sRNA (1): YFP-targeting molecule
Small RNAs (sRNAs) are small double-stranded transcripts capable of silencing mRNAs of complementary sequence (Yoo et al., 2013). The sRNA sequence was designed with two functional parts: a region complementary to the target mRNA (target binding site) and MicC scaffold region for recruitment of Hfq BBa_K1323004, a protein which enhances the hybridization between the sRNA and target mRNA (Yoo et al., 2013). The MicC RNA sequence is the best scaffold sequence as shown by previous studies done by the same research group (Na et al., 2013). The target binding sequence of this sRNA is complementary to the S.epidermidis codon optimized YFP BBa_K1323010.
This part was DNA-synthesized by Bio Basic.
The sRNA binding region.
sRNA 1 targets the YFP gene starting at the end of its Shine-Dalgarno (SD) sequence. The SD region is determined by Yoo et al. (2013) to be the most effective binding region for mRNA silencing. This 26 bp sequence has binding energy of -27.6 kcal/mol as determined using DINAMelt software (http://mfold.rna.albany.edu/?q=DINAMelt/Two-state-melting). For effective binding, sRNA should have a binding energy between -30 kcal/mol to -40 kcal/mol (Yoo et al., 2013). We aligned the sRNA 1 sequence with other constructs and the ATCC 12228 genome to determine the highest binding energy for non-specific RNA binding, which is -21.18 kcal/mol. This shows that sRNA 1 binds more specifically to YFP and should not interfere with other gene expressions in our system.
Sequence and Features
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- 21INCOMPATIBLE WITH RFCIllegal XhoI site found at 140
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Dokyun Na, S. M. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnology, 170-174.
Na, D. et al. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnology 31, 170–174.
Yoo, S.M., Na, D., Lee, S.Y. (2013). Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nature protocol 8(9), 1694-1707.