Part:BBa_K4914000

LIRA_21OR92_A2

Description

LIRA_21OR92 is one type of RNA Toehold switch, LIRA (Loop-Initiated RNA Activator), which can detect at least one type of two miRNAs, miR21 and miR92a, or It can be said to work as OR gate logic.

Usage

This part is used for detecting miR21 and/or miR92a (CRC biomarkers) (Okugawa et al. 2014)[1]and then expressing a coding sequence (CDS). CDS can be changed by changing from the codon start sequence of LIRA and continuing with the appropriate sequence of CDS that we use.

Biology

This LIRA sequence was inspired by research by Ma et al. 2022[2], which designed an RNA toehold that will form a multi-arm RNA junction once transcribed inside the E.coli cell.

Characterization

We characterize our part through Analysis Job NUPACK with LIRA's sequences "GGGGCCATTTTTTATTTTTTTTTTATTCTATCAGTTTCAACATCAGTCTGATAAGCTAAAACATGATATTGGTAGAATATTTT CCACTGGTAACAGGCCGGGACAAGTGCAATAAATAATGTAGCCAGTGGAAAATAGAGGAGATGGAAAATGGCTTC", where the eight last bases are part of reporter aeBlue chromoprotein BBa_K864401 and the results are:

Figure 1. The melting profile of LIRA_21OR92_A2.

Based on the Figure 1. It can be seen that a fraction of bases unpaired tend constants along the human temperature range (36°C-38°C). It means that the secondary structure of LIRA_21OR92_A2 is stable along that temperature range.

Figure 2. The secondary structure probability of LIRA_21OR92_A2.

Based on the Figure 2. It can be seen that this LIRA has an ideal target structure, that is RBS and the start codon remains enclosed when there is no miR21, miR92a, also miR21 and miR92a.

Figure 3. The equilibrium concentration of LIRA_21OR92_A2 when there are no miR21 and miR92a, there is miR21, there is miR92a, there are miR21 and miR92a.

Based on the Figure 3. It can be seen that our LIRA tends to be in the form of LIRA (not in the form of complex LIRA+LIRA) so it is ready to tend to accept miR21 or miR92a as CRC biomarkers. As well as when there are biomarker(s) miR21 and/or miR92a, the LIRA tends to form complexes with biomarker(s) as expected.

Based on the computational calculation of ON/OFF weighted (in silico), this LIRA has the highest value rather than 9 other LIRAs that are generated from web-based automated NUPACK Design Job. The complete calculation and method for ranked can be seen on Toehold LIRA Design Optimization section of our Model Page.

Reference

[1]Okugawa, Y., Toiyama, Y. and Goel, A., 2014. An update on microRNAs as colorectal cancer biomarkers: where are we and what’s next?. Expert review of molecular diagnostics, 14(8), pp.999-1021 https://doi.org/10.1586/14737159.2014.946907
[2]Ma, D., Li, Y., Wu, K., Yan, Z., Tang, A.A., Chaudhary, S., Ticktin, Z.M., Alcantar-Fernandez, J., Moreno-Camacho, J.L., Campos-Romero, A. and Green, A.A., 2022. Multi-arm RNA junctions encoding molecular logic unconstrained by input sequence for versatile cell-free diagnostics. Nature biomedical engineering, 6(3), pp.298-309. https://doi.org/10.1038/s41551-022-00857-7

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]