Fluorescent nucleotides

Labeling of the RNA sample with a fluorescent nucleotide can be done either directly or indirecdy. In a direct labeling, one of the nucleotides (usually dUTP or dCTP) has a fluorescent moiety (either Cy3 or Cy5)... 

R.S. Cozzone, A.J. Gortay, J.G. Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues. Eur. J. Biochem., 237, 247-254 (1996)... 

Despite the wide applicability of TNP-ATP as a useful fluorescent nucleotide analog, the dynamics of its fluorescence is still not fully understood. To find the origin of change in the apparent fluorescence intensity in different environments, it is important to learn how the local environment influences the fluorescence dynamics. Four important fluorescence characteristics of TNP-ATP are summarized in the following manner [23], First, the fluorescence lifetime of TNP-ATP... 

Among fluorescence probes of nucleic acid structure, dynamics, and folding, 2-aminopurine (2AP) is unique in that its introduction into an RNA can often be done with little perturbation of the RNA structure. Whereas other fluorescent nucleotide analogs have higher quantum yields than 2AP, their modifications have the potential to disrupt secondary and... 

In the most recent microarray experiments, multicolor fluorescence labeling is used for simultaneous analysis of two or more samples in a single assay. For this, total RNA or mRNA are labelled with fluorescent nucleotides by a reverse transcription reaction. Cyanines Cy3 and Cy5 are used for dual color analysis. 

Figure 17.21 Single nanometric aperture for real time single molecule DNA sequencing. A DNA polymerase molecule attached to the bottom surface is used to successively Incorporate fluorescent nucleotides complementary to the DNA strand, causing fluorescence bursts for each incorporation process (see text for details). Image copyright of Pacific Biosciences Inc., reprinted with permission.

Cruickshank KA. Quantitation of fluorescent nucleotide incorporation by capillary gel electrophoresis and laser-induced fluorescence detection. Analyt. Biochem. 1999 269 21. 

Guerra CE. Analysis of oligonucleotide microarrays by 3 end labeling using fluorescent nucleotides and terminal transferase. Biotechniques 2006 41 53-56. 

Rist MJ, Marino JP. Fluorescent nucleotide base analogs as probes of nucleic acid structure, dynamics and interactions. Curr. Org. Chem. 2002 6 775-793. 

A tandem Kornblum ox/daf/on/imidazole formation reaction was used during the preparation of new fluorescent nucleotides by B. Fischer and co-workers.The adenosine monophosphate free acid was mixed with 10 equivalents of 2-bromo-(p-nitro)-acetophenone and dissolved in DMSO. The required pH value was maintained with the addition of DBU which also served as a base. The Kornblum oxidation of the alkyl halide yielded the glyoxal, which reacted in situ with the aromatic amine to form the desired imidazole derivative. 

Nucleoside Pyrophosphates. - The synthesis of 8-aryl-3-P-o-ribofuranosylimiazo[2,l-i]purine 5 -phosphates (122) from AMP or ATP has been described. To access these fluorescent nucleotide derivatives, a combination of Kornblum oxidation reaction and imidazole formation was employed. For this conversion, the appropriate adenosine phosphate, present in its free acid form, was treated with p-nitro-acetophenone in DMSO in the presence of DBU. Treatment of a 5-(chloroethyl)-4-(triazole-l-yl)pyrimidine-nucleoside with benzylhydrazine offered the 6,6-bicyclic pyrimido-pyradazin-7-one, the precursor to (123). This triphosphate was used as a substrate for DNA polymerases. ... 

To observe whether GVs could be loaded with nucleotides, a series of experiments was carried out demonstrating that GVs could take up, in an ethanol-dependent way, molecules such as Ca + ions or fluorescent nucleotide triphosphates. In a further attempt, the loadable microreactor could now be designed macromolecules required for mRNA synthesis by the T7 RNA polymerase (the T7 RNA polymerase and the plasmid DNA) were injected into a selected GV and YO-PRO-1 was added externally. After the fluorescence intensity (caused by the DNA template) became stable, ethanol was added and the GVs were allowed to stand for another period. Then ribonucleotides were added with a micropipet in the vicinity of the selected GV and the increase in the fluorescence intensity was followed with time (Figure 13.3). The quantification of the fluorescence increase showed that the fluorescence increased from a starting normalized value of 100 to a value after 40 min of about 220. The corresponding control experiment carried out under the same conditions but in the absence of T7 RNA polymerase shows only a modest increase in fluorescence. 

When comparing hybridization results between two cell types, the same type and quantity of RNA should be used to generate both probes. If sufficient material is available, probes can be directly generated by incorporating fluorescent nucleotides during a reverse-transcription reaction. Otherwise an additional RNA amplification step will be needed. This is not desirable because it could cause problems with transcript representation. 

Inner salts 157, obtained by N-alkylation of the isothiazole, are azomethine dye-based fluorescent nucleotides. They were used as the labels for nucleic acid (03JP2003034696, Scheme 53). 

A related fluorescent nucleotide affinity label, 5 -p-fluorosulfonylbenzoyl-2-aza-1,7V -ethenoadenosine (5 -FSBaeA), in which a nitrogen replaces the carbon atom at the 2-position of 5 -FSBeA, has also been prepared (205, 206). 5 -FSBaeA has different spectral properties, with an emission peak at 490 nm and an excitation maximum at 356 nm. Thus, it can be used in a complementary manner to 5 -FSBeA as a covalently bound chromophore. 5 -FSBaeA reacts at a GTP regulatory site of glutamate dehydrogenase, and energy transfer experiments have led to an estimated distance between the catalytic and GTP sites in... 

Katihus, E., Katihene, Z., Woodbury, N. W. (2006). Signaling aptamers created using fluorescent nucleotide analogues. Anal Chem 78, 6484-6489. 

Li, Z. M. et al. A photocleavable fluorescent nucleotide for DNA sequencing and analysis. Proceedings of the National Academy of Sciences of the United States of America 100, 414-419 (2003). 

This section describes a general method that can be used to label any kind of DNA probe, from oligonucleotides to genomic DNA. It works well to incorporate many different commercially available modified nucleotides. The basic strategy is to fragment probe DNA into small pieces and then to label the 3 ends by addition of hapten-labeled or fluorescent nucleotides. 

Note If the probes are directly labeled with fluorescent nucleotides, steps 10-12 are not performed skip to step 13. 

Rgure 1.18. Ruoco(4)0its for covalent labefing (DNS 0 and F2TC) and fluorescent nucleotide analogs (c-ATP and /iR-beozo AMP). 

Several modes of detecting a single base extension have been investigated, including measuring the incorporation of fluorescent, haptenated, or radioactive ddNTPs [6, 7] or gel electrophoresis based-detection of fluorescent primers extended by non-fluorescent nucleotides [8]. Recently, the Applied Biosystems SNapshot lYimer Extension Kit was introduced. In this assay, a primer is extended by one or more fluorescent- labelled dideoxynucleotides with subsequent detection in a fluorescent-based DNA sequencer. Several primers can be analysed within one lane of the DNA sequencer. 

---