Part:BBa_K3014009

Nearly all biomolecules are quantifiable by established methods. But finding a method to quantify single tRNA species specifically is in vain. Originating from a relatively time-consuming and expensive RT-qPCR protocol published in 2015¹, our derived method is straightforward and cheaper. It simply works with DNA intercalating dye, instead of probes, and uses RNA as a template therefore omitting the necessity of a reverse transcriptase reaction.

DNA-RNA-Hybrid-Adapter for tRNA quantification

Therefore, we designed a linear DNA/RNA hybrid adapter, that specifically binds to the 5’ terminus of mature tRNA.It is complementary to the 3’ overhang of tRNA after the amino acid was removed by diacylation treatment. Using a forward primer, that binds to the adapter and a tRNA specific reverse primer, the desired tRNA can be amplified in a PCR reaction. Individual tRNA levels can be quantified with the use of a DNA intercalating dye.

The adapter is composed of DNA except of the last two 3’-terminal nucleotides, which are composed of RNA. All tRNA molecules show an identical sequence at their 3’ terminus: ACCN, to which the adapter anneals. The last 3’-terminal nucleotide of the adapter should be complementary to the descriminator base of the respective tRNA as shown in figure 1.

Figure 1: Simplified scheme of adapter hybridization to a tRNA.

tRNA quantification

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 adapter 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 adapter 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. <p>To amplify single tRNA species specifically, we used 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 adapter primer.

Adapter test

We tested the adapter in a tRNA dilution series.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 (Ct). If sample A showed a Ct of 8 and sample B showed a Ct of 11, sample A contained 23 = 8 times more template. Therefore, for a 10-fold dilution a difference in Ct values of around 3.16 was expected. Figure 2 shows, that our assumption was fulfilled. The Ct values for AGA increased from 10.94 of the undiluted concentration to 13.17 for 1:10 and for 1:100, the Ct value was 16.56. The results for GAA are quite similar: lowest Ct value was 24.15 (1:1) and increased to 27.35 (1:10) and 31.29 (1:100).

Figure 2: tRNA dilution series 1:1, 1:10, 1:100. V. natriegens tRNA (AGA and GAA) was diluted 1:1, 1:10 and 1:100. AGA 1:1 is represented by the dark blue curve, AGA 1:10 by the red one, AGA 1:100 is coloured in grey, GAA 1:1 in yellow. The light blue curve shows GAA 1:10 and green TGC 1:100. The experiment was carried out in triplicates, the standard deviation is represented by the error bars.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 121
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 121
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 121
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 121
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 121
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. S. Honda, M. Shigematsu, K. Morichika, A. G. Telonis, Y. Kirino (2015) Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA, RNA Biology, Vol. 12, pp 501 – 508