qRT-PCR for the relative quantification of specific tRNA-species

Alongside with the generation of a climate-friendly medium, the goal of our project PhyCoVi was to optimize the strain Vibrio natriegens for a potential use in the biotech industry. The optimization is performed on the genomic level to increase the intracellular availability of tRNA species. As a result, the strain’s performance to express heterologous proteins is enhanced.

A method needs to be developed to quantify individual tRNA species specifically to prove the increased expression not only on the protein level.  Multiple methods can be found to quantify non-coding RNA ^{1, 2} or total tRNA concentration ^{3, 4}. Whereas finding a well-established method to quantify single tRNA species specifically is in vain. The only method paper was published in the journal “RNA biology” in 2015 by Honda et al.: “Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA” ⁵. The authors of this paper removed the amino acid at the 3’ end followed by hybridization and ligation with a DNA/RNA hybrid stem loop creating a “four-leaf clover” shaped appearance of the tRNA ligation product. The stem loop adaptor contained a TaqMan probe binding site. During the qPCR the TaqMan probe was cut by exonuclease function of the used polymerase resulting in emission of fluorescence.

Building 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 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 species specifically, we distinguished between two options. First option was using the specific tRNA primer in a reverse transcription to convert the whole tRNA pool to cDNA. Following RNase H digestion results in pure cDNA of the desired tRNA species.

Later the desired tRNA species is amplified during a qPCR by using the specific reverse primer and the unspecific adaptor primer.

During qPCR a DNA-intercalating fluorescence dye (Green DNA dye) allows for relative quantification: Green DNA dye binds to double stranded DNA and absorbs blue light and emits green light. The more double stranded DNA is generated, the higher the resulting fluorescence. And the higher the concentration of the template in the sample the faster the fluorescence exceeds the threshold. The number of cycles at which this happens is called the threshold cycle (C_t). (e.g. if sample A showed a C_t of 8 and sample B showed a C_t of 11, sample A contained 2³ = 8 times more template.)

After running a DNA gel, we noticed that the obtained amplification products did not show the expected length. This may have been a result of distinct secondary structures of the tRNA species: the reverse transcription reaction was performed at 42 °C which is the enzyme’s optimum working temperature. However, this temperature is not high enough to prevent secondary structures or to break them up. Therefore, areas with secondary structures may have been inaccessible for the reverse transcriptase resulting in shorter cDNA fragments.

For this reason, we tested a second option to amplify single tRNA species specifically. A modified polymerase together with the specific reverse primer can be used to amplify the desired tRNA species using RNA as a template. This modified polymerase works at temperatures around 65 °C and can use both RNA and DNA as a template. The reverse transcription reaction is thus not needed as a consecutive step anymore. Moreover, the modified enzyme creates the specific cDNA from RNA directly and the high temperature prevents secondary structures. The relative quantification based on C_t values is the same as in the option described before.

Autolysis in combination with bead-milling Results

Free amino acid estimation with rFAN assay

Samples from Experiment Cell_extraction_with_autolysis_combined_with_bead-milling.pdf were used for the analysis.

Yeast extract is mostly obtained by autolysis ¹. In autolysis cells digest their own cell compounds with their own enzymes ². The idea was to transfer this commonly used principal on algae. Therefore, C. vulgaris and C. sorokiniana were heated to 50 °C in alkaline or acidic environment for 41 h. To further crack the cell wall, both algae were treated with bead-milling afterwards. To quantify the success of cell wall disruption free amino acids were measured with rFAN-assay.

The yield of free amino acids was set into relation with the amount of biomass used in the experiment (figure 1).

Figure 1 -Autolysis and subsequent bead-milling of algae C. vulgaris and C. sorokiniana. The percentage of free amino acids [%] relates to the biomass used in the experiment.

The highest amounts of free amino acids were reached with yeast at pH 12 with 4.85 %. Both algae showed very low yield in free amino acids. The best results showed C. sorokiniana at pH 12. It is possible, that the amount of glass beads and the size of the glass beads were to little, which led to less cell wall disruption. Therefore, amino acids would have been retained within the cells. This would explain the little amounts of free amino acids achieved with this method. Also, C. vulgaris and C. sorokinia have a cell wall, in contrast to yeast ³>. Cell walls are harder to break, than a plasma membrane. This could explain the difference between the yeast samples and the algae samples. Due to the low yield in free amino acids, it was decided to investigate other methods for cell extraction of algae.

Anthrone assay to Determine Soluble Carbohydrate Concentration

Similar to the rFAN assay the anthrone assay is a method to detect free monosaccharides in a liquid. Therefor samples from the experiment Experiments_AnthroneAssay.pdf were analyzed. Hereby a calibration curve with known amounts of glucose is created (Figure 2, left side). This calibration curve creates the possibility to calculate the sugar concentration of the samples (Figure 2, right side).

Figure 2: Pictures of the anthrone calibration curve as well as the anthrone assay of samples. For the calibration curve known amounts of glucose is dissolved in water and the optical density at 620 nm is measured (left side). This can be used to determine the monosaccharide concentration of anthone treated samples which previously underwent autolysis (pH3 or pH6) with or without subsequent bead-mill treatment (RKM) (right side).

One can tell from the coloring of the samples in figure 2, that the carbohydrate concentration should differ very slightly between the samples pH3, pH6, bead mill extraction +pH3 and bead mill extraction +pH6. Due to the cloudiness of the control sample, a background corrected optical density could not be determined. Therefore, the coloring scheme served as evaluation for successful carbohydrate determination.

Hereby, bead-mill (RKM) with subsequent autolysis at pH3 was determined to be the method of choice.

CDW correlation of algae Chlorella vulgaris Results

By plotting the measured optical densities against the means of the calculated cellular dry weights, a correlation was obtained. It is shown in the following figure.

Figure 1 - OD-CDW correlation of the algae Chlorella vulgaris. Mean of cellular dry weight in g/L (n=2) was plotted against the measured optical density at 750 nm. Trend line was shown in red.

The trend line in figure 1 is poorly matching the trend of the measurement points. For this reason, the correlation curve was rejected. For improvement of this experiment, measurements should be performed only by one experimenter to reduce pipetting errors or other handling mistakes. Also the measurements should be taken over a longer time period to gain more trust worthy results.

CDW-OD correlation by dilution Results

In the following table you can see the calculated cell dry weights with the corresponding optical density of the tubes. Some tubes broke during the experiment so corresponding measurements could not occur.