Nonenzymatic RNA primer extension by activated mononucleotides has long served as a model for the study of prebiotic RNA copying. We have recently shown that the rate of primer extension is greatly enhanced by the formation of an imid-azolium-bridged dinucleotide between the incoming monomer and a second, downstream activated monomer. However, the rate of primer extension is further enhanced if the downstream monomer is replaced by an activated oligonucleotide. Even an unactivated downstream oligonucleotide provides a modest enhancement in the rate of reaction of a primer with a single activated monomer. Here we study the mechanism of these effects through crystallographic studies of RNA com-plexes with the recently synthesized nonhydrolyzable substrate analog, guanosine 5′-(4-methylimidazolyl)-phosphonate (ICG). ICG mimics 2-methylimidazole activated guanosine-5′-phosphate (2-MeImpG), a commonly used substrate in non-enzymatic primer extension experiments. We present crystal structures of primer-template complexes with either one or two ICG residues bound downstream of a primer. In both cases, the aryl-phosphonate moiety of the ICG adjacent to the primer is disordered. To investigate the effect of a downstream oligonucleotide, we transcribed a short RNA oligonucleo-tide with either a 5′-ICG residue, a 5′-phosphate or a 5′-hydroxyl. We then determined crystal structures of primer-template complexes with a bound ICG monomer sandwiched between the primer and each of the three downstream oli-gonucleotides. Surprisingly, all three oligonucleotides rigidify the ICG monomer conformation and position it for attack by the primer 3′-hydroxyl. Furthermore, when GpppG, an analog of the imidazolium-bridged intermediate, is sandwiched between an upstream primer and a downstream helper oligonucleotide, the complex is better pre-organized for primer extension than in the absence of a downstream oligonucleotide. Our results suggest that a downstream helper oligonucle-otide contributes to the catalysis of primer extension by favoring a reactive conformation of the primer-template-intermediate complex.

The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5′-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process.

Nucleic-acid-based small molecule and oligonucleotide therapies are attractive topics due to their potential for effective target of disease-related modules and specific control of disease gene expression. As the non-naturally occurring biomolecules, modified DNA/RNA nucleoside and oligonucleotide analogues composed of _L-(deoxy)riboses, have been designed and applied as innovative therapeutics with superior plasma stability, weakened cytotoxicity, and inexistent immunogenicity. Although all the chiral centers in the backbone are mirror converted from the natural _D-nucleic acids, _L-nucleic acids are equipped with the same nucleobases (A, G, C and U or T), which are critical to maintain the programmability and form adaptable tertiary structures for target binding. The types of _L-nucleic acid drugs are increasingly varied, from chemically modified nucleoside analogues that interact with pathogenic polymerases to nanoparticles containing hundreds of repeating _L-nucleotides that circulate durably in vivo. This article mainly reviews three different aspects of _L-nucleic acid therapies, including pharmacological _L-nucleosides, Spiegelmers as specific target-binding aptamers, and _L-nanostructures as effective drug-delivery devices.