Part:BBa_K4195077

Biology

This sequence is the first part of guide designed for detection of toxin gene pirB. Ribozyme ENabled Detection of RNA (RENDR)
RENDR is a high-performing, plug-and-play RNA-sensing platform(1). RENDR utilizes a split variant of the Tetrahymena thermophila ribozyme by synthetically splitting it into two non-functional fragments (Fig. 1). Two fragments are each appended with designed RNA guide sequences, which can interact with the RNA input of interest. The split ribozyme is then inserted within a desired gene output. When bound with the RNA input, two transcribed split ribozyme fragments are triggered to self-splice and thus the intact transcript of the protein output will form.
T--XMU-China--GFP detection.png\400px
Fig. 1 Schematic illustration of RENDR.

Usage and Design

The conserved region of pirB gene is set as the RNA input. The guide sequences were designed based on NUPACK prediction(2). Based on the model provided (Equation. 1), we calculate the free energy difference of candidate sequences at 37 °C, and select guide pair g1 and g2 with 215.36 kcal/mol and 205.86 kcal/mol (Fig. 2). The optimized ribozyme split sites are selected from the literature, and named α (split site 15) and β (split site 402)(1).
Equation. 1 ln(FL/OD) ~ΔG_{Guide 1} + ΔG_{Guide 2} + ΔG_{RNA input} − ΔG_SC.
[[File:T--XMU-China--pirB g1α Nupack.png|400px]
Fig. 2 The MFE structure of g1 guide-input complex at 37℃. ΔG_Guide1 and ΔG_Guide2 = The minimum free energy (MFE) of the two RNA guide sequences attached to each fragment of the RENDR ribozyme. ΔG_RNAinput = The MFE of the RNA input. ΔG_SC = The duplex binding energy of the complex. ΔG_Guide1 = -13.2 kcal/mol, ΔG_Guide2 = -10.6 kcal/mol, ΔG_RNAinpu = -27.9 kcal/mol, ΔG_SC = -267.06 kcal/mol, ΔG_SC - ΔG_Guide1 + ΔG_Guide2 + ΔG_RNAinput = 215.36 kcal/mol.