FRET-based optical assay for selection of artificial riboswitches.

Artificial riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules and, therefore, can be useful tools to reprogram cellular behavior for different applications. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer with a randomized expression platform followed by in vivo selection and screening. Here, we describe an in vivo selection and screening technique to discover artificial riboswitches in E.

Computational design of RNA libraries for in vitro selection of aptamers.

Selection of aptamers that bind a specific ligand usually begins with a random library of RNA sequences, and many aptamers selected from such random pools have a simple stem-loop structure. We present here a computational approach for designing a starting library of RNA sequences with increased formation of complex structural motifs and enhanced affinity to a desired target molecule. Our approach consists of two steps: (1) generation of RNA sequences based on customized patterning of nucleotides with increased probability of forming a base pair and (2) a high-throughput virtual screening of the generated library to select aptamers with binding affinity to a small-molecule target.

Development of a 2,4-dinitrotoluene-responsive synthetic riboswitch in E. coli cells.

Riboswitches are RNA sequences that regulate expression of associated downstream genes in response to the presence or absence of specific small molecules. A novel riboswitch that activates protein translation in E. coli cells in response to 2,4-dinitrotoluene (DNT) has been engineered.

Orthogonal cell-based biosensing: fluorescent, electrochemical, and colorimetric detection with silica-immobilized cellular communities integrated with an ITO-glass/plastic laminate cartridge.

This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensor's output.

Biofunctionalized zinc oxide field effect transistors for selective sensing of riboflavin with current modulation.

Zinc oxide field effect transistors (ZnO-FET), covalently functionalized with single stranded DNA aptamers, provide a highly selective platform for label-free small molecule sensing. The nanostructured surface morphology of ZnO provides high sensitivity and room temperature deposition allows for a wide array of substrate types. Herein we demonstrate the selective detection of riboflavin down to the pM level in aqueous solution using the negative electrical current response of the ZnO-FET by covalently attaching a riboflavin binding aptamer to the surface.

Theophylline detection using an aptamer and DNA-gold nanoparticle conjugates.

A detection system for theophylline that combined the recognition properties of an aptamer and the plasmonic response of gold nanoparticles (AuNPs) is presented. The aptamer was used as a linker for AuNPs functionalized with complementary sequences to the aptamer (DNA-AuNPs), producing supramolecular complexes that disassemble when exposed to theophylline due to aptamer binding. The detection event was reported as a change in the AuNPs plasmonic peak and intensity.

In silico selection of RNA aptamers.

In vitro selection of RNA aptamers that bind to a specific ligand usually begins with a random pool of RNA sequences. We propose a computational approach for designing a starting pool of RNA sequences for the selection of RNA aptamers for specific analyte binding. Our approach consists of three steps: (i) selection of RNA sequences based on their secondary structure, (ii) generating a library of three-dimensional (3D) structures of RNA molecules and (iii) high-throughput virtual screening of this library to select aptamers with binding affinity to a desired small molecule.

FRET-based optical assay for monitoring riboswitch activation.

Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins.

Cephalopod coloration model. II. Multiple layer skin effects.

A mathematical model of multiple layer skin coloration in cephalopods, a class of aquatic animals, is presented. The model incorporates diffuse and specular reflection from both pigment and structural photonic components found in the skin of these animals. Specific physical processes of this coloration are identified and modeled utilizing available biological materials data.

Cephalopod coloration model. I. Squid chromatophores and iridophores.

We have developed a mathematical model of skin coloration in cephalopods, a class of aquatic animals. Cephalopods utilize neurological and physiological control of various skin components to achieve active camouflage and communication. Specific physical processes of this coloration are identified and modeled, utilizing available biological materials data, to simulate active spectral changes in pigment-bearing organs and structural iridescent cells.

Computational study of the absorption spectra of green fluorescent protein mutants.

In this work, we present a theoretical study of the relationship between molecular structure and the red-shift in absorption spectra of S65G and S65T green fluorescent protein (GFP) mutants. To identify the effects of the protein environment, we combined results from molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics calculations to obtain structural properties, and applied time-dependent density functional theory to calculate the excitation energies. By using results from the MD simulations, we were able to provide a systematic analysis of the structural details that may effect the red-shift in the absorption spectra when taking into account temperature effects.

Peptide-mediated formation of single-wall carbon nanotube composites.

The formation of silica- and titania-coated single-wall carbon nanotubes (SWNTs) using a mutlifunctional peptide to both suspend SWNTs and direct the precipitation of silica and titania at room temperature is demonstrated.

