of Anthrax,â J. Med. Imaging Heal. Informatics, vol. 5, no. 1, pp. 7â16, Feb. 2015, doi: 10.1166/jmihi.2015.1358.
 E. M. Aghdam and M. Esmaeil Hejazi, âRiboswitches as Potential Targets for Aminoglycosides Compared with rRNA Molecules: In Silico Study,â J. Microb. Biochem. Technol., vol. 07, no. 05, 2015, doi: 10.4172/1948-5948.S9-002.
 R. Penchovsky and C. C. Stoilova, âRiboswitch-based antibacterial drug discovery using high-throughput screening methods,â Expert Opin. Drug Discov., vol. 8, no. 1, pp. 65â82, Jan. 2013, doi: 10.1517/17460441.2013.740455.