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Biomedical subjects

Shana O Kelley

Publications and source records attributed to Shana O Kelley.

At least 19 recordsLinked to original sources
PubMed2024

Phenotypic targeting using magnetic nanoparticles for rapid characterization of cellular proliferation regulators.

Genome-wide CRISPR screens have provided a systematic way to identify essential genetic regulators of a phenotype of interest with single-cell resolution. However, most screens use live/dead readout of viability to identify factors of interest. Here, we describe an approach that converts cell proliferation into the degree of magnetization, enabling downstream microfluidic magnetic sorting to be performed. We performed a head-to-head comparison and verified that the magnetic workflow can identify the same hits from a traditional screen while reducing the screening period from 4 weeks to 1 week. Taking advantage of parallelization and performance, we screened multiple mesenchymal cancer cell lines for their dependency on cell proliferation. We found and validated pan- and cell-specific potential therapeutic targets. The method presented provides a nanoparticle-enabled approach means to increase the breadth of data collected in CRISPR screens, enabling the rapid discovery of drug targets for treatment.

Humans↗
PubMed2006

RNA-templated semiconductor nanocrystals.

The use of a biomolecular RNA template for the synthesis of CdS semiconductor nanocrystals is described. Transfer RNA, with a well-defined three-dimensional structure, is used as a scaffold and ligand system in the aqueous synthesis of CdS. We have found that nanocrystal structures can be modulated by the structure of the templating tRNA. When possessing a folded three-dimensional structure, tRNA-templated synthesis yields a single product. If the same tRNA is rendered unstructured through the introduction of destabilizing mutations, a range of products are observed. The results reported indicate that biomolecules can be used to systematically engineer the structures and properties of semiconductor-based materials, and that the synergy between the dimensions of nanostructures and biomolecules provides a means to tune the properties of materials with nanoscale precision.

Cadmium Compounds↗
PubMed2006

Nucleotide-directed growth of semiconductor nanocrystals.

We engineer colloidal quantum dot nanocrystals through the choice of biomolecular ligands responsible for nanoparticle nucleation, growth, stabilization, and passivation. We systematically vary the presence of, and thereby elucidate the role of, phosphate groups and a multiplicity of functionalities on the mononucleotides used as ligands. The results provide the basis for synthesis of nanoparticles using precisely controlled synthetic oligonucleotide sequences.

Adenosine Triphosphate↗
PubMed2006

Tunable DNA cleavage by intercalating peptidoconjugates.

The properties of a novel family of peptide-based DNA-cleavage agents are described. Examination of the DNA-cleavage activities of a systematic series of peptide-intercalator conjugates revealed trends that show a strong dependence on peptide sequence. Conjugates differing by a single residue displayed reactivities that varied over a wide range. The cleavage activity was modulated by the electrostatic or steric qualities of individual amino acids. Isomeric conjugates that differed in the position of the tether also exhibited different reactivities. The mechanism of DNA cleavage for these compounds was also probed and was determined to involve hydrogen-atom abstraction from the DNA backbone. Previous studies of these compounds indicated that amino acid peroxides were the active agents in the cleavage reaction; in this report, the chemistry underlying the reaction is characterized. The results reported provide insight into how peptide sequences can be manipulated to produce biomimetic compounds.

Amino Acids↗
PubMed2005

An aminoacyl-tRNA synthetase with a defunct editing site.

Many aminoacyl-tRNA synthetases (aaRSs) contain two active sites, a synthetic site catalyzing aminoacyl-adenylate formation and tRNA aminoacylation and a second editing or proofreading site that hydrolyzes misactivated adenylates or mischarged tRNAs. The combined activities of these two sites lead to rigorous accuracy in tRNA aminoacylation, and both activities are essential to LeuRS and other aaRSs. Here, we describe studies of the human mitochondrial (hs mt) LeuRS indicating that the two active sites of this enzyme have undergone functional changes that impact how accurate aminoacylation is achieved. The sequence of the hs mt LeuRS closely resembles a bacterial LeuRS overall but displays significant variability in regions of the editing site. Studies comparing Escherichia coli and hs mt LeuRS reveal that the proofreading activity of the mt enzyme is disrupted by these sequence changes, as significant levels of Ile-tRNA(Leu) are formed in the presence of high concentrations of the noncognate amino acid. Experiments monitoring deacylation of Ile-tRNA(Leu) and misactivated adenylate turnover revealed that the editing active site is not operational. However, hs mt LeuRS has weaker binding affinities for both cognate and noncognate amino acids relative to the E. coli enzyme and an elevated discrimination ratio. Therefore, the enzyme achieves fidelity using a more specific synthetic active site that is not prone to errors under physiological conditions. This enhanced specificity must compensate for the presence of a defunct editing site and ensures translational accuracy.

Acylation↗
PubMed2005

Combinatorial analysis of loop nucleotides in human mitochondrial tRNALeu(UUR).