Characterization of microbial contamination in United States Air Force aviation fuel tanks.

Bacteria and fungi, isolated from United States Air Force (USAF) aviation fuel samples, were identified by gas chromatograph fatty acid methyl ester (GC-FAME) profiling and 16S or 18S rRNA gene sequencing. Thirty-six samples from 11 geographically separated USAF bases were collected. At each base, an above-ground storage tank, a refueling truck, and an aircraft wing tank were sampled at the lowest sample point, or sump, to investigate microbial diversity and dispersion within the fuel distribution chain.

Thermally induced alpha-helix to beta-sheet transition in regenerated silk fibers and films.

The structure of thin films cast from regenerated solutions of Bombyx mori cocoon silk in hexafluoroisopropyl alcohol (HFIP) was studied by synchrotron X-ray diffraction during heating. A solid-state conformational transition from an alpha-helical structure to the well-known beta-sheet silk II structure occurred at a temperature of approximately 140 degrees C. The transition appeared to be homogeneous, as both phases do not coexist within the resolution of the current study.

Polypeptide-templated synthesis of hexagonal silica platelets.

Several studies have demonstrated the use of biomimetic approaches in the synthesis of a variety of inorganic materials. Poly-L-lysine (PLL) promotes the precipitation of silica from a silicic acid solution within minutes. The molecular weight of PLL was found to affect the morphology of the resulting silica precipitate.

Constrained iron catalysts for single-walled carbon nanotube growth.

The diameter of single walled carbon nanotubes (SWNTs) determines the electronic properties of the nanotube. The diameter of carbon nanotubes is dictated by the diameter of the catalyst particle. Here we describe the use of iron nanoparticles synthesized within the Dps protein cage as catalysts for the growth of single-walled carbon nanotubes.

Cellular internalization and targeting of semiconductor quantum dots.

Peptide-mediated internalization and organelle targeting of quantum dots.

Polypeptide-mediated silica growth on indium tin oxide surfaces.

Herein, we describe the formation of silica structures on indium tin oxide (ITO) surfaces using poly-L-lysine (PLL) to template the condensation of silicic acid. Precisely controlled electrostatic fields were used to preposition PLL onto ITO surfaces. Subsequent polypeptide-mediated silicification resulted in the formation of silica with concentration gradients that followed the pattern of the externally applied electrostatic field used in the deposition of the PLL.

Dissolution and regeneration of Bombyx mori silk fibroin using ionic liquids.

In this work, the suitability of imidazolium-based ionic liquid solvents is investigated for the dissolution and regeneration of silkworm (Bombyx mori) silk. Within an ionic liquid the anion plays a larger role in dictating the ultimate solubility of the silk. The dissolution of the silk in the ionic liquid is confirmed using wide-angle X-ray scattering.

Engineered protein cages for nanomaterial synthesis.

Self-assembled particles of genetically engineered human L subunit ferritin expressing a silver-binding peptide were used as nanocontainers for the synthesis of silver nanoparticles. The inner cavity of the self-assembled protein cage displays a dodecapeptide that is capable of reducing silver ions to metallic silver. This chimeric protein cage when incubated in the presence of silver nitrate exhibits the growth of a silver nanocrystal within its cavity.

Identification of peptides that promote the rapid precipitation of germania nanoparticle networks via use of a peptide display library.

Peptides that promote the rapid, room-temperature precipitation of amorphous germania nanoparticle networks from solution have been identified via use of a combinatorial peptide display library.

Entrapment of enzymes and nanoparticles using biomimetically synthesized silica.

Entrapment of enzymes and nanoparticles using biosilicification reactions.

Study of the chemical and physical influences upon in vitro peptide-mediated silica formation.

Herein, we report on the ability to create complex 2-D and 3-D silica networks in vitro via polycationic peptide-mediated biosilicification under experimentally altered chemical and physical influences. These structures differ from the sphere-like silica network of particles obtained in vitro under static conditions. Under chemical influences, overall morphologies were observed to shift from a characteristic network of sphere-like silica particles to a sheetlike structure in the presence of -OH groups from additives and to sharp-edged, platelike structures in the presence of larger polycationic peptide matrixes.

Enzyme immobilization in a biomimetic silica support.

Robust immobilization techniques that preserve the activity of biomolecules have many potential applications. Silicates, primarily in the form of sol-gel composites or functionalized mesoporous silica, have been used to encapsulate a wide variety of biomolecules but the harsh conditions required for chemical synthesis limit their applicability. Silaffin polypeptides from diatoms catalyze the formation of silica in vitro at neutral pH and ambient temperature and pressure.