Probes chemical labeling

The chemical modification of nucleic acids at specific sites within individual nucleotides or within oligonucleotides allows various labels to be incorporated into DNA or RNA probes. This labeling process can produce conjugates having sensitive detection properties for the localization or quantification of oligo binding to a complementary strand using hybridization assays. 

Forster, A.C., Mclnnes, J.L., Skingle, D.C., and Symons, R.H. (1985) Non-radioactive hybridization probes prepared by the chemical labeling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acid Res. 13, 745-761. 

References [52-54] do not include any data directly comparing squaraine rotaxanes with common cyanine dyes such as Cy5 (GE Healthcare) and Alexa 647 (Life Technologies). Nevertheless, from the available data it can be concluded that squaraine rotaxanes are remarkably resistant to chemical and photochemical degradation, and likely to be very useful as a versatile fluorescent scaffold for constructing various types of highly stable, red and near infrared (NIR) imaging probes and labels. 

Chemically modified DNAs can also be used as hybridization probes, provided that the modification does not interfere with the formation of hybrid DNA molecules. A psoralen biotin label has also been developed. Psoralen is a photoactivable agent that can intercalates into single- or double-stranded nucleic acids. On irradiation at 365 nm, it will covalently bind to the probes. This labeling reaction is simple and straightforward. However, the reagents for labeling and detection are only available in a kit format. 

In order to probe chemical constituents and follow their biochemical reaction in cells and tissues, there is a need to make fluorescent labels more specific, brighter, and more robust. This will require greater understanding of the photophysics and photochemistry of fluorescent probes and the mechanisms of their photobleaching. 

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

In reverse hybridization, unlabeled probes are immobilized on nitrocellulose membranes (0.5 xg per spot), the test sample is photo-chemically labeled (Section 7.8.2) and hybridized simultaneously to the panel of probes (Dattagupta et al., 1989). It is not necessary to purify sample DNA before or after labeling and a 300 xl sample is added to 1.1 ml of the prewarmed hybridization solution (3 X SSC, 20 mM pyrophosphate, 5% BLOTTO, 10% PEG, 1 mg/ml carDNA prehybridization 30 min, hybridization 2 h). The haptens are then detected with labeled antibody or streptavidin. 

Figure 21 Chemical probes for monitoring glycan-processing enzymes, (a) Mechanism-based probe for labeling active exo-glycosidases in cell lysates/ (b) FRET-based reporter for OGT activity in cells. ...

Fig. 7.3-3 General strategy for activity-based protein profiling (ABPP). Proteomes are treated with chemical probes that label active enzymes of a particular class (or classes) in a manner that allows for their detection, isolation, and identification. Active enzymes are denoted by open/unshaded active sites, with their inactive counterparts (e.g., zymogen or inhibitor-bound forms) shaded in black.

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