Nucleic acid fluorophor labeling

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Overall, the significant Stokes shift of 100 nm and the good quantum yields make the coumarin dye a powerful fluorescent probe for nucleic acids assays or cell biology. The postsynthetic click chemistry makes this fluorophore readily accessible for fluorescent labeling of nucleic acids. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

Transfer RNA (tRNA) molecules mediate translation of the nucleic acid genetic code into the amino acid building blocks of proteins, thus ensuring the survivability of cells. The dynamic properties of tRNA molecules are crucial to their functions in both activity and specificity. This chapter summarizes two methods that have been recently developed or improved upon previous protocols to introduce fluorophores to site-specific positions in tRNA. One method enables incorporation of fluorophores carrying a primary amine (such as proflavin or rhodamine) to dihydrouridine (D) residues in the tRNA tertiary core, and a second method enables incorporation of pyrroloC and 2-aminopurine to positions 75 and 76, respectively, of the CCA sequence at the 3 end. These site-specific fluorophore labeling methods utilize tRNA transcripts as the... 

Fluorescent dyes, especially the cyanine dyes Cy3 and Cy5, are the most popular choice for dual colour microarray analysis. The main benefit of using Cy dye fluorophors in particular is that the two dyes can be excited and detected from the same slide. Fluorescent dyes can be directly incorporated into nucleic acid by either enzymatic or chemical methods. Basically there are two fluorescent dye-labeling strategies ... 

While the capture on DNA chips of fluorophore-labelled targets, and the extension of arrayed primers with fluorophore-labelled nucleotides has been widely used for some time, it is only more recently that assay formats have developed that utilize immobilized nucleic acids already modified with fluorophores. Fundamental analyses of surface monolayer structures and chemistries can be readily performed by immobilizing such modified oligonucleotides into SAM structures [105,106], but it is those interactions that can be monitored using fluorescence quenching or fluorescence resonance energy transfer (FRET) that have gained the most attention. 

The effect of temperature on fluorescence has been studied, as has the effect of salt concentration and water-soluble conjugated polymers. A method for the quantification of ssDNA dsDNA is described, as well as kinetics of mismatch hybridization and the kinetics of collision in short ss-nucleic acids. Fluorescence quenching of Cy-5 labelled oligonucleotides by poly(phenylene ethynylene) particles has been shown to be a more sensitive method than excitation of the Cy-5 fluorophore. An ultrasensitive method for the detection of DNA uses highly fluorescent conjugated nanoparticles, and detection limits below IfM were achieved. DNA transport through a carbon nanotube has also been observed using fluorescence microscopy. " ... 

Nazarenko, I. Lowe, B. Darfler, M. Ikonomi, P. Schuster, D. Rashtchian, A. Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res. 2002, 30, e37. 

Reisfeld et al. (1987) described a hydrazide biotin labeling method (Table 7.20D) by which they could detect 1 pg of target DNA. Hydrazide compounds can be used similarly for a large number of other labels (enzymes, chromo- or fluorophores, electron-dense markers, etc.). Since the reaction is specific for ss nucleic acid, loops in duplices can be specifically labeled. 

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