Synthesis and Nonenzymatic Template-Directed Polymerization of 2′-Amino-2′-deoxythreose Nucleotides

Threose nucleic acid (TNA) is a potential alternative genetic material that may have played a role in the early evolution of life. We have developed a novel synthesis of 2′-amino modified TNA nucleosides (2′-NH₂-TNA) based on a cycloaddition reaction between a glycal and an azodicarboxylate, followed by direct nucleosidation of the cycloadduct. Using this route, we synthesized the thymine and guanine 2′-NH₂-TNA nucleosides in seven steps with 24% and 12% overall yield, respectively. We then phosphorylated the guanine nucleoside on the 3′-hydroxyl, activated the phosphate as the 2-methylimidazolide, and tested the ability of the activated nucleotide to copy C₄ RNA, DNA, and TNA templates by nonenzymatic primer extension. We measured pseudo-first-order rate constants for the first nucleotide addition step of 1.5, 0.97, and 0.57 h^–1 on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C with 150 mM NaCl, 100 mM <i>N</i>-(hydroxylethyl)­imidazole catalyst, and 5 mM activated nucleotide. The activated nucleotide hydrolyzed with a rate constant of 0.39 h^–1, causing the polymerization reaction to stall before complete template copying could be achieved. These extension rates are more than 1 order of magnitude slower than those for amino-sugar ribonucleotides under the same conditions, and copying of the TNA template, which best represented a true self-copying reaction, was the slowest of all. The poor kinetics of 2′-NH₂-TNA template copying could give insight into why TNA was ultimately not used as a genetic material by biological systems

Synthesis and Nonenzymatic Template-Directed Polymerization of 2′-Amino-2′-deoxythreose Nucleotides

Threose nucleic acid (TNA) is a potential alternative genetic material that may have played a role in the early evolution of life. We have developed a novel synthesis of 2′-amino modified TNA nucleosides (2′-NH₂-TNA) based on a cycloaddition reaction between a glycal and an azodicarboxylate, followed by direct nucleosidation of the cycloadduct. Using this route, we synthesized the thymine and guanine 2′-NH₂-TNA nucleosides in seven steps with 24% and 12% overall yield, respectively. We then phosphorylated the guanine nucleoside on the 3′-hydroxyl, activated the phosphate as the 2-methylimidazolide, and tested the ability of the activated nucleotide to copy C₄ RNA, DNA, and TNA templates by nonenzymatic primer extension. We measured pseudo-first-order rate constants for the first nucleotide addition step of 1.5, 0.97, and 0.57 h^–1 on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C with 150 mM NaCl, 100 mM <i>N</i>-(hydroxylethyl)­imidazole catalyst, and 5 mM activated nucleotide. The activated nucleotide hydrolyzed with a rate constant of 0.39 h^–1, causing the polymerization reaction to stall before complete template copying could be achieved. These extension rates are more than 1 order of magnitude slower than those for amino-sugar ribonucleotides under the same conditions, and copying of the TNA template, which best represented a true self-copying reaction, was the slowest of all. The poor kinetics of 2′-NH₂-TNA template copying could give insight into why TNA was ultimately not used as a genetic material by biological systems

Synthesis and Nonenzymatic Template-Directed Polymerization of 2′-Amino-2′-deoxythreose Nucleotides

Threose nucleic acid (TNA) is a potential alternative genetic material that may have played a role in the early evolution of life. We have developed a novel synthesis of 2′-amino modified TNA nucleosides (2′-NH₂-TNA) based on a cycloaddition reaction between a glycal and an azodicarboxylate, followed by direct nucleosidation of the cycloadduct. Using this route, we synthesized the thymine and guanine 2′-NH₂-TNA nucleosides in seven steps with 24% and 12% overall yield, respectively. We then phosphorylated the guanine nucleoside on the 3′-hydroxyl, activated the phosphate as the 2-methylimidazolide, and tested the ability of the activated nucleotide to copy C₄ RNA, DNA, and TNA templates by nonenzymatic primer extension. We measured pseudo-first-order rate constants for the first nucleotide addition step of 1.5, 0.97, and 0.57 h^–1 on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C with 150 mM NaCl, 100 mM <i>N</i>-(hydroxylethyl)­imidazole catalyst, and 5 mM activated nucleotide. The activated nucleotide hydrolyzed with a rate constant of 0.39 h^–1, causing the polymerization reaction to stall before complete template copying could be achieved. These extension rates are more than 1 order of magnitude slower than those for amino-sugar ribonucleotides under the same conditions, and copying of the TNA template, which best represented a true self-copying reaction, was the slowest of all. The poor kinetics of 2′-NH₂-TNA template copying could give insight into why TNA was ultimately not used as a genetic material by biological systems

