Oligonucleotide fluorescein labeling

Oligonucleotides bearing nido-carborane have been synthesized as phospho-diesters using an automated DNA synthesizer (see Scheme 2.2-14) [39]. These oligophosphates are homogeneous, very hydrophilic and are readily taken up into cells. Fluorescein-labeled nido-carboranyl oligomeric phosphate diesters accumulate in the cell nucleus [40]. 

Fig. 6. Electropherogram of fluorescein labeled phosphorothioate oligonucleotides (PS pd (T)io-25) recorded in 10% T,0% C polyacrylamide matrix at 2000 V/cm,L = 38 mm (reprinted with permission from [23]. Copyright 1994 American Chemical Society).

FIGURE 4.3 Repetitive sample injection and separation. Cycle conditions injection time, 5 s dead time, 1 s separation time, 45 s dead time, 1 s. The insets show two separation events on an expanded time scale. Sample fluorescein-labeled phosphorothioate oligonucleotide mixture, poly(dT)10 25. Separation conditions buffer, 100 mM Tris, 100 mM boric acid, 2 mM EDTA, 7 M urea, pH 8.5 Electric field strength, 2300 V/cm separation length, 3.8 cm [547]. Reprinted with permission from the American Chemical Society. 

Free-solution (gel-free) CE separation has been used in the detection of a target gene sequence amplified using the CPT reaction (see Chapter 9, section 9.2.1.7 for details). The intact chimeric oligonucleotide probe (fluorescein-labeled at the 5 end and biotin-labeled at the 3 end) was separated from a cleaved probe (only fluorescein-labeled at the 5 end). Owing to the presence of biotin in the intact probe, it migrated later than the cleaved probe, even in the absence of a gel sieving matrix [606]. 

Zhang, J., Malicka, J., Gryczynski, I., and Lakowicz, J. R. (2005). Surface-enhanced fluorescence of fluorescein-labeled oligonucleotides capped on silver nanoparticles, yowrno/ of Physical Chemistry B 109 7643-7648. 

Real-time hybridization of 5 -fluorescein-labelled target oligonucleotides was described using a fibre-optic DNA sensor by Ehrat [32], Lu [33] and coworkers, and a fibre-optic DNA sensor array capable of positively identifying a point mutation of a biotin-primer-labelled PCR product was reported by Walt [34]. 

Crockett AO, Wittwer CT. Fluorescein-labeled oligonucleotides for real-time PCR using the inherent quenching of deoxyguanosine nucleotides. Anal Biochem 2001 290 89-97. 

The functionalisation at the N1-position of 2 -deoxy-pseudouridine by Michael addition of methyl acrylate offered access to the phosphoramidites (61). The precursor to (61) was also functionalised as an amine derivative, which was transformed into the fluorescein-labelled phosphoramidites (62a-b). Fluorescent oligonucleotides were synthesised either from these latter building blocks or by post-synthetic modifications of oligomers containing the 2 -deoxy-pseudouridine-1 -propanoate units. ... 

Figure S.1S. Fluorescence intensity titration curve. Fluorescence intensity decreases wMi recombinant, ftjll-lergth Xenopus XPA concentration increases due to XPA binding to the single fluorescein labeled at the 5 -end of a ds 50-mer oligonucleotide. The titral on curve is saturated (levels off) at 100 nM XPA Cutlosey from William R. Wiley tnvironmental Molecular Sciences Laboratories.

We have developed a hybridization approach to monitor the intracellular uptake and distribution of ASOs in cell culture experiments [R. Berg et al., unpublished]. Cells treated with ASOs were hxed in paraformaldehyde and then biotin-labeled complementary oligonucleotides were added in hybridization buffer. Detection was with an avidin-horseradish peroxidase conjugate, which cleaves diaminobenzidine to produce a colored precipitate within the cells. An alternative detection approach uses fluorescein-labeled streptavidin, which is visualized by fluorescence and/or confocal microscopy [S. Fard et al., manuscript submitted]. We are currently adapting this protocol for use on tissue and tumor sections taken from mice treated sys-temically with ASOs. 

Lapos and Ewing have performed 80 ms injections every 5 s for the separation of 4-choloro-7-nitrobenzofurazan (NBD)-labeled amino acids. They have continued to improve upon this method by utilizing optical gating to inject analytes in a parallel channel format and have achieved five serial separations of four NBD-labeled amino acids in four parallel channels every 10 s. In addition, they have performed three serial separations of five fluorescein-labeled oligonucleotides (10,20,30, 60, and 80 basepairs in length) within 20 s. ... 

Hybridization of Fluorescein-Labeled Oligonucleotide Probes and Enhanced Chemiluminescence Detection... 

The hybridization buffer may be stored subaliquoted at -20°C for at least 6 mo. The fluorescein-labeled oligonucleotide may be stored at -20°C, in the dark, for at least 6 mo. The antibody conjugate, BSA, and the ECL detection reagents should all be stored between 2 and 8°C. All the other reagents should be stable at room temperature. Membrane blots should be stored at room temperature in a vacuum desiccator for maximum sensitivity. 

Abel and co-workers [106] at Ciba-Geigy Ltd. have reported an automated optical biosensor system. Their device utilizes 5 -biotinylated-16-mer oligonucleotide probes bound to an optical fiber functionalised with avidin to detect complementary oligonucleotides pre-labeled with fluorescein moieties in a total internal reflection fluorescence (TIRF) evanescent wave motif similar to that of Squirrell. Immobilization of nucleic acid probes onto the optical fiber substrate was achieved by functionalisation of the surface with (3-aminopropyl)triethoxysilane (APTES) or mercaptomethyldimethylethoxysilane (MDS). Onto the short alkylsilane layer was... 

Figure 4.10 Flow cytometry analysis of silica particles surface-modified with protein and DNA. Epoxy-organosilica nanoparticles (a, c) and MPMS particles (b, d) modified with GFP (a, b) or fluorescein-labeled oligonucleotide (c, d) were analyzed. Red lines and green lines indicated before and after reaction, respectively (Reproduced with permission from Ref [52] 2008, American Chemical Society.).

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

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