A prerequisite for constructing a protocell is supplying a mechanism for replicating the nucleic acids that fill structural, functional or informational roles. Two approaches under active investigation in our laboratory are the in vitro selection of ribozyme polymerases (replicases) and the non-enzymatic replication of nucleic acids using chemically-activated nucleotides. In both cases, the goal is a high-fidelity system for replicating essentially any nucleotide sequence.

Ribozyme polymerases are being selected from fully random or partially structured libraries of RNA or by modification of natural ribozymes with reversible phosphodiesterase bond cleaving activity.

Non-enzymatic replication utilizes chemically-activated monomers or condensing agents to effect polymerization of monomers on template sequences. Chemical activation requires substituting a preferred leaving group in place of one of the hydroxyl groups of the 5´phosphate in a nucleoside monophosphate. Although this chemistry is well established in the literature, it has been limited by the slow reaction rates of naturally-occurring 3´OH-terminated nucleotides. We have recently found that substitution of the 3´OH with an amino group dramatically increases the reaction rate with activated monomers. The product, phosphoramidate DNA, is a well-known polymer with properties similar to naturally-occurring DNA and RNA. We are currently synthesizing a variety of chemically-activated nucleotides with 3´NH2 substitutions and are evaluating the kinetics and fidelity of non-enzymatic polymerization.

Ultimately, we hope to establish a robust and efficient system for template-directed synthesis of NP DNA that can be incorporated into fatty acid vesicles. Based on the known properties of NP DNA, we expect that it can adopt the types of compact structures characteristic of RNA aptamers and ribozymes and will be a rich source of functionally active sequences. Combined with a membrane component for compartmentalization and a system for non-enzymatic replication, we believe that NP DNA may be able to provide the functional and information-carrying needs of a simple cell. To reach this goal we are optimizing the structures of 3´NH2 substituted nucleotides, establishing methods for the in situ activation of nucleotides using condensing agents, evolving protein enzymes capable of synthesizing NP DNA from DNA templates and vice versa to facilitate in vitro selections, and gaining a thorough biochemical and biophysical understanding of NP DNA through experimentation.