Alonso Ricardo

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.

mRNA display: from basic principles to macrocycle drug discovery.

We describe a new discovery technology that uses mRNA-display to rapidly synthesize and screen macrocyclic peptide libraries to explore a valuable region of chemical space typified by natural products. This technology allows high-affinity peptidic macrocycles containing modified backbones and unnatural side chains to be readily selected based on target binding. Success stories covering the first examples of these libraries suggest that they could be used for the discovery of intracellular protein-protein interaction inhibitors, highly selective enzyme inhibitors or synthetic replacements for monoclonal antibodies. The review concludes with a look to the future regarding how this technology might be improved with respect to library design for cell permeability and bioavailability.

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 N-(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.

Abstract C212: Discovery of high affinity cyclic peptidomimetics binding Mcl-1 and Ras.

Many well-validated oncology targets regulate signaling pathways through interactions with other proteins. Addressing these targets, which often lack suitable small molecule binding sites, requires larger and more complex molecules that are capable of binding relatively large or flat surfaces. We are testing whether this target class can be addressed by using in vitro selection to screen extremely large cyclic peptidomimetic libraries with novel design features. Peptide-mRNA display libraries comprised of approximately 1 trillion cyclic peptidomimetics were created by in vitro translation using a mixture of natural, unnatural, and N-methylated amino acids. Pooled libraries were subjected to multiple rounds of selection and amplification to identify sequences that bound with high affinity to immobilized Mcl-1 or Ras. Selected Mcl-1 cyclic peptidomimetics were synthesized and their binding and specificity were studied by surface plasmon resonance (SPR) using immobilized Mcl-1 and Bcl-2. Fluorescence polarization (FP) was used to demonstrate that isolated peptidomimetics competed with Bim-derived peptides (IC50≈100-400 nM), a measure of their ability to block the interactions between Mcl-1 and binding partners. The co-crystal structure of one Mcl-1 inhibitor revealed that the cyclic peptidomimetic bound the BH3 helix-binding grove by forming a β-hairpin, an unprecedented type of interaction with Bcl-2 family members. The cell permeability of analogs of this compound was verified by fluorescence microscopy and by induction of apoptosis. In a second example, a cyclic peptidomimetic selected against mutant H-Ras was shown to bind both H-Ras and K-Ras with high affinity by SPR (KD=2.5 nM) and FP (Ki≈£10 nM). Despite its high affinity, this compound did not significantly impact Ras activity in several in vitro assays (GST-Raf-RBD binding, GTPase, and GDP/GTP exchange). A co-crystal structure with mutant K-Ras, however, revealed that this compound binds a novel site in the C-terminus of the Ras G-domain. Remarkably, this compound also forms a β-hairpin, and like the Mcl-1 compound, also appears to enter cells. Experiments to test how this compound may affect Ras signaling or localization, and the function of this novel site in cells or in vitro with full-length lipid modified Ras are in progress. The target-bound conformation of both inhibitors reveled a network of intramolecular hydrogen bonds that are predicted to reduce interactions with water and may help to explain the intrinsic cell permeability of these molecules. The use of extremely large peptidomimetic libraries built with novel chemical diversity appears to be an extremely efficient platform for isolating high affinity binders for hard-to-target intracellular targets, yielding novel binding modes and binding sites that are unlikely to be identified by typical small molecule approaches or rational design. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C212. Citation Format: Kristopher Josephson, Zhong Ma, Zhaolin Wang, Yili Sun, Sylvia Tobe, Sarah Perlmutter, Rohit Vyasamneni, Ping Ye, Nicolas Boyer, Micelle Arata, Kelli Pattavina, Kathleen Seyb, Hong Zheng, Imelda Sollomoni, Ezekiel Nims, Emily de Koning, Alonso Ricardo, Douglas Treco. Discovery of high affinity cyclic peptidomimetics binding Mcl-1 and Ras. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C212.

Der Ursprung irdischen Lebens

Allmahlich lichtet sich der Nebel um eines der grosten Geheimnisse der Natur. Im Labor wiederholen Forscher die tastenden Schritte, mit denen einst aus unbelebter Materie die ersten Organismen entstanden.

The Nature of Life: Classical and Contemporary Perspectives from Philosophy and Science: Is there a common chemical model for life in the universe?

A review of organic chemistry suggests that life, a chemical system capable of Darwinian evolution, may exist in a wide range of environments. These include non-aqueous solvent systems at low temperatures, or even supercritical dihydrogen-helium mixtures. The only absolute requirements may be a thermodynamic disequilibrium and temperatures consistent with chemical bonding. A solvent system, availability of elements such as carbon, hydrogen, oxygen and nitrogen, certain thermodynamic features of metabolic pathways, and the opportunity for isolation, may also define habitable environments. If we constrain life to water, more specific criteria can be proposed, including soluble metabolites, genetic materials with repeating charges, and a well defined temperature range.