Synthesis and Optical Characterization of a Rhodamine B Spirolactam Dimer.

Amide derivatives of xanthene dyes such as rhodamine B are useful in a variety of sensing applications due to their colorimetric responses to stimuli such as acidity changes and UV light. The optical properties of these molecules can be influenced by intermolecular associations into dimeric structures, but the exact impact can be hard to predict. We have designed a covalently linked intramolecular dimer of the dye rhodamine B utilizing -phenylenediamine to link the two dyes via amide bonds.

Human Tat-specific factor 1 binds the HIV-1 genome and selectively transports HIV-1 RNAs.

Human immunodeficiency virus type 1 (HIV-1) propagation requires many human cofactors. Multiple groups have demonstrated that Tat-specific factor 1 (Tat-SF1) is an HIV-1 dependency factor. Depletion of this protein lowers HIV-1 infectivity, however, it does not affect the overall levels of viral RNA.

Interrelationship between cytoplasmic retroviral Gag concentration and Gag-membrane association.

The early events in the retrovirus assembly pathway, particularly the timing and nature of Gag translocation from the site of protein translation to the inner leaflet of the plasma membrane, are poorly understood. We have investigated the interrelationship between cytoplasmic Gag concentration and plasma membrane association using complementary live-cell biophysical fluorescence techniques in real time with both human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) Gag proteins. In particular, dual-color, z-scan fluorescence fluctuation spectroscopy in conjunction with total internal reflection fluorescence and conventional, epi-illumination imaging were utilized.

New insights into HTLV-1 particle structure, assembly, and Gag-Gag interactions in living cells.

Human T-cell leukemia virus type 1 (HTLV-1) has a reputation for being extremely difficult to study in cell culture. The challenges in propagating HTLV-1 has prevented a rigorous analysis of how these viruses replicate in cells, including the detailed steps involved in virus assembly. The details for how retrovirus particle assembly occurs are poorly understood, even for other more tractable retroviral systems.

Characterization of cytoplasmic Gag-gag interactions by dual-color z-scan fluorescence fluctuation spectroscopy.

Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness analysis. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation volume, cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts.

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stochiometry.

Background: Human T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells.

Probing the ionic atmosphere of single-stranded DNA using continuous flow capillary electrophoresis and fluorescence correlation spectroscopy.

Two-beam fluorescence cross-correlation spectroscopy coupled with continuous flow capillary electrophoresis (2bFCCS-CFCE) was used to study the relationship between diffusion and effective charge of a fluorescently labeled 40-base polythymine single-stranded DNA (ssDNA) as a function of Mg2+ concentration. Cross-correlation analysis of the fluorescence monitored from two spatially offset microscopic detection volumes revealed the diffusion and electrophoretic migration of ssDNA at a range of Mg2+ concentrations and electric field strengths. The effective charge of the ssDNA could then be determined using simple calculations.

Fluorescence correlation spectroscopy for ultrasensitive DNA analysis in continuous flow capillary electrophoresis.

Continuous flow capillary electrophoresis (CFCE) is non-separations based analytical technique based on the free solution electrophoretic mobility of biological molecules such as DNA, RNA, peptides, and proteins. The electrophoretic mobilities and translational diffusion constants of the analyte molecules are determined using single molecule detection methods, including fluorescence correlation spectroscopy (FCS). CFCE is used to resolve multiple components in a mixture of analytes, measure electrophoretic mobility shifts due to binding interactions, and study the hydrodynamic and electrostatic properties of biological molecules in solution.

Two-beam fluorescence cross-correlation spectroscopy for simultaneous analysis of positive and negative ions in continuous-flow capillary electrophoresis.

Two-beam fluorescence cross-correlation spectroscopy was used for multicomponent electrophoretic analysis of positive and negative ions flowing continuously, and in opposite directions, through a polymer-coated electrophoresis capillary. Cross-correlation analysis of the fluorescence monitored from two spatially offset microscopic detection volumes revealed the magnitude and direction of the electrophoretic flow velocity of each analyte. This enabled resolution of a three-component mixture containing nanomolar concentrations of cationic rhodamine 6G and anionic 5-carboxytetramethylrhodamine (TAMRA) and TAMRA-labeled single-stranded DNA in aqueous buffer.