RNA self-processing towards changed topology and sequence oligomerization

Abstract

Reversible chemistry, allowing for chain-forming as well as chain-breaking steps, is important for biological self-organization. In this context, ribozymes, catalyzing both RNA cleavage and ligation, may have significantly contributed to extending the sequence space and length of RNA molecules in early life forms. Here we present an engineered RNA that self-processes by passing through a number of cleavage and ligation steps. Intermolecular reactions compete with intramolecular reactions, resulting in a variety of products. Our results demonstrate that RNA can undergo self-oligomerization, which may have been important for extending the RNA genome size in RNA world scenarios.

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

“…Hairpin ribozymes that form a stable structure, such that fragments remain bound, favor ligation, whereas hairpin ribozymes that are less stable, such that cleavage fragments can easily dissociate, favor cleavage (Fedor 1999, Welz et al 2003. These characteristic features distinguish the hairpin ribozyme from other small ribozymes, and we have shown in previous work that structural manipulation of hairpin ribozyme variants allows tuning of cleavage and ligation activity (Welz et al 2003;Ivanov et al 2005;Vauleon et al 2005;Drude et al 2007Drude et al , 2011Pieper et al 2007;Müller 2013, Balke et al 2014). Among these variants is a hairpin ribozyme that can cleave off its 5 ′ -and 3 ′ -end (Pieper et al 2007).…”

“…These characteristic features distinguish the hairpin ribozyme from other small ribozymes, and we have shown in previous work that structural manipulation of hairpin ribozyme variants allows tuning of cleavage and ligation activity (Welz et al 2003;Ivanov et al 2005;Vauleon et al 2005;Drude et al 2007Drude et al , 2011Pieper et al 2007;Müller 2013, Balke et al 2014). Among these variants is a hairpin ribozyme that can cleave off its 5 ′ -and 3 ′ -end (Pieper et al 2007). The cleaved product has two reactive ends that can ligate to circular species or concatemers of two or more molecules.…”

“…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).…”

In vitro circularization of RNA

“…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.…”