A series of disease-related mutations are known to affect the hs mt tRNA(Leu(UUR)) gene, and the molecular-level properties of this tRNA may underlie the effects of pathogenic sequence changes. A combinatorial approach has been used to explore the importance of the D, TPsiC, and anticodon loops of hs mt tRNA(Leu(UUR)) in the structure and function of this molecule. A tRNA library was constructed with 20 randomized nucleotides in the loop regions of hs mt tRNA(Leu(UUR)), and tRNA variants that were aminoacylated by hs mt LeuRS were isolated using an in vitro selection approach. Analysis of 26 selected sequences revealed that a stabilized anticodon stem significantly enhances aminoacylation activity. However, anticodon loop nucleotides were not conserved in the active sequences, indicating that this region of hs mt tRNA(Leu(UUR)) is not involved in recognition by LeuRS. Within the D and TPsiC loops, only two nucleotides conserved their identities, while new sequences were selected that likely mediate interloop interactions. The results indicate that hs mt tRNA(Leu(UUR)), which is known to have structurally weak D and anticodon stems, benefits functionally from the introduction of stabilizing interactions. However, the locations of individual nucleotides that govern discrimination of this tRNA by hs mt LeuRS still remain obscure.

Anticodon↗
PubMed2005

Synthesis, characterization, and cellular uptake of DNA-binding rose bengal peptidoconjugates.

Peptide conjugates of the xanthene dye rose bengal (RB) are described featuring sequences that promote DNA binding. The complexation of these conjugates with DNA causes efficient quenching of the fluorophore singlet state and suppresses singlet oxygen production. When incubated with human cells, the RB conjugates pass through the cell membrane but are not visualized in the nucleus. This behavior is in stark contrast to that exhibited by structurally analogous conjugates containing the unhalogenated xanthene dye fluorescein. These results highlight the marked sensitivity of cell permeability characteristics to subtle structural differences.

Amino Acid Sequence↗
PubMed2005

Oxidative DNA strand scission induced by peptides.

Cellular oxidative stress promotes chemical reactions causing damage to DNA, proteins, and membranes. Here, we describe experiments indicating that reactive oxygen species, in addition to degrading polypeptides and polynucleotides through direct reactions, can also promote damaging biomolecular cross reactivity by converting protein residues into peroxides that cleave the DNA backbone. The studies reported show that a variety of residues induce strand scission upon oxidation, and hydrogen abstraction occurring at the DNA backbone is responsible for the damage. The observation of peptide-promoted DNA damage suggests that crossreactions within protein/DNA complexes should be considered as a significant cause of the toxicity of reactive oxygen species.

Amino Acids↗
PubMed2005

Amplified electrocatalysis at DNA-modified nanowires.

Arrayed gold nanowires are a novel and useful platform for electrochemical DNA detection. Pilot studies testing the use of these templated structures with an electrocatalytic reporter system revealed that very low detection thresholds for target DNA sequences can be obtained. One factor contributing to the heightened sensitivity is the high signal-to-noise ratio achieved with the large electrocatalytic signals observed at DNA-modified nanowires. Here, we explain the improved sensitivity with evidence illustrating that electrocatalysis at DNA-modified nanostructures generates amplified signals that are significantly larger than those observed at bulk gold surfaces. The results presented strongly suggest that the three-dimensional architectures of the nanowires facilitate the electrocatalytic reaction because of enhanced diffusion occurring around these structures. Effects unique to the nanoscale are shown to underlie the utility of nanowires for DNA biosensing.

Biosensing Techniques↗
PubMed2005

Structural probing of a pathogenic tRNA dimer.

The A3243G mutation within the human mitochondrial (hs mt) tRNALeuUUR gene is associated with maternally inherited deafness and diabetes (MIDD) and other mitochondrial encephalopathies. One of the most pronounced structural effects of this mutation is the disruption of the native structure through stabilization of a high-affinity dimeric complex. We conducted a series of studies that address the structural properties of this tRNA dimer, and we assessed its formation under physiological conditions. Enzymatic probing was used to directly define the dimeric interface for the complex, and a discrete region of the D-stem and loop of hs mt tRNALeuUUR was identified. The dependence of dimerization on magnesium ions and temperature was also tested. The formation of the tRNA dimer is influenced by temperature, with dimerization becoming more efficient at physiological temperature. Complexation of the mutant tRNA is also affected by the amount of magnesium present, and occurs at concentrations present intracellularly. Terbium probing experiments revealed a specific metal ion-binding site localized at the site of the A3243G mutation that is unique to the dimer structure. This metal ion-binding site presents a striking parallel to dimeric complexes of viral RNAs, which use the same hexanucleotide sequence for complexation and feature a similarly positioned metal ion-binding site within the dimeric structure. Taken together, these results indicate that the unique dimeric complex formed by the hs mt tRNALeuUUR A3243G mutant exhibits interesting similarities to biological RNA dimers, and may play a role in the loss of function caused by this mutation in vivo.