Efficient and Rapid Template-Directed Nucleic Acid Copying Using 2′-Amino-2′,3′-dideoxyribonucleoside−5′-Phosphorimidazolide Monomers

The development of a sequence-general nucleic acid copying system is an essential step in the assembly of a synthetic protocell, an autonomously replicating spatially localized chemical system capable of spontaneous Darwinian evolution. Previously described nonenzymatic template-copying experiments have validated the concept of nonenzymatic replication, but have not yet achieved robust, sequence-general polynucleotide replication. The 5′-phosphorimidazolides of the 2′-amino-2′,3′-dideoxyribonucleotides are attractive as potential monomers for such a system because they polymerize by forming 2′→5′ linkages, which are favored in nonenzymatic polymerization reactions using similarly activated ribonucleotides on RNA templates. Furthermore, the 5′-activated 2′-amino nucleotides do not cyclize. We recently described the rapid and efficient nonenzymatic copying of a DNA homopolymer template (dC₁₅) encapsulated within fatty acid vesicles using 2′-amino-2′,3′-dideoxyguanosine−5′-phosphorimidazolide as the activated monomer. However, to realize a true Darwinian system, the template-copying chemistry must be able to copy most sequences and their complements to allow for the transmission of information from generation to generation. Here, we describe the copying of a series of nucleic acid templates using 2′-amino-2′,3′-dideoxynucleotide−5′-phosphorimidazolides. Polymerization reactions proceed rapidly to completion on short homopolymer RNA and LNA templates, which favor an A-type duplex geometry. We show that more efficiently copied sequences are generated by replacing the adenine nucleobase with diaminopurine, and uracil with C5-(1-propynyl)uracil. Finally, we explore the copying of longer, mixed-sequence RNA templates to assess the sequence-general copying ability of 2′-amino-2′,3′-dideoxynucleoside−5′-phosphorimidazolides. Our results are a significant step forward in the realization of a self-replicating genetic polymer compatible with protocell template copying and suggest that N2′→P5′-phosphoramidate DNA may have the potential to function as a self-replicating system

Crystal Structure Studies of RNA Duplexes Containing s²U:A and s²U:U Base Pairs

Structural studies of modified nucleobases in RNA duplexes are critical for developing a full understanding of the stability and specificity of RNA base pairing. 2-Thio-uridine (s²U) is a modified nucleobase found in certain tRNAs. Thermodynamic studies have evaluated the effects of s²U on base pairing in RNA, where it has been shown to stabilize U:A pairs and destabilize U:G wobble pairs. Surprisingly, no high-resolution crystal structures of s²U-containing RNA duplexes have yet been reported. We present here two high-resolution crystal structures of heptamer RNA duplexes (5′-uagc<b><u>s</u></b>^{<b><u>2</u></b>}<b><u>U</u></b>cc-3′ paired with 3′-aucg<b><u>A</u></b>gg-5′ and with 3′-aucg<b><u>U</u></b>gg-5′) containing s²U:A and s²U:U pairs, respectively. For comparison, we also present the structures of their native counterparts solved under identical conditions. We found that replacing O2 with S2 stabilizes the U:A base pair without any detectable structural perturbation. In contrast, an s²U:U base pair is strongly stabilized in one specific U:U pairing conformation out of four observed for the native U:U base pair. This s²U:U stabilization appears to be due at least in part to an unexpected sulfur-mediated hydrogen bond. This work provides additional insights into the effects of 2-thio-uridine on RNA base pairing

Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms

Mass spectrometry (MS) is a powerful technique for characterizing noncanonical nucleobases and other chemical modifications in small RNAs, yielding rich chemical information that is complementary to high-throughput indirect sequencing. However, mass spectra are often prohibitively complex when fragment ions are analyzed following either solution phase hydrolysis or gas phase fragmentation. For all but the simplest cases, ions arising from multiple fragmentation events, alternative fragmentation pathways, and diverse salt adducts frequently obscure desired single-cut fragment ions. Here we show that it is possible to take advantage of predictable regularities in liquid chromatographic (LC) separation of optimized RNA digests to greatly simplify the interpretation of complex MS data. A two-dimensional analysis of extracted compound chromatograms permits straightforward and robust de novo sequencing, using a novel Monte Carlo algorithm that automatically generates bidirectional paired-end reads, pinpointing the position of modified nucleotides in a sequence. We demonstrate that these advances permit routine LC–MS sequencing of RNAs containing noncanonical nucleotides, and we furthermore examine the applicability of this approach to the study of oligonucleotides containing artificial modifications as well as those commonly observed in post-transcriptionally modified RNAs