Part:BBa_K3657050:Design

Double Split-Tetrahymena Ribozyme 3'Guide + mScarlet

Assembly Compatibility:

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  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 540
  
• 21
  COMPATIBLE WITH RFC[21]
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  COMPATIBLE WITH RFC[23]
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  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Considerations

Over the years, the Group I intron from Tetrahymena thermophila as well as related self-splicing RNAs have enjoyed diverse engineering attempts to repurpose them as in vivo RNA editing tools . The main step towards engineering these self-splicing ribozymes consist of turning the ribozymes' self-splicing activity into trans-splicing activity . Conveniently, the structure of T. thermophila group I intron is fairly amenable to such engineering attempts by virtue of its modular structure . The ribozyme consists of a compact catalytic core (Figure 1, core) – this should not be disturbed when attempting to engineer the ribozyme – flanked on the 5' and 3' ends respectively by a pair of hairpin loops (Figure 1, 5': P2 and P2.1; 3': P9.2 and P9.1), The P2 substructure is followed by an internal guide sequence (IGS) directly interacting with the ribozyme core and an external guide sequence (EGS), which positions the 5' exon for the splicing reaction (Figure 1, EGS). On the 3' end, the P9 substructure is followed by the 3' splice site and the 3' exon itself (Figure 1, 3' active site). For a properly functioning ribozyme, the P2 and P9 substructures may be engineered, but must be able to form a tertiary interaction between P2.1 and P9.1 for high activity .

Engineering a trans-splicing version of the T. thermophila group I intron is almost trivially easy – intuitively all one has to do is to detach the 5' exon from the group I intron and extend the EGS to include at least 10 bases complementary to the target RNA sequence. This usually suffices to yield at least minimal trans-splicing activity .

EGS / IGS Design

To achieve high-efficiency trans-splicing using the T. thermophila group I intron requires careful design of the external guide sequence (EGS). First and foremost, a valid target sequence for trans-splicing needs to contain an uracil (U) base at the splice site. This is a hard, non-negotiable sequence requirement – no U, no activity. Secondly, the target RNA sequence needs to be accessible – it must not form stable secondary structures in the region complementary to the ribozyme EGS as well as at the splice site itself . This can be checked in silico by predicting the secondary structure of the target RNA within a sliding window around potential splice sites. Experimentally, splice site preferences may be measured by preparing a splice site library of ribozymes with different target sites and measuring splicing efficiency .

A further consideration to make trans-splicing as efficient as possible is the length of the EGS, together with the size of the bulge formed between the IGS and EGS upon 5' substrate binding . Here, the literature tells us that the optimal size of the EGS-bulge should be in the range between 5 and 6 bases . In addition, the bulge on the side of the ribozyme should be able to form base base pairs with the start of the 3' exon, just after the 3' splice site (P10 extension). For our experiments we designed a fixed EGS/IGS and corresponding splice-site for the sake of simplicity. These were designed according to all design principles mentioned above to ensure high trans-splicing activity.

Split Trans Splicing Ribozymes

Taking another look at our overview of the T. thermophila ribozyme in Figure 1, we may notice that the both the P2.0 and P9.2 stems are not structurally essential for ribozyme activity. Therefore, we may safely split the ribozyme across these two stems to yield a split ribozyme core ( BBa_K3657048 ) a 5' guide sequence, directing the ribozyme to a target 5' exon ( BBa_K3657047 for our choice of IGS/EGS and target sequence ) and a 3' exon guide sequence tethering a chosen 3' exon to the trans-splicing ribozyme ( for example BBa_K3657050 for a 3' exon containing the sequence of the mScarlet fluorescent protein ). Splitting the ribozyme in this way makes the base Tetrahymena ribozyme more modular, allowing a single ribozyme core to do the work of splicing on multiple 3' and 5' exons, thereby improving on earlier T. thermophila ribozymes in the iGEM registry (e.g. BBa_I4285 ).

Single Guide Trans Splicing Ribozymes

Taking note of the property of the tertiary interaction between P2.1 and P9.1, we make take our engineering strategy one step further. We may split out ribozyme along the P2.1 and P9.1 stem and insert a linker between the split 5' and 3' parts, which results in P9.1 and P2.1 being kept in spatial proximity by base-pairing interactions and fusing the 5' and 3' guide sequences into a single-guide sequence for a split trans-splicing ribozyme core. We provide parts for this type of ribozyme core and single-guide sequence as well ( BBa_K3657041 for the core and BBa_K3657042 for the guide. )

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