Closing the circle: replicating RNA with RNA

Abstract

How life emerged on this planet is one of the most important and fundamental questions of science. Although nearly all details concerning our origins have been lost in the depths of time, there is compelling evidence to suggest that the earliest life might have exploited the catalytic and self-recognition properties of RNA to survive. If an RNA based replicating system could be constructed in the laboratory, it would be much easier to understand the challenges associated with the very earliest steps in evolution and provide important insight into the establishment of the complex metabolic systems that now dominate this planet. Recent progress into the selection and characterization of ribozymes that promote nucleotide synthesis and RNA polymerization are discussed and outstanding problems in the field of RNA-mediated RNA replication are summarized.

Figure 1.

RNA-catalyzed polymerization and cross-replication by ligation. (A) Cartoon schematic of cross-replication of RNA ligase ribozymes: A ligase ribozyme (colored in two shades of green) catalyzes the ligation of two orange oligonucleotides (Rz′-1 and Rz′-2) to generate a ligase ribozyme that catalyzes the ligation of two green oligonucleotides (Rz-1 and Rz-2) to regenerate the first ligase ribozyme. (Adapted from Lincoln and Joyce 2009.) (B) Cartoon schematic scheme of ribozyme-catalyzed RNA polymerization. The complete extension of an RNA primer (pink) according to the sequence of a template (blue) by an RNA polymerase produces an RNA duplex.

Figure 2.

Genomic elements, their asymmetrical transcription resulting in genomic replication and synthesis of functional RNAs by ligation. (A) Short double-stranded elements define the total RNA genome together with guide sequences important for the later synthesis of full length functional RNAs. (B) Asymmetrical transcription from either strand of the duplex genomic element results in the synthesis of multiple genomic element copies together with an excess of one strand. This asymmetry could be generated by having one end of the genomic element fray with a higher propensity than the other. (C) Excess single-stranded transcripts can in turn hybridize to guide element sequences making them substrates for ligase enzymes and allowing the synthesis of long highly functional RNAs.

Figure 3.

In vitro encapsulated selection scheme for trans acting RNA polymerase ribozymes. A DNA pool was generated that contained a T7 promoter allowing transcription of mutagenized Round 18 ribozyme sequence library. After ligating a RNA primer (orange)–template (green) complex to the DNA pool, genomes were encapsulated and RNA transcribed by T7 RNAP. Active RNA polymerase ribozymes extend the RNA primer tethered to their DNA genome and in the process incorporate 4-thiouridine residues in the growing strand. This allows selection of functional genomes using thiol-sensitive mercury gels and hybridization-based capture using biotinylated oligonucleotides. The captured DNA was then PCR amplified and used in a further round of selection (Zaher and Unrau 2007 and reprinted here with permission by the author).

Figure 4.

RNA polymerase ribozyme modularity: The B6.61 Ligase core and accessory domain in a range of equimolar (0.5 µm) contexts. (A) Comparison of polymerization activity of trans bimolecular constructs (L.1 + A.1) with that of B6.61. (B) Polymerization activity assay of different assemblies of the two hybridized trans bimolecular constructs as shown in panel C. (C) Cartoon schematic of the four bimolecular assemblies.

Figure 5.

Comparison of initiation mechanisms. (A) Primer-independent (de novo) initiation B, C, and D—Primer-dependent initiation strategies: (B) “Borrowing” a hydroxyl from a nearby protein residue (C) Use of a short oligonucleotide from abortive cycling in de novo initiation or from a cleaved mRNA; (D) Template folds back to form a stable hairpin that is then extended. Adapted from (Paul et al. 1998; van Dijk et al. 2004 and reprinted with permission from The Journal of General Virology ©2004; Ng et al. 2008 and reprinted with express permission from the authors).

Figure 6.

Evolving a processive strand-displacing RNA polymerase ribozyme from the B6.61 RNA polymerase. (A) After formation of an initiation complex, which exposes the template strand, a short RNA primer sequence is either synthesized de novo or allowed to hybridize. (B) Elongation requires that nucleotides immediately upstream of the site of nucleotide incorporation are single-stranded. If interactions between the ligase core (L) and the accessory domain (Circled A) can be improved so that the freshly synthesized strand together with template are held robustly and nonspecifically the polymerase should become much more processive. If in addition these two domains can be rigidly connected to a third domain (Circled B) that ensures dehybridization of the incoming duplex, transcription from dsRNA as well as ssRNA templates would be possible. This elongation complex should remain invariant in shape as it slides along the template. (C) Transcription ends with the release of one strand from the original duplex RNA and the duplex RNA being regenerated with a freshly synthesized RNA strand.

Figure 7.

Selecting for strand displacement in an RNA polymerase ribozyme. A pool of potential strand displacing RNA polymerase ribozymes is tethered by a long flexible linker to a RNA (red strand) that is partially complementary to the template strand (bottom black strand). The unhybridized nucleotides in the red strand are not able to hybridize to the template strand resulting in the formation of an elongation complex mimic. Transcription by the tethered polymerase that is able to displace the red strand results in the polymerase disassociating from the extending primer template duplex (black strands). Freed polymerases can then be differentially recovered and amplified.