BBa_K1165002 1 BBa_K1165002 PenI-cleaving Ribozyme 1 of XOR Gate 1 2013-09-26T11:00:00Z 2015-05-08T01:09:33Z The target-binding flanking arms of the ribozyme were obtained from the complement of the PenI Repressor sequence. 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 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 represent any nucleotide except guanine (Mir 2001), although with GUC as the hammerhead consensus cleavage site (Lieber, 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 2008) as is depicted below. [image] The synthetic extended arms are designed to bind to the complementary extended arms of another similar PenI-cleaving ribozyme (Part _____), but do not contribute to any additional secondary structure as confirmed via the Hairpin tool in IDT's OligoAnalyzer (Owczarzy 2008), as both arms of a particular ribozyme only contain a purine and pyrimidine pair that do not bind to each other (in this case, A and G). 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. <strong>References</strong> Mir, A. A., Lockett, T. J. & Hendry, P. Identifying ribozyme-accessible?? sites using NUH triplet-targeting gapmers. Nucleic Acids Res 29, 1906???1914 (2001). 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). Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163???W169 (2008). false false _1477_ 0 18237 9 Not in stock false Although this particular part is likely to only cleave the PenI repressor mRNA due to its sequence specificity, the simple <em>in silico</em> design of this ribozyme allows for the cleavage of virtually any RNA sequence containing a cleave site (NUH). This particular sequence was obtained by first scanning our target PenI mRNA for the optimal cut site GUC. The 10 nucleotides surrounding this cut site were obtained and the complement of this area was generated for the ribozyme flanking arms. The length of this region was decided by ideal melting temperature (51.4 degrees Celsius). Additional synthetic extended arms were subsequently added to the 5' and 3' ends of the sequence for binding to the complement ribozyme in an XOR gate. This region of non-pairing purines and pyrimidines was staggered in base repeats to avoid slippage of binding and was defined in length by significantly higher melting temperatures (approximately 68 degrees Celsius). Finally, the catalytic core of the Y-type hammerhead ribozyme was introduced at the appropriate region. Throughout this entire process, secondary was continually checked and minimized using IDT OligoAnalyzer's Hairpin tool (Owczarzy 2008). The complete ribozyme sequences was also BLASTed again the <em>E.coli</em> genome, which was our chassis and yielded insignificant results. Other factors such as differential length or target binding regions due to varying melting temperature and GC content were also considered during the <em>in silico</em> design of this ribozyme and its pair in the XOR logic gate. <strong>References</strong> Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163???W169 (2008). false iGEM Concordia 2013 annotation2364286 1 TAA Bump range2364286 1 36 38 annotation2364285 1 Target-Binding Arms range2364285 1 39 43 annotation2364282 1 Synthetic Extended Arms range2364282 1 1 30 annotation2364283 1 Synthetic Extended Arms range2364283 1 82 111 annotation2364287 1 Catalytic Core range2364287 1 44 69 annotation2364284 1 Target-Binding Arms range2364284 1 31 35 annotation2364288 1 Target-Binding Arms range2364288 1 70 81 BBa_K1165002_sequence 1 aaaaagggggaaaaggggaaagggaaggagaaatgtaaagtgtctgatgagtcgctgaaatgcgacgaaacttaacctctagaggaagggaaaggggaaaagggggaaaaa igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 James Alastair McLaughlin Chris J. Myers 2017-03-06T15:00:00.000Z