Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition

Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.     

Cellular microfabrication: observing intercellular interactions using lithographically-defined DNA capture sequences

Previous reports have shown that synthetic DNA strands can be attached to the plasma membrane of living cells to equip them with artificial adhesion "receptors" that bind to complementary strands extending from material surfaces. This approach is compatible with a wide range of cell types, offers excellent capture efficiency, and can potentially be used to create complex multicellular arrangements through the use of multiple capture sequences. In this work, we apply an aluminum "lift off" lithography method to allow the efficient generation of complex patterns comprising different DNA sequences. The resulting surfaces are then demonstrated to be able to capture up to three distinct types of living cells in specific locations. The utility of this approach is demonstrated through the observation of patterned cells as they communicate by diffusion-based paracrine signaling. It is anticipated that the ability of this technique to create virtually any type of 2D heterogeneous cell pattern should prove highly useful for the examination of key questions in cell signaling, including stem cell differentiation and cancer metastasis.     

Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.     

Axisymmetric Surface Diffusion: Dynamics and Stability of Self-Similar Pinchoff

The dynamics of surface diffusion describes the motion of a surface with its normal velocity given by the surface Laplacian of its mean curvature. This flow conserves the volume enclosed inside the surface while minimizing its surface area. We review the axisymmetric equilibria: the cylinder, sphere, and the Delaunay unduloid. The sphere is stable, while the cylinder is long-wave unstable. A subcritical bifurcation from the cylinder produces a continuous family of unduloid solutions. We present computations that suggest that the stable manifold of the unduloid forms a separatrix between states that relax to the cylinder in infinite time and those that tend toward finite-time pinchoff. We examine the structure of the pinchoff, showing it has self-similar structure, using asymptotic, numerical, and analytical methods. In addition to a previously known similarity solution, we find a countable set of similarity solutions, each with a different asymptotic cone angle. We develop a stability theory in similarity variables that selects the original similarity solution as the only linearly stable one and consequently the only observable solution. We also consider similarity solutions describing the dynamics after the topological transition.     

Detection of human and rodent 5-HT3B receptor subunits by anti-peptide polyclonal antibodies

Background  

The 5-HT₃ receptor is a member of a neurotransmitter-gated ion channel family which includes nicotinic acetylcholine, GABA_A, and glycine receptors. While antibodies specific for the 5-HT_3A receptor subunit are plentiful, and have revealed a wealth of structural and functional information, few antisera exist for the detection of 5-HT_3B receptor subunits. Here we describe the generation and characterisation of a rabbit polyclonal antiserum that specifically recognises 5-HT_3B receptor subunits     

Dibenzyl Structuresin a Macromolecular Chain. III. Dibenzyldiisocyanates Reactivity

The reactivity of the 2,2′-, 2,4′-, 4,4′-dibenzyldiisocyanate (2,2′-, 2,4′-, 4,4′-DBDI) with n-butanol in benzene has been studied. The concentrations of all species were monitored by using high performance liquid chromatography (HPLC). The reactivity of 4,4′-DBDI is similar to that of 4,4′-diphenylmethanediisocyanate (4,4′-MDI). Very strong intramolecular catalytic effects were noticed in the case of 2,2′-DBDI, probably due to the variable molecular geometry. These effects are responsible for the whole reaction pattern. The 2,4′-DBDI NCO ortho and para groups reactivities are different and comparable to that of 2,4-toluylenediisocyanate (2,4-TDI).     

Lp theory for the multidimensional aggregation equation

We consider well‐posedness of the aggregation equation ∂_tu + div(uv) = 0, v = −▿K * u with initial data in \input amssym ${\cal P}_2 {\rm (\Bbb R}^d {\rm )} \cap L^p ({\Bbb R}^d )$ in dimensions 2 and higher. We consider radially symmetric kernels where the singularity at the origin is of order |x|^α, α > 2 − d, and prove local well‐posedness in \input amssym ${\cal P}_2 { (\Bbb R}^d {\rm )} \cap L^p ({\Bbb R}^d )$ for sufficiently large p < p_s. In the special case of K(x) = |x|, the exponent p_s = d/(d = 1) is sharp for local well‐posedness in that solutions can instantaneously concentrate mass for initial data in \input amssym ${\cal P}_2 { (\Bbb R}^d {\rm )} \cap L^p ({\Bbb R}^d )$ with p < p_s. We also give an Osgood condition on the potential K(x) that guarantees global existence and uniqueness in \input amssym ${\cal P}_2 { (\Bbb R}^d {\rm )} \cap L^p ({\Bbb R}^d )$ . © 2010 Wiley Periodicals, Inc.