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In biosensors that use DNA- or RNA-based enzymes, the catalytic nucleic acid is frequently designed such that its activity depends upon target-induced conformational changes; however, this can be hampered by unacceptably high background. To reduce spurious signals, the enzyme can be split into separate oligonucleotides that function only in the presence of ligand, but dividing the parent oligonucleotide into subcomponents that can be successfully reconstituted has necessitated rational design or in vitro evolution strategies. Writing in JACS, Mokany et al. describe versatile multicomponent nucleic acid enzymes (MNAzymes) that are modular versions of the previously described DNAzymes known as 10-23 and 8-17. An MNAzyme consists of two “partzymes,” each comprising a substrate arm, a partial catalytic core, and a sensor arm. In the presence of target, the sensor arms of the partzymes anneal side-by-side; likewise, the two substrate arms bind adjacently. By binding in proximity, the partzymes restore the enzyme's catalytic core. In the simplest incarnation, the sensor detects a target sequence and triggers cleavage (and fluorescence) of a readout oligonucleotide bearing both fluorophore and quencher. MNAzymes can detect target sequences in isothermal assays, or, if a portion of one of the substrate arms is split off into a separate “stabilizer arm,” discriminate single-base mismatches. In contrast to previously described multipart DNAzymes, MNAzymes are enzymatically active at PCR annealing temperatures, freeing them for quantification of targets in real-time PCR. The authors describe successfully mixing and matching several partzyme arms and cores to produce 12 different MNAzymes, whose catalytic properties resembled those of the parent DNAzymes. Future development of MNAzymes will include broadening the range of enzymatic reactions, and adapting them for use as molecular switches, with possible application to molecular computers and DNA nanomachines.
Mokany et al. MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches. J. Am Chem Soc. 2009 Dec 28. [Epub ahead of print; doi: 10.1021/ja9076777].
Tagging Sugars in Mice: Click HereA recent advance in the field of glycobiology has been the ability to study glycosylation in vivo through metabolic labeling of cell surface sialoglycoconjugates using N-acyl–modified analogs of N-acetyl mannosamine. This “chemical reporter” can then be tagged for fluorescence or affinity purification by selectively reacting with a probe reagent. An ideal chemical reporter is the azide group, which is small, inert under normal cellular conditions, and has multiple modes of reactivity. By using the Staudinger ligation reaction, which forms an amide bond between azides and triarylphosphines, azidosugar-labeled glycoconjugates have been tagged in cultured cells and live mice. Phosphine reagents suffer several drawbacks, however, such as oxidation and a rather slow reaction rate, which hinders analysis of more rapid in vivo processes. As an alternative, Chang et al. describe in the Proceedings of the National Academy of Science their modification of the Huisgen 1,3-dipolar cycloaddition of azides and alkynes to form triazole products. While the copper–catalyzed version of this reaction—referred to as “click chemistry”—is well known, its use in vivo is precluded by the toxicity of copper. In the authors' “copper-free click chemistry,” the alkyne is located in a strained cyclooctyne ring with added propargylic fluorine atoms, which greatly increases the reaction rate such that it is comparable to the “click” reaction. They first examined the reactivity of several different derivatives of cyclooctyne (OCT): the more water-soluble aryl-less cyclooctyne (ALO) and dimethoxy azacyclooctyne (DIMAC), as well as two more reactive cyclooctynes containing propargylic fluorine atoms, monofluorinated cyclooctyne (MOFO) and difluorinated cyclooctyne (DIFO). These derivatives were conjugated to the FLAG tag for detection of the ligation product by a fluorescein-labeled anti-FLAG antibody. In cultured cells, the relative order of labeling efficiencies of the different FLAG-conjugated cyclooctynes matched that of the reagents' intrinsic reactivities. For in vivo analysis, they first metabolically labeled mice for a week, then injected a cycloctyne-FLAG for a 3-h treatment and performed ex vivo analysis of the splenocytes. Although OCT-FLAG did not label at all, both ALO-FLAG and DIMAC-FLAG did, which appears to be due to their superior bioavailability. DIFO-FLAG showed even better labeling, but MOFO-FLAG did not, a surprising result since both have higher intrinsic reactivities than OCT-FLAG. Therefore, it appears DIFO-FLAG is efficient for tagging serum or tissue glyco-proteins in live mice.
