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Since real-world patient samples contain only traces of pathogens (attomolar at the lowest), any detection method relying on binding affinity (such as Cas13a with target RNA) needs prior amplification of the detected molecule to concentrations within the range of the Kd.
After we confirmed that the detection limit of Cas13a is in the nanomolar region, we had to tackle the problem
of low target concentrations in medical samples.
We explored different amplification methods that would be simple and stable on our transportable platform. We wanted to incorporate isothermal reactions on paper with lyophilized reaction mixes.
Therefore, we explored isothermal PCR methods coupled to in vitro transcription . We chose isothermal methods for signal amplification since isothermal techniques
come with the potential of simplified hardware design which would lead to decreased production and development costs. PCR using a proper thermocycler and well-defined cycling times also require a trained personnel to use them, which does not fit to our goal of a distributable diagnosis device.
Eventually, we would need to detect both DNA and RNA samples by using either a
reverse transcriptase coupled isothermal recombinase polymerase amplification and subsequent
in vitro transcription for RNA targets or leaving out the step of reverse transcription for DNA targets. We term this amplification process RT-RPA-TX.
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Recombinase Polymerase Amplification
The Recombinase Polymerase Amplification (RPA) developed by TwistDx is an isothermal amplification method for DNA.
Rather than melting the double strand and annealing the primers through temperature cycles, it uses a recombinase
that binds the primers and assists in the annealing process. Another protein, single-strand DNA binding protein (SSB)
promotes the binding of the primers to the recombinase in this process.
Hence, the first step in the development of an isothermal amplification method of an RNA signal via RT-RPA-TX was testing the
Recombinase Polymerase Amplification (RPA) itself in bulk. For this, we took our previously cloned His-tagged TEV Protease (BBa_K2323002 improved from BBa_K1319008)
and ran dummy PCR reactions using the TwistDx RPA Kit and the universal biobricks primers VF2 (BBa_G00100) and VR (BBa_G00101). We ran
the provided test reaction of the TwistDx RPA Kit as a positive control (TwistAmp exo). The results are shown in Figure 1.
The first point to note is that PCR primers and RPA primers have different requirements, due to the need for affinity of the recombinase to the primers (RPA primers tend to be shorter with a higher GC content). Therefore VF2 and VR might not be ideal primers for RPA. The second point is that RPA's optimum amplification length is 500bp. For longer amplifications, it is possible that the polymerase falls off the strand. This could explain why we see at maximum a band of 500bp, and shorter bands, although our amplicon should be 1262bp. We could therefore see that amplification using RPA was possible in bulk, but that primer design would need to be optimized for our final product.
To better assess the RPA reaction, we therefore constructed a benchmark plasmid coding for 3 of our target sequences (E. coli, B. subtilis and Norovirus) under the control of a T7 promoter. It is flanked by VF2 and VR primers binding sequences, so that it can be amplified similarly to our first His-TEV construct, but the amplicon is only 194bp. This was a great benchmarking sequence, as the plasmid could be used for amplification from lyzed cells (as would be the case with a real sample), the sequence was short enough, could be coupled with transcription and consisted of relevant targets for our read-out.
RPA on paper
The next step was to bring the RPA reaction on paper. For this, we lyophilized the reaction mixture provided by TwistDx
on paper and tried to run RPA reactions directly from it by inserting the blotting paper into the PCR tube.
Activity could be shown on an agarose gel but time-stability experiments showed that the RPA kit activity decreases quickly.
In the user's manual, it is stated that after breaking the seal, one should use up the reaction mix in the next hour.
We can confirm this statement since activity declined rapidly after as little as two hours and was non-existent after
24 hours, as is visible in Figure 2.
Furthermore, temperature seems to be a very important factor during amplification using RPA. The samples incubated at of 20 °C, though still active, showed dramatically decreased activity compared to the same samples at 37°C.
For usage of RPA on a detection device that does not require purification of the sample, the reaction needs to take
place in the environment of a lysate with cell debris and released proteins from the cell. In order to see whether this
is achievable, we performed a colony-PCR like setting of RPA using a E. coli culture in LB-medium with OD=1.0
and ran a normal RPA of purified plasmid as a control. As our first PCR produced too long of an amplicon, we changed to a shorter PCR scheme, which should lead to an amplification of 194bp. The results are shown in Figure 3.
