Synthetic Genetic Polymers Capable of Heredity and Evolution

Abstract

Genetic information storage and processing rely on just two polymers, DNA and RNA. Whether their role reflects evolutionary history or fundamental functional constraints is unknown. Using polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers (XNAs) based on simple nucleic acid architectures not found in nature. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and for folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.The nucleic acids DNA and RNA provide the molecular basis for all life through their unique ability to store and propagate information. To better understand these singular properties and discover relevant parameters for the chemical basis of molecular information encoding, nucleic acid structure has been dissected by systematic variation of nucleobase, sugar and backbone moieties (1-7).These studies have revealed the profound influence of backbone, sugar and base chemistry on nucleic acid properties and function. Crucially, only a small subset of chemistries allows information transfer through base pairing with DNA or RNA, a prerequisite for crosstalk with extant biology. However, base pairing alone cannot conclusively determine the capacity of a given chemistry to serve as a genetic system, as hybridization need not preserve information content (8). A more thorough examination of candidate genetic polymers' potential for information storage, propagation and evolution requires a system for replication which would allow a systematic exploration of the informational, evolutionary and functional potential of synthetic genetic polymers and open up applications ranging from biotechnology to material science.In principle, informational polymers can be synthesized and replicated chemically (9) with advances in the non-enzymatic polymerization of mononucleotides (10) (11, 12) enabling model selection experiments (13). Nevertheless, chemical polymerization remains relatively inefficient. On the other hand, enzymatic polymerization has been hindered by the stringent substrate selectivity of polymerases. Despite progress in understanding the determinants of polymerase substrate specificity and in engineering polymerases with expanded substrate spectra (7), most unnatural nucleotide analogues are poor polymerase substrates at full substitution, both as nucleotides for polymer synthesis and as templates for reverse transcription. Notable exceptions are 2′OMe-DNA and TNA. 2′OMe-DNA is present in eukaryotic rRNAs, is well-tolerated by natural reverse transcriptases (RTs) and has been shown to support heredity and evolution at near full substitution (14). TNA allowed polymer synthesis and evolution in a three letter system (15) but only limited re...

A short adaptive path from DNA to RNA polymerases

“…Recent work in our group has focused on the engineering of polymerases for the synthesis and replication of unnatural nucleic acid polymers (23). This line of investigation led to the serendipitous identification of a polymerase (D4) with enhanced RNA polymerase activity, which is the starting point of the work described herein (Fig.…”

“…We randomized position 664 in TNE and screened for enhanced RNA polymerase activity using our high-throughput polymerase activity assay [PAA (23)]. The PAA is based on capture of primer extension products and their quantification via hybridization to a specific antisense probe (Fig.…”

“…Together, these enable efficient synthesis of both long RNAs and highly modified nucleic acid polymers through modification of key aspects of the molecular interaction of the polymerase with nascent duplex and substrate NTP. The second gate thus pinpoints a previously undescribed postsynthetic determinant of polymerase substrate specificity with implications for the development of strategies for the enzymatic synthesis and evolution of unnatural nucleic acid polymers (23).…”

Synthesis, Functionalization, and Design of Magnetic Nanoparticles for Theranostic Applications

“…[530] In fact, aptamers are artificially selected sequences of short single-stranded nucleic acids such as DNA, RNA or modified oligonucleotides which can fold into numerous unique 3D conformations. [477,531] These conformations which originate from intramolecular Watson-Crick interactions, [462] have extremely high affinity in binding small and large biochemical targets. [532] However, finding the appropriate sequence able to participate in ligand binding is akin to finding a needle in a haystack.…”

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′‐5′ Linkages

“…However, none of these efficiently synthesized RNA with mixed 3′‐5′/2′‐5′ linkages when GTP was replaced by 3’O‐methyl‐GTP (3′OMe‐GTP). To this end, we prepared a TGK variant with two further mutations known to expand the polymerase substrate spectrum (I521L,8b F545L11) yielding polymerase TGLLK (TgoT: Y409G, I521L, F545L, E664K; Figure S2), which proved effective at synthesizing both DNA and RNA with defined 2′‐5′ linkages (Figure 2). …”