549 research outputs found
Evaluation of the governing parameters in oligonucleotides separation by hydrophilic interaction liquid chromatography using fractional factorial designs followed by surface response optimization
Hyphenating chromatographic, electrophoretic and on-line immobilized enzymatic strategies for improved oligonucleotide analysis
Schiff base complexes derived from N,N’-bis(3-methoxysalicylidene)ethylene-1,2-diamine ligands for NIR luminescence and RNA cleavage
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A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids
The physicochemical properties of nucleic acids are dominated by their highly charged
phosphodiester backbone chemistry. The polyelectrolyte structure decouples information
content (base sequence) from bulk properties such as solubility and has been proposed as a
defining trait of all informational polymers. However, this conjecture has not been tested
experimentally. Here, we describe the encoded synthesis of a genetic polymer with an
uncharged backbone chemistry: alkyl-phosphonate nucleic acids (phNA), in which the
canonical, negatively charged phosphodiester is replaced by an uncharged P-alkylphosphonodiester
backbone. Using synthetic chemistry and polymerase engineering, we
describe the enzymatic, DNA-templated synthesis of P-methyl- and P-ethyl-phNAs, and the
directed evolution of specific streptavidin-binding phNA aptamer ligands directly from
random-sequence, mixed P-methyl- / P-ethyl-phNA repertoires. Our results establish a first
example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic
polymer and provide a foundational methodology for their exploration as a source of novel,
functional molecules
Smoothing a rugged protein folding landscape by sequence-based redesign
The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as -antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create , a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.BTP is a Medical Research Council Career Development Fellow. AAN and JJH are supported by the Wellcome Trust (grant number WT 095195). SM acknowledges fellowship support from the Australian Research Council (FT100100960). NAB is an Australian Research Council Future Fellow (110100223). GIW is an Australian Research Council Discovery Outstanding Researcher Award Fellow (DP140100087). AMB is a National Health and Medical Research Senior Research Fellow (1022688). JCW is an NHMRC Senior Principal Research fellow and also acknowledges the support of an ARC Federation Fellowship. We thank the Australian Synchrotron for beam-time and technical assistance. This work was supported by the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE) (www.massive.org.au). We acknowledge the Monash Protein Production Unit and Monash Macromolecular Crystallization Facilit
Smoothing a rugged protein folding landscape by sequence-based redesign
The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation.
Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and
misfolding. Serpins exist in a metastable state that undergoes a major conformational change in
order to inhibit proteases. However, conformational labiality of the native serpin fold renders them
susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To
investigate how serpins balance function and folding, we used consensus design to create conserpin,
a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant.
Characterization of its structure, folding and dynamics suggest that consensus design has remodeled
the folding landscape to reconcile competing requirements for stability and function. This approach
may offer general benefits for engineering functional proteins that have risky folding landscapes,
including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein
therapeutics
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