Base Sequence↗
PubMed2004

Site-specific assembly of DNA and appended cargo on arrayed carbon nanotubes.

We describe a strategy that permits discrete regions of arrayed carbon nanotubes (CNTs) to be functionalized simultaneously and specifically with DNA oligonucleotides. The different chemical properties of two regions on single CNTs and orthogonal chemical coupling strategies have been exploited to derivatize CNTs within highly ordered arrays with multiple DNA sequences. Through duplex hybridization, we then targeted different DNA sequences with appended metal nanoparticles to distinct sites on the CNT architecture with precise spatial control. The materials generated from these studies represent the first CNTs with bipartite functionalization. The approach described provides a high level of precision in parallel and directed assembly of DNA sequences and appended cargo and is useful for the preparation of novel hybrid bionanomaterials.

DNA↗
PubMed2004

Ultrasensitive electrocatalytic DNA detection at two- and three-dimensional nanoelectrodes.

Electrochemical DNA detection systems are an attractive approach to the development of multiplexed, high-throughput DNA analysis systems for clinical and research applications. We have engineered a new class of nanoelectrode ensembles (NEEs) that constitute a useful platform for biomolecular electrochemical sensing. High-sensitivity DNA detection was achieved at oligonucleotide-functionalized NEEs using a label-free electrocatalytic assay. Attomole levels of DNA were detected using the NEEs, validating the promise of nanoarchitectures for ultrasensitive biosensing.

Biosensing Techniques↗
PubMed2004

Thiazole orange-peptide conjugates: sensitivity of DNA binding to chemical structure.

[structure: see text] Derivatives of the highly fluorescent and DNA-binding dye thiazole orange (TO) are described that feature appended peptides. Functionalization of TO can be achieved at either of the endocyclic nitrogens, and the photophysical properties and DNA-binding modes are sensitive to the position of the tethered peptide. A series of TO-peptide conjugates are described, demonstrating the utility of a solid-phase synthesis approach to their preparation and illustrating how the photophysical and DNA-binding properties of the compounds are influenced by chemical structure.

Amino Acid Sequence↗
PubMed2004

Interdomain communication between weak structural elements within a disease-related human tRNA.

The structure of the human mitochondrial (hs mt) tRNALeu(UUR) features several domains that are predicted to exhibit limited thermodynamic stability. An elevated frequency of disease-related mutations within these domains suggests a link between structural instability and the functional effects of pathogenic mutations. A series of tRNAs featuring mutations within the D and anticodon stems were prepared and investigated using nuclease probing. Structural mapping studies indicated that these domains were partially denatured for the wild type (WT) hs mt tRNALeu(UUR) and were significantly stabilized by mutations introducing additional or stronger base pairs into the stem regions. In addition, trends in the aminoacylation activities of the D stem mutants suggested that the loose structure is required for function, with mutants displaying the most ordered structures exhibiting the lowest levels of aminoacylation activity. A pronounced interdependence of the structures of the anticodon and D stems was observed, with mutations strengthening the D stem stabilizing the anticodon stem and vice versa. The existence of strong interdomain communication was further elucidated with a mutant of hs mt tRNALeu(UUR) containing a stabilized D stem and a pathogenic mutation that disrupted the anticodon stem. Strengthening the structure of the D stem completely restored the function of the disease-related mutant to WT levels, indicating that propagated structural weaknesses contribute to the functional deactivation of this tRNA by mutations.

Acylation↗
PubMed2003

Electrocatalytic detection of pathogenic DNA sequences and antibiotic resistance markers.

The detection of specific DNA sequences using electrochemical readout would permit the rapid and inexpensive detection and identification of bacterial pathogens. A new assay developed for this purpose is described that harnesses a sensitive electrocatalytic process to monitor DNA hybridization. Two sequences belonging to the pathogenic microbe Helicobacter pylori are used to demonstrate the versatility and specificity of the assay: one that codes for an unique H. pylori protein and one that represents a small portion of the 23S rRNA from this organism. Both sequences can be monitored into the nanomolar concentration range. Target sequences introduced to the electrode surface as synthetic oligonucleotides, PCR products, and RNA transcripts are all detected with high specificity. In addition to reporting the presence of pathogen-related sequences, this assay can accurately resolve single-base changes in target sequences. An A2143C substitution within the H. pylori rRNA that confers antibiotic resistance significantly attenuates hybridization to an immobilized probe corresponding to the WT sequence. The single-base mismatch introduced by this mutation slows the kinetics of hybridization and permits discrimination of the two sequences when short hybridization times are employed. The remarkable sensitivity of this label-free assay to small sequence changes may provide the basis of a new method for the detection and genotyping of infectious bacteria using electrochemical methods.

Bacterial Proteins↗