A band at the expected length is visible in both the control condition and the E.coli environment. We observed that the yield measured by gel quantification was approximately the same.
Since the stability of RPA on paper for a long period of time would be crucial to our diagnosis device, we considered the possible reasons for the unstability of our RPA mix. We could think of two factors affecting the stability: humidity and temperature. As we want our platform to be distributable across the world, we needed to test those hypothesis. We found out, that the unstability is mainly cause by exposure to air, presumably humidity. We could dramatically increase stability when covering the paperstrip in a plastic Petri dish and sealing it with Parafilm. The stability was increased when keeping the paperstrip at 4°C, but the RPA was anyway still active after 24 hours storage at room temperature (when protected in a sealed environment). The results are shown in Figure 4. We did not have time to conduct long-term storage tests, but we are hopeful that with the right sealing strategy, the RPA could still be active on paper after a year.
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Figure 1: RPA reaction at 37 °C of Control by TwistDx and His6-TEV using VF2 and VR primers.
Figure 2: RPA reaction of lyophilised RPA mixture at 37 °C or 20 °C of His6-TEV using VF2 and VR primers.
The control is a reaction mixture without the lyophilisation step.
Figure 3: Colony-PCR of Cas13a Benchmark plasmid using RPA. Control is a standard RPA reaction of purified plasmid
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Figure 4: RPA reaction after freeze-dried storage on paper at different conditions. Conditions that were taken
into consideration are temperature and air accessibility. Air determines the samples that were accessible to air.
The other samples were stored in a Petri dish sealed with parafilm.
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RPA on paper time-stability optimisation
Coupling to in vitro transcription
The coupling of TX to RPA is in theory feasible because both reaction are conducted at 37°C, and still active at room temperature. We thus proceeded to try RPA-TX on the His6-TEV construct. However, we never succeeded to get the expected bands in
the urea-PAGE following phenol-chloroform extraction. As our initial amplicon from the His-TEV construct was too long (1262bp compared to 500bp optimal amplification length from RPA), we believe that the enzyme did not manage to produce double-stranded DNA, and therefore transcription was not completed. At this point, we decided to create our benchmark plasmid (described above), which has an amplicon of only 194bp. We could characterize it for RPA, but unfortunately we did not have time to conduct RPA-TX experiments.
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Bringing RPA and TX on paper
The final step was stabilizing all the prior tested work on paper and stabilise it on there so it could be sealed
into the sample processing unit developed
by the Hardware Team to automatise the amplification process and enable
subsequent detection on a paperstrip using Cas13a. For this we performed experiments
and showed that RPA-Tx lyophilised on paper worked judged by the 15%-Urea-PAGE. The gel for RNA qunatification is shown in Figure 5.
The gel showed a band at the approximate size of 130 bp of the Benchmark construct transcript that
increased in concentration during the RPA-Tx reaction.
We were able to perform the experiment in a time dependent measurement ending the RPA-Tx reaction after 30 minutes,
60 minutes and 2 hours. The experiment suggested that the transcription of DNA template into RNA is mainly restricted by
the speed of the T7 RNA Polymerase since the reaction speed seemed to be constant over the observation time. The concentration
of RNA at overnight transcription was not measurable since the gel was completely overloaded which is most probably a result
of incomplete DNase I digestion after the transcription due to immense amount of DNA after running the RPA this long.
Finally, we proceeded to test the ability of our product from RPA-Tx to trigger Cas13a collateral RNase activity.
Due to time limitations, we were not able to show time stability of the RPA-Tx mixture anymore.
Figure 1: RPA reaction at 37 °C of Control by TwistDx and His6-TEV using VF2 and VR primers.
Discussion
We showed here the first steps of establishing an amplification circuit for our detection device.
Since we were able to screen for storage conditions and do colony PCR-like experiments with the RPA mixture on paper,
the reproducibility of the RPA reaction after dry-freezing it on paper is proven. Time-stability has
also been shown for 24 hours, though this will need to be extended to longer time-scales to provide
it to customers.
One issue that can still needs to be solved is that so far, the coupled RPA-Tx reaction was only tested for purified samples.
The fact of having lysate in the sample is likely to have distorting effects on the efficiency on both reactions.
Since RPA worked in a colony PCR-like set up with prior lysis of the cells, that is already a good start but the
In-Vitro Transcription is usually more delicate, therefore performing it in a lysate environment
might prove to be more difficult.
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