Kinetic Explanations for the Sequence Biases Observed in the Nonenzymatic Copying of RNA Templates

ABSTRACTThe identification of nonenzymatic pathways for nucleic acid replication is a key challenge in understanding the origin of life. We have previously shown that nonenzymatic RNA primer extension using 2-aminoimidazole (2AI) activated nucleotides occurs primarily through an imidazolium-bridged dinucleotide intermediate. The reactive nature and preorganized structure of the intermediate increase the efficiency of primer extension but remain insufficient to drive extensive copying of RNA templates containing all four canonical nucleotides. To understand the factors that limit RNA copying, we synthesized all ten 2AI-bridged dinucleotide intermediates and measured the kinetics of primer extension in a model. The affinities of the ten dinucleotides for the primer/template/helper complexes vary by over 7,000-fold, consistent with nearest neighbor energetic predictions. Surprisingly, the reaction rates at saturating intermediate concentrations still vary by over 15-fold, with the most weakly binding dinucleotides exhibiting a lower maximal reaction rate. Certain noncanonical nucleotides can decrease sequence dependent differences in affinity and primer extension rate, while monomers bridged to short oligonucleotides exhibit enhanced binding and reaction rates. We suggest that more uniform binding and reactivity of imidazolium-bridged intermediates may lead to the ability to copy arbitrary template sequences under prebiotically plausible conditions.

Structural interpretation of the effects of threo-nucleotides on nonenzymatic template-directed polymerization

The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3′-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands.

Structural interpretation of the effects of threo-nucleotides on nonenzymatic template-directed polymerization

ABSTRACTThe prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3’-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer ten-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands.

Deoxyinosine and 7-Deaza-2-Deoxyguanosine as Carriers of Genetic Information in the DNA of Campylobacter Viruses

ABSTRACT Several reports have demonstrated that Campylobacter bacteriophage DNA is refractory to manipulation, suggesting that these phages encode modified DNA. The characterized Campylobacter jejuni phages fall into two phylogenetic groups within the Myoviridae: the genera Firehammervirus and Fletchervirus. Analysis of genomic nucleosides from several of these phages by high-pressure liquid chromatography-mass spectrometry confirmed that 100% of the 2′-deoxyguanosine (dG) residues are replaced by modified bases. Fletcherviruses replace dG with 2′-deoxyinosine, while the firehammerviruses replace dG with 2′-deoxy-7-amido-7-deazaguanosine (dADG), noncanonical nucleotides previously described, but a 100% base substitution has never been observed to have been made in a virus. We analyzed the genome sequences of all available phages representing both groups to elucidate the biosynthetic pathway of these noncanonical bases. Putative ADG biosynthetic genes are encoded by the Firehammervirus phages and functionally complement mutants in the Escherichia coli queuosine pathway, of which ADG is an intermediate. To investigate the mechanism of DNA modification, we isolated nucleotide pools and identified dITP after phage infection, suggesting that this modification is made before nucleotides are incorporated into the phage genome. However, we were unable to observe any form of dADG phosphate, implying a novel mechanism of ADG incorporation into an existing DNA strand. Our results imply that Fletchervirus and Firehammervirus phages have evolved distinct mechanisms to express dG-free DNA. IMPORTANCE Bacteriophages are in a constant evolutionary struggle to overcome their microbial hosts’ defenses and must adapt in unconventional ways to remain viable as infectious agents. One mode of adaptation is modifying the viral genome to contain noncanonical nucleotides. Genome modification in phages is becoming more commonly reported as analytical techniques improve, but guanosine modifications have been underreported. To date, two genomic guanosine modifications have been observed in phage genomes, and both are low in genomic abundance. The significance of our research is in the identification of two novel DNA modification systems in Campylobacter-infecting phages, which replace all guanosine bases in the genome in a genus-specific manner.

2′,3′-cGMP exists in vivo and comprises a 2′,3′-cGMP-guanosine pathway

The discovery in 2009 that 2′,3′-cAMP exists in biological systems was rapidly followed by identification of 2′,3′-cGMP in cell and tissue extracts. To determine whether 2′,3′-cGMP exists in mammals under physiological conditions, we used ultraperformance LC-MS/MS to measure 2′,3′-cAMP and 2′,3′-cGMP in timed urine collections (via direct bladder cannulation) from 25 anesthetized mice. Urinary excretion rates (means ± SE) of 2′,3′-cAMP (15.5 ± 1.8 ng/30 min) and 2′,3′-cGMP (17.9 ± 1.9 ng/30 min) were similar. Mice also excreted 2′-AMP (3.6 ± 1.1 ng/20 min) and 3′-AMP (9.5 ± 1.2 ng/min), hydrolysis products of 2′,3′-cAMP, and 2′-GMP (4.7 ± 1.7 ng/30 min) and 3′-GMP (12.5 ± 1.8 ng/30 min), hydrolysis products of 2′,3′-cGMP. To validate that the chromatographic signals were from these endogenous noncanonical nucleotides, we repeated these experiments in mice ( n = 18) lacking 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), an enzyme known to convert 2′,3′-cyclic nucleotides to their corresponding 2′-nucleotides. In CNPase-knockout mice, urinary excretions of 2′,3′-cAMP, 3′-AMP, 2′,3′-cGMP, and 3′-GMP were increased, while urinary excretions of 2′-AMP and 2′-GMP were decreased. Infusions of exogenous 2′,3′-cAMP increased urinary excretion of 2′,3′-cAMP, 2′-AMP, 3′-AMP, and adenosine, whereas infusions of exogenous 2′,3′-cGMP increased excretion of 2′,3′-cGMP, 2′-GMP, 3′-GMP, and guanosine. Together, these data suggest the endogenous existence of not only a 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP/3′-AMP → adenosine), which was previously identified, but also a 2′,3′-cGMP-guanosine pathway (2′,3′-cGMP → 2′-GMP/3′-GMP → guanosine), observed here for the first time. Because it is well known that adenosine and guanosine protect tissues from injury, our data support the concept that both pathways may work together to protect tissues from injury.