Ser was replaced at position 286 of firefly luciferase (Luciola mingrelica) by a series of naturally occurring and unnatural amino acids. The effect of these substitutions on the properties of luciferase, such as thermostability, pH dependence, and color of light emitted, was investigated. For these purposes, the Ser286 codon (AGT) was replaced by an amber stop codon (TAG) within the luciferase gene and transformed into Escherichia coli strains producing specific amber suppressor tRNA's to express luciferase with different substitutions at this position. The incorporation of Leu, Lys, Tyr, or Gln at this position reduced the thermostability of mutated luciferases. The color of emitted light changed upon substitution from yellow-green (λmax 582 nm) for the wild-type enzyme having Ser286 to, for example, red (λmax 622 nm) for luciferase having Leu286. For further evaluation of the structural relationship between the amino acid position at 286 and the wavelength of emitted light, we used the method of in vitro incorporation of unnatural amino acids, which involves readthrough of a nonsense (UAG) codon by a misacylated suppressor tRNA. The amino acids incorporated at position 286 in this fashion included O-glucosylated serine, serine phosphonate, tyrosine phosphate, and tyrosine methylenephosphonate. The wavelength of light emitted by the luciferase analogues was measured. While the introduction of serine phosphonate and glucosylated serine did not change the λmax of light produced by luciferase, the incorporation of tyrosine phosphate and tyrosine methylenephosphonate into position 286 altered the spectra of emitted light compared with those of Ser286 and Tyr286. The pH dependence of the wavelength of light emitted by the luciferases containing the negatively charged phosphorylated Tyr analogues was demonstrated and could be rationalized in terms of the pK a's of the phosph(on)ate oxygens.

Optogenetics relies on the expression of specific microbial rhodopsins in the neuronal plasma membrane. Most notably, this includes channelrhodopsins, which when heterologously expressed in neurons function as light-gated cation channels. Recently, a new class of microbial rhodopsins, termed anion channel rhodopsins (ACRs), has been discovered. These proteins function as efficient light-activated channels strictly selective for anions. They exclude the flow of protons and other cations and cause hyperpolarization of the membrane potential in neurons by allowing the inward flow of chloride ions. In this study, confocal near-infrared resonance Raman spectroscopy (RRS) along with hydrogen/deuterium exchange, retinal analogue substitution, and site-directed mutagenesis were used to study the retinal structure as well as its interactions with the protein in the unphotolyzed state of an ACR from Guillardia theta (GtACR1). These measurements reveal that (i) the retinal chromophore exists as an all-trans configuration with a protonated Schiff base (PSB) very similar to that of bacteriorhodopsin (BR), (ii) the chromophore RRS spectrum is insensitive to changes in pH from 3 to 11, whereas above this pH the Schiff base (SB) is deprotonated, (iii) when Ser97, the homologue to Asp85 in BR, is replaced with a Glu, it remains in a neutral form (i.e., as a carboxylic acid) but is deprotonated at higher pH to form a blue-shifted species, (iv) Asp234, the homologue of the protonated retinylidene SB counterion Asp212 in BR, does not serve as the primary counteranion for the protonated SB, and (v) substitution of Glu68 with an Gln increases the pH at which SB deprotonation is observed. These results suggest that Glu68 and Asp234 located near the SB exist in a neutral state in unphotolyzed GtACR1 and indicate that other unidentified negative charges stabilize the protonated state of the GtACR1 SB.

Many applications in pharmaceutical development, clinical diagnostics, and biological research demand rapid detection of multiple analytes (multiplexed detection) in a minimal volume. This need has led to the development of several novel array-based sensors. The most successful of these so far have been suspension arrays based on polystyrene beads. However, the 5 microm beads used for these assays are incompatible with most microfluidic chip technologies, mostly due to clogging problems. The challenge, then, is to design a detection particle that has high information content (for multiplexed detection), is compatible with miniaturization, and can be manufactured easily at low cost. DNA is a solid molecular wire that is easily produced and manipulated, which makes it a useful material for nanoparticles. DNA molecules are very information-rich, readily deformable, and easily propagated. We exploit these attributes in a suspension array sensor built from specialized recombinant DNA, Digital DNA, that carries both specific analyte-recognition units, and a geometrically encoded identification pattern. Here we show that this sensor combines high multiplexing with high sensitivity, is biocompatible, and has sufficiently small particle size to be used within microfluidic chips that are only 1 microm deep. We expect this technology will be the foundation of a broadly applicable technique to identify and quantitate proteins, nucleic acids, viruses, and toxins simultaneously in a minimal volume.

The filoviruses, Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SEBOV), cause severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Monovalent recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode a filovirus glycoprotein (GP) in place of the VSV glycoprotein, have shown 100% efficacy against homologous filovirus challenge in rodent and NHP studies. Here, we examined the utility of a single-vector, single-injection trivalent rVSV vector expressing MARV, ZEBOV, and SEBOV GPs to protect against MARV-, ZEBOV-, and SEBOV-induced disease in outbred Hartley guinea pigs where we observed protection from effects of all 3 filoviruses.

Opsin‐based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino‐modified dimethylphenyl ring results in over a 100‐nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT‐Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all‐trans protonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single‐/double‐bond system similar to the O‐intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13‐cis PSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR‐OTVSs, especially for applications such as deep brain imaging.