Naturally Occurring Modified RNA Base Analogs
Cellular RNAs consist of four major nucleosides and a large number of minor nucleosides (~100) (Limbach
et al., 1994, Cantara
et al., 2011, Abeydeera
et al., 2008) which are generated post-transcriptionally by enzymatic modification of the four major ones, or in exceptional cases, by base replacement (Grosjean
et al., 1998, Czerwoniec
et al., 2009). These natural minor nucleotides perform a large number of functions (Grosjean
et al., 1998, Grosjean
et al., 2005), some of which are yet to be discovered.
One function of minor nucleotides is stabilization of functional RNA structure (especially tRNAs and ribosomal RNAs) (Helm
, 2006). The presence of modified minor nucleoside often stabilizes secondary and tertiary folding structure resulting in augmented thermal stability and reduced dynamics of the RNA molecule. Under certain circumstances, the presence of one or several minor nucleotides can induce an alternative folding, often comprising significant alterations in the secondary structure.
A substantial number of minor nucleoside 5’ triphosphate (mNTP) allows for in vitro preparation of modified RNA with RNA polymerases (Chelliserrykattil
et al., 2004, Lauridsen
et al., 2012) for new RNA function and folding structure. Research is required to determine which RNA polymerases are compatible with each mNTP.
Pseudouridine-5’-triphosphate and T7-RNA polymerase have been used to successfully synthesize mRNA with reduced innate immune response and toxicity compared to unmodified mRNA. (Kariko
et al., 2007, Nallagatla
et al., 2008, Kariko
et al., 2005)
mNTPs can also be used in the preparation of RNA aptamers or ribozymes to modulate their stability and function (Kariko
et al., 2005, Keefe
et al., 2008, Klussmann
et al., 2006). Such aptamers and ribozymes are expected to exhibit low, or none, in vivo toxicity since they are prepared from only naturally occurring standard and modified nucleosides.