5 research outputs found
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′-NH<sub>2</sub>-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′-NH<sub>2</sub>-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 C<sub>4</sub> 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<sup>–1</sup> on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C
with 150 mM NaCl, 100 mM <i>N</i>-(hydroxylethyl)Âimidazole
catalyst, and 5 mM activated nucleotide. The activated nucleotide
hydrolyzed with a rate constant of 0.39 h<sup>–1</sup>, 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′-NH<sub>2</sub>-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′-NH<sub>2</sub>-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′-NH<sub>2</sub>-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 C<sub>4</sub> 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<sup>–1</sup> on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C
with 150 mM NaCl, 100 mM <i>N</i>-(hydroxylethyl)Âimidazole
catalyst, and 5 mM activated nucleotide. The activated nucleotide
hydrolyzed with a rate constant of 0.39 h<sup>–1</sup>, 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′-NH<sub>2</sub>-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′-NH<sub>2</sub>-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′-NH<sub>2</sub>-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 C<sub>4</sub> 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<sup>–1</sup> on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C
with 150 mM NaCl, 100 mM <i>N</i>-(hydroxylethyl)Âimidazole
catalyst, and 5 mM activated nucleotide. The activated nucleotide
hydrolyzed with a rate constant of 0.39 h<sup>–1</sup>, 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′-NH<sub>2</sub>-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 (dC<sub>15</sub>) 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
DataSheet_1_Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.docx
IntroductionThe complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885.MethodsThe interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers.ResultsZilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies.ConclusionOur findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.</p