A Study On RNA And Double-Standed RNA Functions
Introduction
The earliest studies performed on RNA were conducted as early as 1933. (Brachet 1933) Yet, possibly to this day, academic community cannot be assured that all properties of RNA have been investigated. Double-stranded RNA (dsRNA) is found in some viruses in a form of small interfering RNA (siRNA). (Dana et al. 2017) It is a variation of RNA that science knows a lot about and many attributes are still being investigated. Frequent use in modern therapeutics was made possible by heavy research on the subject in the 1990s.
Potent and Specific genetic interference by double-stranded RNA in Caenorhabditis elegans, by Fire, Xu, Montgomery and others, is one of many papers published around 1998 covering this matter. The scientists tried to address multiple issues RNA was presenting at the time: what difference in effect sense and antisense preparations of RNA had on the subject and why interference effects were present in the future generations, although most RNA samples degraded in the embryo. It was discovered that while dsRNA had both, potent and specific interference, RNA had rather intermediate effects. (Fire et al. 1998).
Methods, Results and Discussion
Effects of ‘sense’, ‘antisense’ and ‘mixed’ RNA were examined throughout the experiments. Synthesis of RNA was performed with T3 and T7 polymerase reactions and two DNase treatments. Further purification on agarose gel prevented interference between ‘sense’ and ‘antisense’ strands. Further investigation showed that purified and non-purified gels had identical effects on the test subjects. Pre-annealed “sense/antisense” solution was used to test for “mixed” effect of the RNA. Final injection mixes were measured out in 0.5 x 106 to 1.0 x 106 RNA molecules per dose.
The volume of injected material was estimated from a displacement on the site of the injection. Even though the volume injected varied, authors claimed that it would not have much effect on the conclusions drawn. (Fire et al. 1998) Body shape, movement, hatching, feeding and sexual identity were examined for effects of the foreign RNA. RNA solutions were injected into head and tail regions aiming at the cytoplasm of the intestinal region. This method was proven the least disruptive to the animal. (Melo and Fire, 1995) Following the injection animals were transferred to the fresh culture plates and moved every 16 hours to separate adults from the progeny and isolate the phenotypic cohorts.
For comparison of the RNA activities unc-22 gene was chosen. Interference in unc-22 region caused visible twitching phenotype and made it easier to observe the effects of the interference through the generations. ‘Sense’ and ‘antisense’ RNA rendered a recognizable phenotype only in high concentrations (~60 000 molecules per region), while the ‘mixed’ or double-stranded RNA caused complete interference. Moreover, an injection of ‘sense’ and ‘antisense’ RNA separately within a short period of time had the same effect as injecting pre-mixed dsRNA. Authors noted that injection of the dsRNA was unrelated to the unc-22 alongside the single strands and did not affect the interference of a single strand. The effect of dsRNA was identical to the known null-mutant phenotype. This finding was reinforced by additional experimentation with unc-54, fem-1 and hlh-1 genes and respective dsRNA.
In all 3 cases an effect of null-mutant was observed. Trials performed with single strands had no such effect on any of the genes. Though the mechanisms of interference were not completely understood in 1998, authors claimed that the findings added to the debate. Mainly, the fact that dsRNA did not create any changes on the genetic level. All changes were observed on the post-transcriptional level. This was caused by almost complete elimination of endogenous mRNA transcripts in the cells. Another surprising ability of the dsRNA was fast and efficient transport throughout the body of the animal. An injection in the head or the tail regions still had an effect on the opposing regions of the body. Some implications about the possible uses of the dsRNA were made.
The use of dsRNA as a tool for genetic research in nematodes, invertebrates and vertebrates bared high potential. Some assumptions about the mechanism of the dsRNA-mediated interference had to be made in order to recommend it for future research. Yet, the following experimentation proved that dsRNA and siRNA are viable tools not only for research, but also patient treatment. (Cathew and Sontheimer 2010)Conclusion, future work and commentary Concluding the work, authors made multiple notes on the potential of dsRNA research and applications. Simple synthesis and high degree of interference gave molecular biology a new powerful tool for gene expression related experimentation. They evaluated the point by noting that many coding regions of DNA contained unknown functions and dsRNA interference is a sufficient tool to explore the functions of said regions. Though some cells might not be affected by dsRNA-mediated interference due to predominant low-level expression, the tool remained viable for genetic research.
It is important to add that the full function of the dsRNA-mediated interference was not discovered, though some suggestions for future experimentation were stated. Like understanding the function of the dsRNA mechanism, especially post-transcriptional aspects of it. Authors also suggested that dsRNA had a physiological function in organisms; for example, gene silencing. This hypothesis is supported by the fact that dsRNA molecules were efficiently transported throughout the body of the animal. This suggests that there was an effective transport mechanism.
Overall the methodology outlined in the article was sufficient for the adequate conclusion, yet many inaccuracies were allowed in the execution of the experiment. The greatest issue was the volume of RNA-salt solution injected into the animals. The amount injected was judged by the displacement on the site of the injection, which may have possibly led to a great difference in dosage. Authors themselves noted that the amount injected contained 0.5 x 106 to almost 3.0 x 106 molecules of RNA. The 20-40% difference would have been acceptable, since single strands did not have any effect on the organisms. Yet a 100% difference may have affected the results. Tools required for more precise measurements were available at the time of the experimentation.
A reiteration of the experiment with high precision tools would be reasonable. Despite the poor methodology, all arguments were well supported. Main findings were reinforced by additional experiments, for example, the silencing effect of dsRNA was tested on 3 additional genes with various functions and was proven in all cases. Single strands were introduced to the experiment to support the hypothesis that only the dsRNA had any visible effect. The conclusion was drawn out without much support from existing material on the subject and was based on assumptions. The statement that dsRNA may work in other invertebrates was not supported with any other published works. At the same time the basis for the experimentation was well braced with previous findings on C. elegans biology and chemical dynamics of RNA molecules allowing for simple and efficient synthesis of RNA and dsRNA.
References:
- Brachet, J, 1933. Recherches sur la synthese de l'acide thymonucleique pendant le developpement de l'oeuf d'Oursin. Archives de Biologie 44: 519-576.
- Carthew, W & Sontheimer, J, 2009. Origins and Mechanisms of miRNAs and siRNAs. Cell, 136(4), 642–655.
- Dana H, Chalbatani GM, et al., 2017 Molecular Mechanisms and Biological Functions of siRNA. International Journal of Biomedical Science : IJBS. 2017;13(2):48-57 PMID: 28824341.
- Fire, A, et al., 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391(6669), pp. 806–811. doi: 10.1038/35888.
- Mello, C & Fire, A, 2015. DNA transformation. Methods Cell Biol. 48, 451–482.
