Introduction
In our quest for total control over all RNA interactions, we may want to have a way to modify our RNAs within
a living cell. Luckily, nature has got us covered! Ribozymes – catalytic RNAs – with the power of
replacing parts of another RNA are readily available for us to use. For the remainder of our project, we will
focus on one specific ribozyme the function of which is particularly suited for our task – the self-splicing
group I intron from
Tetrahymena thermophila TransSplicing1.
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 TransSplicing1TransSplicing2. The main step
towards engineering these self-splicing ribozymes consist of turning the ribozymes' self-splicing
activity into trans-splicing activity TransSplicing1. Conveniently, the structure
of T. thermophila group I intron is fairly amenable to such engineering attempts by virtue of
its modular structure TransSplicing1. 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 TransSplicing1.
Figure 1: Schematic of the Group I intron of T. thermophila
The T. thermophila self-splicing ribozyme is composed of a catalytic ribozyme core which should
not be disturbed by engineering, flanked by the distal hairpin structures P9 and P2, which should be able to
form tertiary interactions bringing P2.1 and P9,1 into spatial proximity. Finally, the ribozyme contains an internal
guide sequence (IGS) positioning the 5' and 3' exons for the splicing reaction, as well as an external guide sequence
(EGS) to aid binding and positioning of the 5' exon.
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 TransSplicing1.
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 TransSplicing1.
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 TransSplicing1.
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 TransSplicing1. Here, the
literature tells us that the optimal size of the EGS-bulge should be in the range between 5 and 6 bases TransSplicing2.
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.
)
Experiments
To test the trans-splicing activity of our various split trans-splicing constructs, we set up a series of straight-forward
experiments with a fluorescence readout. We design our experiments in such a way that all three types of our trans-splicing
ribozymes may be assayed in the very same setup.
Start Codon Rescue
Figure 2: Start codon rescue
Schematic of the start codon rescue experiment. We grow cells containing two plasmids – one high-copy plasmid
containing all components of our (split) trans-splicing ribozymes under a strong promoter (ribozyme plasmid). The other (splice donor plasmid)
contains a cassette containing a constitutive promoter followed by a strong RBS and a start codon followed by the target sequence for our
ribozyme's EGS and a terminator. Controls include cells without either the ribozyme plasmid or the splice donor plasmid.
If successful trans-splicing occurs, mScarlet RNA without a start codon is inserted after the RBS and start codon from the donor sequence,
resulting in mScarlet translation and red fluorescence. This can be measured and compared to controls using a fluorescence plate reader.
We grow cells containing two plasmids – one high-copy plasmid
containing all components of our (split) trans-splicing ribozymes under a strong promoter (ribozyme plasmid). The other (splice donor plasmid)
contains a cassette containing a constitutive promoter followed by a strong RBS and a start codon followed by the target sequence for our
ribozyme's EGS and a terminator. Controls include cells without either the ribozyme plasmid or the splice donor plasmid.
If successful trans-splicing occurs, mScarlet RNA without a start codon is inserted after the RBS and start codon from the donor sequence,
resulting in mScarlet translation and red fluorescence. This can be measured and compared to controls using a fluorescence plate reader.
Presence of fluorescence indicates successful trans-splicing.
Fluorescent Fusion Protein Generation
We grow cells containing two plasmids – one high-copy plasmid
containing all components of our (split) trans-splicing ribozymes under a strong promoter (ribozyme plasmid). The other (splice donor plasmid)
contains a cassette containing a constitutive promoter followed by a strong RBS and a start codon followed by sfGFÜ and the target sequence for our
ribozyme's EGS and a terminator. Controls include cells without either the ribozyme plasmid or the splice donor plasmid.
If successful trans-splicing occurs, mScarlet RNA without a start codon is inserted after the RBS and start codon from the donor sequence,
resulting in mScarlet translation and red fluorescence. This can be measured and compared to controls using a fluorescence plate reader.
Presence of fluorescence indicates successful trans-splicing. Furthermore, we can attempt to measure the efficiency of trans-splicing by
comparing the ratio of red (mScarlet) to green (sfGFP) fluorescence to control samples of purified mScarlet and sfGFP.