RNA self‐processing: Formation of cyclic species and concatemers from a small engineered RNA

Abstract

a b s t r a c tWe have engineered a self-processing RNA, derived from the hairpin ribozyme that runs through a cascade of cleavage and ligation reactions thereby changing its topology. The first two cleavage events leave the resulting RNA with a 5 0 -OH group and a 2 0 ,3 0 -cyclic phosphate. Thus, upon refolding, intramolecular ligation delivers a cyclic species. In addition, we demonstrate formation of concatemers resulting from multiple intermolecular ligations. Our results demonstrate the potential of RNA for self-supported topology changes and support the suggestion of 2 0 ,3 0 -cyclic phosphates being suitable activated building blocks for reversible phosphodiester bond formation in the RNA world.

In vitro circularization of RNA

“…26,27 This protocol has been mostly used with T7-RNA-Polymerase and therefore requires guanosine conjugated via a 5 0 -phosphate with the desired functionality, as initiator nucleotide (the 5 0 -phosphate is necessary for recognition/ acceptance of the modified building block by the polymerase). In our laboratory, we have made use of this strategy for enzymatic preparation of RNAs carrying a biotinylated nucleoside 28 or a monophosphate 29 at the 5 0 -end. 3 0 -terminal functionalization As for 5 0 -terminal modification, 3 0 -end modification can be easily achieved by chemical synthesis provided that a suitable modified building block attached to a solid support is available or alternatively, can be made in the laboratory.…”

“…62 We have successfully used T4 Rnl2 to circularize a 104nt linear precursor RNA with donor and acceptor ends positioned in a double stranded region. 29 Recently, circular RNAs ranging in size from 129 to 387 nucleotides were prepared by intramolecular ligation of the respective linear precursor in the presence of a 20nt DNA helper oligonucleotide. 63 In summary, RNA circularization by enzymatic ligation is a valuable option.…”

“…Previously, we have engineered hairpin ribozyme variants that fold into 2 alternative cleavage active conformations, thus cutting off their 5 0 -and 3 0 -terminus, and thereby producing a 5 0 -OH group and a 3 0 -terminal 2 0 ,3 0 -cyclic phosphate. 29,75,76 These characteristic ends are required for ligation, which then can take place in an intramolecular manner as depicted in Fig. 3.…”

“…We have demonstrated hairpin ribozyme supported RNA circularization for the generation of 83mers. 29,75,76 However, as shown in Fig. 3, the hairpin ribozyme structure may be embedded in RNA sequences of variable length (gray lines), thus paving the way toward preparation of large RNA circles.…”

Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM

“…Using the ribozyme CRZ-2 (Scheme 2), which was developed previously (Pieper et al 2007) and recently analyzed in detail (Petkovic and Müller 2013), as template, we computationally optimized sequences using the program switch.pl (Flamm et al 2001) of the Vienna RNA package (Lorenz et al 2011). Four variants with different behavior according to our scoring functions were selected and analyzed in detail by polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM).…”