Mini Review December 2011, 2(2):32-35 GERF Bulletin of Biosciences *Corresponding author: sagarowmr@gmail.com Copyright © 2011 Green Earth Research Foundation www.gerfbb.com RNA-RNA ligation: Methods, Prospects and Applications Sagar Sridhara* and Suparna Sanyal Department of Cell and Molecular Biology, BMC, 75124, Uppsala University, Sweden Abstract Ligation is a widely used molecular biology tool, used for the purpose of joining the ends of linear DNA or RNA molecules, either to the same (intramolecular ligation) or different molecule (intermolecular ligation). The 5’-3’ end to end ligation of a linear, single-stranded mRNA molecule results in a circular mRNA, which has many advantages in comparison to its linear counterpart. The methods for circularization of mRNA have been discussed with special emphasis on enzyme mediated ligation. Keywords: Ligation, T4 DNA ligase, T4 RNA ligase, Circular mRNA. In the field of molecular biology, researchers are curious to know the detailed mechanism of central dogma, with regard to the structure and functions of the key components including DNA, RNA and the proteins. In the majority of living cells, DNA is the genetic material, whereas in several viruses and bacteriophages, RNA is the genetic material. The three types of RNA found in the cells are mRNA (messenger RNA), rRNA (ribosomal RNA) and tRNA (transfer RNA) (Carpi, 2003). Generally, mRNA is a linear, single-stranded molecule. But, circular RNAs can also be found in nature. The non- linear form of RNA is found, although not frequently, in case of viroids, where the RNA molecules exist as single stranded covalently closed circles (Sanger et al., 1976). Such naturally occurring RNA circles are also found in plant pathogens, virusoids and the satellite viruses (Puttaraju and Been, 1996). Circular mRNA offers many unique advantages over linear ones; viz., increased stability, allowance of topology investigations, creation of long repeating polypeptides and usage as a model system (Chan et al., 1988; Umekage and Kikuchi, 2009; Ford and Ares, 1994). Hence, circular mRNA molecule can be constructed for effective use as a molecular biology tool. Circular mRNA The main features and advantages of circular mRNA are discussed below. The circular form of mRNA possesses higher stability than their linear counterparts making them more resistant to nuclease degradations (Puttaraju and Been, 1996). The half-life of excised group I intron circularized RNA is much higher, in comparison to the half-life of a typical E. coli mRNA at 37°C (Chan et al., 1988). Moreover, the topological information of RNA can be obtained in its circular form, which is not possible in its linear form as the information may be lost upon analysis of linear products. As the circular mRNA does not possess free 5’ or 3’ends, this property can be effectively utilized to study the functions of the free ends of a linear mRNA (Ford and Ares, 1994). Furthermore, the potential changes in the functions of certain regulatory proteins, involved with the mRNA could be analyzed by the circularization of mRNA. Circular mRNA can also have strong implication in protein research. In one study, circular mRNA was used to create a very long, repeating polypeptide on a stop codon deficient open reading frame (Perriman and Ares, 1998). In eukaryotes, translating mRNA is shown to be circular by molecular interaction between the proteins binding to 5’ cap and 3’ poly A tail (Wells et al., 1998). It has been proposed that circularization ensures that the mRNA has been correctly processed and thus aids in proof-reading. The recent knowledge of different initiation modes of translation in bacteria suggests that the circularization of mRNA may facilitate direct recycling of ribosomes or the ribosomal subunits after the first-round of translation termination. This has not been experimentally proved and hence still is a theoretical model (Preiss, 2003). Thus, putting these evidences and predictions together, the circular mRNA serves as a suitable model to analyze the effect of variations Introduction