Efficient and rapid template-directed nucleic acid copying using 2'-amino-2',3'-dideoxyribonucleoside-5'-phosphorimidazolide monomers

Abstract

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 (dC(15)) 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.

Figure 1

N2′→P5′-DNA genetic polymer system. (a) 2′-Amino-2′,3′-dideoxyribonucleoside−5′-phosphorimidazolides. Clockwise from upper-left: 2′-NH₂−ImpddA, R¹ = -H or 2′-NH₂−ImpddD, R¹ = -NH₂; 2′-NH₂−ImpddC; 2′-NH₂−ImpddU, R¹ = -H or 2′-NH₂−Impdd-C5-(1-propynyl)-U, R¹ = -C≡C−CH₃; 2′-NH₂−ImpddG. (b) Structure of O2′→P5′-phosphodiester-linked DNA (right) compared to N2′→P5′-phosphoramidate−DNA (left). The 2′-internucleotide bridging atom substitution is shown in red. (c) Primer-extension reaction scheme. A 5′-Cy3-labeled 2′-amino-terminated DNA primer anneals to a complementary template. 2′-NH₂−ImpddN analogues form Watson−Crick base pairs on a complementary template and participate in a chemical chain reaction forming the N2′→P5′-DNA polymer product. (d) Chemistry of the template-directed copying reaction using the activated 2′-NH₂−ImpddN monomer. The attacking nucleophile is shown in red.

Figure 2

Primer-extension reaction using a 2′-NH₂−ImpddG as monomer. (a) Primer-extension reaction scheme showing a 5′-Cy3-labeled 2′-amino-terminated DNA primer annealed to a complementary template. 2′-NH₂−ImpddG monomer participates in a chemical chain reaction extending the primer by four (4) nucleotides on the complementary template forming a N2′→P5′-DNA polymer product. * indicates new phosphoramidate bond. (b) High-resolution gel electrophoresis analysis of primer-extension products on indicated templates. Primer-extension reactions contained 0.1 μM Cy3-labeled-2′-amino-terminated DNA primer, 0.5 μM template, 100 mM MES-CAPS-HEPES, pH 7.5, 100 mM 1-(2-hydroxyethyl)imidazole, and 5 mM 2′-NH₂−ImpddG. The reaction was initiated by addition of 2′-NH₂−ImpddG, thermal cycling as described in the , and incubation at 4 °C for the indicated time. Arrows indicate primer and full-length product. (c) MALDI-TOF MS analysis of primer-extension product. 100−200 pmol amino-terminated primer was extended on an RNA template followed by base hydrolysis of the RNA template and preparation for MALDI-TOF MS as detailed in the . DNA control oligonucleotide (left): calcd mass 4999.87 and observed mass 4999.67; N2′→P5′-DNA extension product (right): calcd mass 4994.95 and observed mass 4994.91. (d) Graphical summary of primer-extension reactions. Primer-extension reaction efficiency is expressed as percent full-length product (≥+4) achieved at the given time point.

Figure 3

Primer-extension reaction using a 2′-NH₂−ImpddC as monomer. (a) Primer-extension reaction scheme. * indicates new phosphoramidate bond. (b) High-resolution gel electrophoresis analysis of primer-extension products on indicated templates. Primer-extension reactions were completed as previously described with 5 mM 2′-NH₂−ImpddC. Arrows indicate primer and full-length product. (b) MALDI-TOF MS analysis of primer-extension product performed as described in the . DNA control oligonucleotide (left): calcd mass 4839.84 and observed mass 4839.66; N2′→P5′-DNA extension product (right): calcd mass 4834.92 and observed mass 4835.08. (c) Graphical summary of primer-extension reaction efficiency.

Figure 4

Primer-extension reaction using a 2′-NH₂−ImpddA as monomer. (a) Primer-extension reaction scheme. * indicates new phosphoramidate bond. (b) High-resolution gel electrophoresis analysis of primer-extension products on indicated templates. Template-copying reactions were run as previously described with 5 mM (upper) or 30 mM (lower) 2′-NH₂−ImpddA. Arrows indicate primer and full-length extension product.

Figure 5

Primer-extension reaction using the 2′-NH₂−ImpddU as monomer. (a) Primer-extension reaction scheme. * indicates new phosphoramidate bond. (b) High-resolution gel electrophoresis analysis of primer-extension products on indicated templates. Primer-extension reactions were run as described with 5 mM (upper) or 30 mM (lower) 2′-NH₂−ImpddU. Arrows indicate primer and full-length product.

Figure 6

Primer-extension reaction using the 2′-NH₂−ImpddD as monomer. (a) Primer-extension reaction scheme. * indicates new phosphoramidate bond. (b) High-resolution gel electrophoresis analysis of primer-extension products on indicated templates. Primer-extension reactions were completed as previously described with 5 mM 2′-NH₂−ImpddD. (c) MALDI-TOF MS analysis of primer-extension product. DNA control oligonucleotide (left): calcd mass 4935.89 and observed mass 4936.00; N2′→P5′-DNA extension product (right): calcd mass 4991.01 and observed mass 4990.86. (d) Graphical summary of primer-extension reactions.

Figure 7

Primer-extension reaction using a 2′-NH₂−ImpddU^p as monomer. (a) Primer-extension reaction scheme. * indicates new phosphoramidate bond. (b) High-resolution gel electrophoresis analysis of primer-extension products on indicated templates. Primer-extension reactions were completed as previously described with 5 mM 2′-NH₂−ImpddU^p. (c) MALDI-TOF MS analysis of primer-extension product. DNA control oligonucleotide (left): calcd mass 4899.84 and observed mass 4899.84; N2′→P5′-DNA extension product (right): calcd mass 4990.92 and observed mass 4990.94. (d) Graphical summary of primer-extension reactions.

Figure 8

High-resolution gel electrophoresis analysis of mixed-sequence RNA template-copying reaction. (a) Primer-extension reaction scheme. A 5′-Cy3-labeled 2′-amino-terminated DNA primer annealed to a complementary RNA template. 2′-NH₂−ImpddN monomers participate in a chemical chain reaction extending the primer by 15 nucleotides on the complementary template, forming a N2′→P5′-DNA polymer product. * indicates new phosphoramidate bond. (b) Primer-extension reactions were completed as previously described with 0.5 μM template (5′-CDDCCDGU^pCDDCDCG-3′-primer binding site), 5 mM 2′-NH₂−ImpddG, 5 mM 2′-NH₂−ImpddC, 10 mM 2′-NH₂−ImpddD, and 10 mM 2′-NH₂−ImpddU^p. The reaction was initiated with addition of the monomers, thermal cycling as described in the , and incubation at 4 °C for the indicated time. Arrows indicate primer, full-length product, and stalled products.