Bioluminescent imaging (BLI) has been widely applicable in the imaging of process envisioned in life sciences. As the most conventional technique for BLI, the firefly luciferin-luciferase system is exceptionally functional in vitro and in vivo. The state-of-the-art strategy in such a system is to cage the luciferin, in which free luciferin is conjugated with distinctive functional groups, thus accommodating an impressive toolkit for exploring various biological processes, such as monitoring enzymes activity, detecting bioactive small molecules, evaluating the properties of molecular transporters, etc. This review article summarizes the rational design of caged luciferins towards diverse biotargets, as well as their applications in bioluminescent imaging. It should be emphasized that these caged luciferins can stretch out the applications of bioluminescence imaging and shed light upon understanding the pathogenesis of various diseases.

The first general method for the selection of boronic acid-based aptamers that allow for glycan substructure focusing is described. Using fibrinogen as a model, we have selected boronic acid-modified DNA aptamers that have high affinities (low nM K d ) and the ability to recognize changes in the glycosylation site. The method developed should also be applicable to the development of aptamers for other glyco-products, such as glycolipids and glycopeptides. E-mail: wang@gsu.edu Supporting Information Available: Description of the experimental procedure and detailed results. This material is available free of charge via the Internet at http://pubs.acs.org. Glycosylation profoundly affects the function and activities of many proteins. 1,2 However, detecting and differentiating variations in glycosylation as an integral part of a glycoprotein is not a trivial matter, mostly due to a lack of good tools. Two most powerful methods exist for developing "binders" for glycoproteins: antibody production and nucleic acid-based aptamer selection. 2 However, none of these methods has the intrinsic ability to specifically focus on the glycosylation site, which include both the glycan and the surrounding structures, in epitope selection. We are interested in examining the possibility of directing the selection of aptamers to preferentially go after the glycosylation site of a glycoprotein (the sweet spot). By taking advantage of many published methods on incorporating modified nucleotide into DNA/RNA for aptamer selection, 3 we decided to incorporate a boronic acid-modified thymidine-5′-triphosphate (B-TTP, Figure 1) into DNA for aptamer selection. Because of the intrinsic ability for the boronic acid moiety to interact with diols 4 and single hydroxyl groups, 5 we hypothesized that the incorporation of the boronic acid moiety into DNA would allow the selection to gravitate toward the glycosylation site and therefore for the specific recognition of the glycosylation site. When necessary, counter selection can be used to eliminate unwanted cross-reactivity for binders as described in literatures. 3, 6 Herein, we report our work that demonstrates the feasibility by using a model protein, fibrinogen, which was chosen because of its commercial availability in large quantities and its known glycan structures. NIH Public AccessWe used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach for aptamer selection. 2,3 Introduction of the boronic acid moiety was accomplished through tethering to the 5-position of TTP ( Figure 1) because (1) modification at this position has long been known to have minimal effect on polymerase-catalyzed incorporation; 3 (2) 5-position modified TTP has been widely used in aptamer selections to tune their affinity and bestow novel properties, 3 and (3) we have demonstrated that the B-TTP can be successfully incorporated into DNA using DNA polymerases, and the synthesized boronic acid-modified DNA (B-DNA) can serve as templates for further amplification. 7For the aptamer sel...

The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD a and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3 ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.