Part:BBa_K5038003

LIRA_H05

Usage

H05 is a type of ribocomputing system that utilizes a loop-initiated RNA activator (LIRA) motif. This motif binds to an input RNA strand via extended loop domains, which will expose downstream functional domains for subsequent reactions. Since the loop of H05 can bind to RNA strands, we can modify the sequence on the loop to detect specific RNAs.

Biology

1.In the absence of a target RNA, the system adopts the structure shown in Figure 1. This structure features a loop and a stem. The loop contains a sequence complementary to the target RNA strand, and the stem houses the ribosome binding site (RBS) and the initiation codon AUG. Both the RBS and AUG are closed due to the pairing within the stem, thereby blocking translation.

(Figure 1. Schematic of structure 1, LIRA_H05 without input RNA strand)

2.When the target RNA is present, H05 undergoes a conformational change as shown in Figure 2, leading to the formation of the structure shown in Figure 3. In this configuration, the hairpin structure opens, exposing the RBS and AUG. This allows the ribosome to bind to the RBS and start translation at AUG.

(Figure 2. Changes on H05)

(Figure 3. Schematic of structure 2, LIRA_H05 with input RNA strand)

3.A reporter gene is located downstream of H05. By comparing the expression of the reporter gene before and after the loop opens, we can infer the opening of hairpin structure, thus we could detect the target RNA.

Characterization

In our experiments, we used EGFP as the reporter gene and input01 as the target RNA, and inferred the opening of the hairpin through the fluorescence intensity. A detailed information is given in BBa_K5038020and BBa_K5038022.

H01 and H05 are the two RNAs that are more efficient as molecular switches of LIRA system. The two RNAs are not fundamentally different, but the differences in the base sequences give rise to different sensitivities and translation leakage of the two LIRAs, which have been experimentally tested with the following results:

(Figure 4. Fluorescence intensity results of H01 and H05)
*** indicates p <0.001

As can be seen from Figure 4, the fluorescence intensity of H01+input01 is much larger than that of H05+input05. To further illustrate that the sensitivity of H01 is stronger than that of H05, we evaluated the sensitivity of both by ON/OFF fluorescence ratio. That is, the fluorescence intensity value of input+ divided by the fluorescence intensity value of input-. The results are shown below:

(Figure 5. ON/OFF fluorescence ratio results of H01 and H05)
*** indicates p <0.001

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]