Part:BBa_K1165003

PenI-cleaving Ribozyme 2 of XOR Gate 1

Description

This is the DNA sequence of a Y-type hammerhead ribozyme that targets and cleaves the PenI repressor (Part BBa_C0074). This ribozyme has target-binding flanking arms adjacent to the Y-type hammerhead ribozyme catalytic core and synthetic extended arms located towards the 5' and 3' ends. The catalytic core allows for the sequence-specific cleavage of sites within the coding sequence of PenI. Hammerhead ribozymes are able to cleave at any NUH within an RNA sequence, where H represents any nucleotide except guanine (Mir et al, 2001), although with GUC as the hammerhead consensus cleaving site (Lieber et al, 1995). The binding of the flanking arms adjacent to the hammerhead catalytic core favours the recognition and cleavage of only one target site. Additional secondary structure within the flanking arms was detected to a small degree by using IDT OligoAnalyzer's Hairpin tool (Owczarzy et al, 2008) as is depicted [http://i.imgur.com/ui96T6v.jpg here] for a catalytic core and a set of flanking arms.

The synthetic extended arms are designed to bind to the complementary extended arms of another similar PenI-cleaving ribozyme (Part BBa_K1165002), but do not contribute to any additional secondary structure as confirmed via the Hairpin tool in IDT's OligoAnalyzer (Owczarzy et al, 2008), as both arms of a particular ribozyme only contain either a purine or pyrimidine pair that do not bind to each other. The two-nucleotide extended arms are also staggered in number to avoid slippage in binding and bind to its complement pair at significantly higher melting temperatures than the adjacent flanking arms. This feature allows for the sequestering of the two ribozymes when both are present and significantly decreasing any ribozyme activity. Regions of complementarity between the two target-binding regions of both ribozymes are also present to ensure that the functional catalytic cores are not able to cleave their mRNA targets, even when bound.

The characteristics of this expressed pair of ribozymes were considered to create a functional RNA-based XOR logic gate.

References

1. Lieber, A. & Strauss, M. Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library. Mol. Cell. Biol. 15, 540–551 (1995).

2. Mir, A. A., Lockett, T. J. & Hendry, P. Identifying ribozyme-accessible  sites using NUH triplet-targeting gapmers. Nucleic Acids Res 29, 1906–1914 (2001).

3. Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163–W169 (2008).

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]