Part:BBa_K5038045

PT7-LIRA_H01-linker-lacZα-T7TE

Usage and Biology

H01 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 H01 can bind to RNA strands, we can modify the sequence on the loop to detect specific RNAs.

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_H01 without input RNA strand)

2.When the target RNA is present, H01 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 H01)

(Figure 3. Schematic of structure 2, LIRA_H01 with input RNA strand)

3.A reporter gene is located downstream of H01. 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 lacZ as the reporter gene and input01 as the target RNA. LacZα can perform α-complementation with LacZω, then form β-gal and convert the yellow‐orange CPRG substrate into the chlorophenol red (CPR) ,and yield a dark red solution, thus infers the existence of input01.

T7 promoter could be induced by IPTG, so the intracellularly concentration of LIRA_H01 produces a large difference between the two conditions of IPTG presence/absence.

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]