Douglas A. Treco

Template-directed Synthesis of a Genetic Polymer in a Model Protocell

Contemporary phospholipid-based cell membranes are formidable barriers to the uptake of polar and charged molecules ranging from metal ions to complex nutrients. Modern cells therefore require sophisticated protein channels and pumps to mediate the exchange of molecules with their environment. The strong barrier function of membranes has made it difficult to understand the origin of cellular life and has been thought to preclude a heterotrophic lifestyle for primitive cells. Although nucleotides can cross dimyristoyl phosphatidylcholine membranes through defects formed at the gel-to-liquid transition temperature, phospholipid membranes lack the dynamic properties required for membrane growth. Fatty acids and their corresponding alcohols and glycerol monoesters are attractive candidates for the components of protocell membranes because they are simple amphiphiles that form bilayer membrane vesicles that retain encapsulated oligonucleotides and are capable of growth and division. Here we show that such membranes allow the passage of charged molecules such as nucleotides, so that activated nucleotides added to the outside of a model protocell spontaneously cross the membrane and take part in efficient template copying in the protocell interior. The permeability properties of prebiotically plausible membranes suggest that primitive protocells could have acquired complex nutrients from their environment in the absence of any macromolecular transport machinery; that is, they could have been obligate heterotrophs.

Effect of Stalling after Mismatches on the Error Catastrophe in Nonenzymatic Nucleic Acid Replication

The frequency of errors during genome replication limits the amount of functionally important information that can be passed on from generation to generation. During the origin of life, mutation rates are thought to have been quite high, raising a classic chicken-and-egg paradox: could nonenzymatic replication propagate sequences accurately enough to allow for the emergence of heritable function? Here we show that the theoretical limit on genomic information content may increase substantially as a consequence of dramatically slowed polymerization after mismatches. As a result of postmismatch stalling, accurate copies of a template tend to be completed more rapidly than mutant copies and the accurate copies can therefore begin a second round of replication more quickly. To quantify this effect, we characterized an experimental model of nonenzymatic, template-directed nucleic acid polymerization. We found that most mismatches decrease the rate of primer extension by more than 2 orders of magnitude relative to a matched (Watson−Crick) control. A chemical replication system with this property would be able to propagate sequences long enough to have function. Our study suggests that the emergence of functional sequences during the origin of life would be possible even in the face of the high intrinsic error rates of chemical replication.

Preparation of Yeast Media

Preparation of sterile media of consistently high quality is essential for the genetic manipulation of yeast. Recipes for media needed in the protocols in this chapter are provided in this unit. Specific suppliers are recommended for specific ingredients.

Growth and Manipulation of Yeast

This unit describes preparation of selected media for growing yeast and also discusses strain storage and revival. Protocols are provided for the assay of β‐galactosidase in liquid culture and for transformation using lithium acetate.

UNIT 13.2 Growth and Manipulation of Yeast

Preparation of sterile media of consistently high quality is essential for the genetic manipulation of yeast. Recipes for media needed in the protocols in this chapter are provided in this unit. Specific suppliers are recommended for specific ingredients.

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.