Replication of Genetic Information with Self‐Encoded Replicase in Liposomes

Abstract

In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromolecules termed a "self-encoding system", in which the genetic information is replicated by self-encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information molecule and an in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qbeta replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its production. This in-liposome self-encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different molecules and are compartmentalized into the microenvironment for evolvability.

Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment

“…To date, several replication systems have been constructed either from various sets of RNA and proteins 19,20 or from RNA ribozymes only 21,22 , although these replication systems lack the process by which the genetic information is translated into proteins. To construct an artificial system that replicates in the same manner as natural organisms, through the translation of a replication enzyme, we combined an artificial genomic RNA that encodes an RNA-dependent RNA polymerase, the Qb replicase, with a reconstituted translation system 23,24 . In this translationcoupled RNA replication (TcRR) system, the genomic plus-strand RNA (2,125 nucleotides) replicates using an RNA replicase translated from its own sequence via the synthesis of the complementary minus-strand (Genomic RNA replication, Fig.…”

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

“…The next step along the road map for SSMC construction is the realization of nucleic acidbased reactions inside liposomes. These have been already carried out, as in the case of DNA synthesis (PCR inside liposomes, Oberholzer et al, 1995), DNA transcription (RNA synthesis, Tsumoto et al, 2001), RNA replication (Kita et al, 2008), and the coupled transcription/translation reactions (protein synthesis). In particular, the protein synthesis is currently the most well studied reaction inside liposomes (for references, see paragraph Nr.…”

“…The correct production of (-)RNA is confirmed by the fact that it is designed to codify for -galactosidase, whose ribosomal production was revealed by the increase of fluorescence due to the action of -galactosidase on a fluorogenic substrate. This is an example of self-encoding genetic material, which codifies for its production inside lipid compartments (Kita et al, 2008). a different approach was tested.…”

“…7B), and concerns the replication of RNA inside vesicles, operated by an enzyme (the Q-replicase) whose production was ultimately encoded in the RNA sequence itself (Kita et al, 2008). The aim of this study was the coupling between informationcarrying molecules and the production of molecular machinery that replicate the information (RNA that codifies for an enzyme whose role is to replicate RNA).…”

Are in vivo selections on the path to extinction?

“…Nevertheless, recursivity at the DNA level is the most common solution in nature but not the only solution. Recursivity can also take place at the RNA level, using an RNA‐dependent RNA polymerase (Kita et al ., 2008), which, if compatible with IVTT, may obviate the need for RCA, but it may also complicate the analysis of the selected individuals and introduce bias by imposing a reverse transcription step prior to amplification (Fig. 1B).…”