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.

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Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

Rhodamine dyes are widely used as fluorescent probes owing to their high absorption coefficient and broad fluorescence in the visible region of electromagnetic spectrum, high fluorescence quantum yield and photostability. A great interest in the development of new synthetic procedures for preparation of Rhodamine derivatives has arisen in recent years because for most applications the probe must be covalently linked to another (bio)molecule or surface. In this critical review the strategies for modification of Rhodamine dyes and a discussion on the variety of applications of these new derivatives as fluorescent probes are given (108 references).

Synthesis and applications of Rhodamine derivatives as fluorescent probes

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

https://www.pnas.org/content/pnas/109/12/E690.full.pdf

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins

Bioorthogonale Chemie – oder: in einem Meer aus Funktionalität nach Selektivität fischen

There is considerable interest in novel cell imaging tools that avoid the use of fluorescent proteins. One widely used class of such reagents are “pro-fluorescent” biarsenical dyes such as FlAsH, ReAsH, CrAsH, and Cy3As. Despite their utility, biarsenicals are plagued by high background labeling and cytotoxicity and are challenging to apply in oxidizing cellular locale. Here we demonstrate that [(3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthene]-3′,6′-diyl)bis(iminomethylene-2,1-phenylene)]bis-(9CI), a rhodamine-derived bisboronic acid (RhoBo) described initially as a monosaccharide sensor, functions as a cell-permeable, turn-on fluorescent sensor for tetraserine motifs in recombinant proteins. RhoBo binds peptides or proteins containing Ser-Ser-Pro-Gly-Ser-Ser with affinities in the nanomolar concentration range and prefers this sequence to simple monosaccharides by >10 000-fold. RhoBo fails to form fluorescent complexes with constituents of the mammalian cell surface, as judged by epifluorescent, confocal...

/pdf/selective-recognition-of-protein-tetraserine-motifs-with-a-2l46uju8d3.pdf

Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid.

Phosphorylation of cortactin downstream of the EGF receptor–Src-Arg kinase cascade triggers maturation of invadopodia, actin-rich protrusions that breast cancer cells use to invade the extracellula...

Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells

Aspects of the invention relate to nanoscale constructs and related methods and compositions thereof. The compositions of the invention are useful for treating disorders that are sensitive to levels of immune cell activation, such as autoimmune disease or other inflammation based disease or disorder.

Spherical nucleic acid-based constructs as immunoregulatory agents

Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin

TNFα antisense oligonucleotides are provided herein. Methods of treating TNFα diseases or disorders using the TNFα antisense oligonucleotides and related products are provided.

Tiffany L. Halo

Papers

Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid.

Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells

Spherical nucleic acid-based constructs as immunoregulatory agents

Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin

Anti-tnf compounds