Gwendolyn M. Stovall

Widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation

Proteins are likely to organize into complexes that assemble and disassemble depending on cellular needs. When ≈800 yeast strains expressing GFP-tagged proteins were grown to stationary phase, a surprising number of proteins involved in intermediary metabolism and stress response were observed to form punctate cytoplasmic foci. The formation of these discrete physical structures was confirmed by immunofluorescence and mass spectrometry of untagged proteins. The purine biosynthetic enzyme Ade4-GFP formed foci in the absence of adenine, and cycling between punctate and diffuse phenotypes could be controlled by adenine subtraction and addition. Similarly, glutamine synthetase (Gln1-GFP) foci cycled reversibly in the absence and presence of glucose. The structures were neither targeted for vacuolar or autophagosome degradation nor colocalized with P bodies or major organelles. Thus, upon nutrient depletion we observe widespread protein assemblies displaying nutrient-specific formation and dissolution.

Technical and Biological Issues Relevant to Cell Typing with Aptamers

A number of aptamers have been selected against cell surface biomarkers or against eukaryotic tissue culture cells themselves. To determine the general utility of aptamers for assessing the cell surface proteome, we developed a standardized flow cytometry assay and carried out a comprehensive study with 7 different aptamers and 14 different cell lines. By examining how aptamers performed with a variety of cell lines, we identified difficulties in using aptamers for cell typing. While there are some aptamers that show excellent correlation between cell surface binding and the expression of a biomarker on the cell surface, other aptamers showed nonspecific binding by flow cytometry. For example, it has recently been claimed that an anti-PTK7 (protein tyrosine kinase 7) aptamer identified a new biomarker for leukemia cells, but data with the additional cell lines shows that it is possible that the aptamer instead identifies a propensity for adherence. Better understanding and controlling for the role of background and nonspecific binding to cells should open the way to using arrays of aptamers for describing and quantifying the cell surface proteome.

Automated Optimization of Aptamer Selection Buffer Conditions

Optimizing the buffer conditions of the selection of nucleic acid binding species (aptamers), increases the likelihood of producing a target aptamer. Aptamers, with high target affinity and specificity, are often compared to antibodies, as aptamers emerge in the industry as diagnostic and therapeutic tools. The increased demand for aptamers encourages high-throughput aptamer generation. The selection buffer conditions may vary as widely as the selection targets, and therefore buffer optimization is helpful if not required for effective aptamer selections. Such optimization work is time consuming and repetitious, which bodes well for high-throughput applications. To accommodate this, an automated buffer testing protocol has been developed to test target-to-unselected RNA pool binding in the presence of 96 different buffer conditions. The dynamic program may vary the monovalent salt(s) identity, monovalent salt(s) concentration, divalent salt(s) identity, divalent salt concentration, buffer identity, buffer concentration, and pH. The optimized buffer conditions likely increase the probability of a successful selection and therefore promote higher ratios of successful aptamer selections against a variety of targets. Preliminary results show trends with the buffer matrix solutions and lysozyme:unselected pool binding. In general, an inverse relationship between lysozyme binding and monovalent salt concentration is observed. (JALA 2004;9:117-22)

Continuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule.

Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self‐ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the populations ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5′ end of the ribozyme displaces paired fluorophore‐quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule.

UNIT 9.6 In Vitro Selection Using Modified or Unnatural Nucleotides

The use of modified nucleotides in an RNA or DNA pool to be used for in vitro selection offers many potential advantages, such as the increased stability of the selected nucleic acid against nuclease degradation. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for preparing a modified RNA pool and verifying its suitability for in vitro selection; and protocols for selecting and amplifying a functionally enriched pool.