Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between product and template

Abstract

Glycerol nucleic acid (GNA) is an interesting alternative basepairing system based on an acyclic, glycerol-phosphate backbone repeat unit. The question of whether DNA polymerases can catalyze efficient template-dependent synthesis using GNA as the template is of particular interest because GNA is unable to form a stable duplex with DNA. In the present study, we screened a variety of DNA polymerases for GNA-dependent DNA synthesis. We find that Bst DNA polymerase can catalyze full-length DNA synthesis on a dodecamer GNA template. The efficiency of DNA synthesis is increased by replacing adenine with diaminopurine in both the GNA template and the DNA monomers and by the presence of manganese ions. We suggest that the BstDNA polymerase maintains a short, transient region of base-pairing between the DNA product strand and the GNA template, but that stable duplex formation between product and template strands is not required for template-dependent polymerization.information transfer ͉ polymerase N ucleic acid analogs with altered backbones or bases are of significant interest in the search for biopolymers with novel chemical and biological properties, and many such analogs have been designed and synthesized (1-3). Evaluation of the hybridization and nuclease-resistance properties of these synthetic nucleic acids has led to several nucleic acid analogues with potential biological applications (4-6). However, much less is known about the potential for information transfer between synthetic nucleic acid systems and the modern DNA/RNA system via templatedependent polymerization, a crucial aspect of the chemical etiology of nucleic acids and directed evolution of functional biopolymers based on a synthetic nucleic acid system. Recently, our group and others have studied the enzymatic synthesis of (3Ј 3 2Ј) ␣-L-threose nucleic acid (TNA; Fig. 1) (7-13). TNA was discovered by Eschenmoser and coworkers (7, 8) during a systematic evaluation of the base-pairing properties of nucleic acids containing alternative sugar-phosphate backbones. TNA was found to be capable of forming stable, antiparallel duplexes with RNA, DNA, and itself, a surprising property for a nucleic acid analog with a shorter backbone repeat unit than DNA or RNA (8,14,15). This remarkable intersystem duplex formation has been considered to be the basis of possible information transfer between TNA and DNA/RNA. Later studies by our group revealed that ␣-L-threose nucleoside 3Ј-triphosphates were substrates for efficient template-dependent enzymatic polymerization by Therminator DNA polymerase, a mutated archaeal family B DNA polymerase (10,11,16). Therminator DNA polymerase was also shown to be an efficient and accurate TNA-dependent DNA polymerase (9). The information transfer between TNA and DNA catalyzed by polymerases provides support for a possible role of TNA as a progenitor of DNA/RNA.The search for nucleic acid analogs with even simpler backbones led to studies of the glycerol nucleic acids (GNAs), which have a three-carbon, acyclic backbone (17-19) (Fi...

Non-natural nucleic acids for synthetic biology

“…This strategy worked for cytosine but not for thymine. In a separate study by Szostak, polymerases were also able to use GNA as a template to make DNA [91]. Aminopropyl versions of GNA have also been reported recently, but compatibility with polymerases has not yet been investigated [92].…”

Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase

“…When used in combinationw ith Kod-RI, an engineered TNA polymerase created by combinatorial librarys creening and scaffold sampling, the aggregate fidelity for acomplete replication cycle of DNA!TNA!DNAi s~1.5 10 À2 ,w hich is slightly higher than one misincorporation per 100 nucleotides. More recently,B st was shown to transcribe and reverse transcribe limited stretches of glyceroln ucleic acid (GNA) polymers, [17,18] as well as copy RNA templatesi nto DNA, [19] suggesting that this enzyme could have broader template specificity than is typical of most naturallyo ccurring DNA polymerases. [12] In that study,t he Geobacillus stearothermophilus DNA polymerase I (Bst) large fragment was identified as one of as mall number of DNA polymerasest hat were capable of extending aD NA primera nnealed to as hort synthetic TNAt emplate with dNTP substrates.…”

Primitive Genetic Polymers

“…The Szostak laboratory recently showed that a GNA region of a DNA-GNA chimeric strand could act as a template for Bst polymerase (Tsai et al 2007). Despite little or no hybridization between GNA and DNA, full-length polymerization of the dodecamer GNA template was achieved.…”