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Measurements

Potential of quantifying tRNA species

The potential of quantifying specific tRNA species is enormous and can make a significant contribution to the further development of metabolic engineering and systems biology. For the first time, it would be possible to identify bottlenecks in tRNA availability specifically of existing systems and strains. Moreover, it would be possible to develop solutions regarding these bottlenecks.

Furthermore, the newly generated data sets consisting of intracellular, relative tRNA concentrations, can be used bioinformatically and in systems biology for the extension of models and mathematical prognoses. This can increase the predictive power of existing systems and specify model assumptions.

Finally, limitations in heterologous protein expression triggered by codon usage of the metabolites to be produced can be avoided. It is already common to perform codon optimization of the sequences of heterologous proteins, replacing rarely used codon triplets by more common codon triplets of the host organism. While this may lead to an increase in expression velocity, it does not provide the optimal solution to bypass the existing bottleneck. It is likely that the existing bottleneck will be slightly shifted to another tRNA species. The reason for this is that no changes are made to the supply of specific tRNA, only the demand is shifted to another tRNA species, resulting in a possible limitation of this specific tRNA. If we succeed in generating a method for the relative quantification of intracellular tRNA, optimization can be performed directly on the host organism prior to heterologous protein expression. Thus, the supply of specific tRNA can be specifically increased during the expression phase. This could lead to an industrial advantage due to an increased protein expression by specifically adapting the tRNA needs of the cells.

Method

Based on the work of Honda et al. we developed a new and simplified method for relative quantification of specific tRNA species without the necessity of TaqMan probes. Instead using a DNA/RNA hybrid stem loop we used a linear DNA/RNA construct as adaptor.

The first step of our protocol is to isolate RNA with a length of < 200 nt from cultured V. natriegens cells. Then, the amino acid bound to the 3’ end needs to be removed by a deacylation reaction. This results in a sticky end, where a linear RNA/DNA hybrid adaptor can be ligated, which is complementary to the 3’ end overhang. Although different tRNAs show differences in length and sequence, the last three nucleotides at the 3’ end are the same for all tRNA species. The ligated adaptor contains a binding site for the forward-primer, which is identical for all tRNAs (unspecific primer). We used T4-RNA-ligase 2 that requires ATP. For this reason, a polynucleotide kinase was necessary to carry out a phosphorylation reaction at the 5’ end. To amplify single tRNA specifically, a reverse transcription needs to carry out with specific tRNA reverse primer, followed by a RNase H digestion to obtain pure cDNA of the desired tRNA species. Later each tRNA species is amplified during a qPCR by using the specific reverse primer and the unspecific adaptor primer.

Because the low temperatures, at which the reverse transcription is carried out (42 °C), can not break down tRNA secundary structures, we decided to use HotScriptase instead. This modified enzyme works at 65 °C and is able to perform reverse transcription and the amplification of the target gene in one step. By using HotScriptase, tRNA secundary structures should be reduced. Furthermore time is saved due to less steps.

Results

With our method we were able to obtain reproducible results regarding different tRNA concentrations. After the protocol was established, we tried to find out which tRNA concentrations could be distinguished and how sensitive the method is. We showed that this method is able to distinguish tRNA concentrations up to a dilution of 1:2 significantly, depending on the codon. Results of a tRNA dilution series with the dilutions 1:1, 1:10 and 1:100 are shown in figure 1. The graphs 2 and 3 show the melting curves of the amplified tRNA species AGA and GAA. The overall results with the lower dilution steps as well as the other corresponding melting curves of the qPCR can be seen completely in our results .

Figure 1: Quantification of diluted tRNAs of the two codons AGA und AGG in V. natriegens with RT-qPCR: Fluorescence over cycle number is shown. AGA 1:1 diluted is represented by pink color. AGA 1:10 diluted by dark blue and 1:100 diluted by light blue. GAA 1:1 is represented by the orange curve, GAA 1:10 by red and GAA 1:100 by dark grey. Also, negative controls are shown: AGA in green and GAA in black. The experiment was carried out in triplicates. Average and standard deviation are shown.

Figure 2: The melting curve of the amplification product of the AGA tRNA in V. natriegens is shown. The generated product has a melting temperature of about 77.5 ° C.

Figure 3: The melting curve of the amplification product of the GAA tRNA in V. natriegens is shown. The generated product has a melting temperature of about 77.5 ° C.

Conclusion