Characterization of fluorescein–oligonucleotide conjugates and measurement of local electrostatic potential

Abstract

The properties of fluorescein are substantially altered upon conjugation to nucleic acids, affecting not only the molar absorptivities and fluorescence quantum yields but also the protolytic equilibrium constant and fluorescence lifetimes. Around neutral pH, the fluorescein moiety is present as both mono‐ and dianion, and the pKa relating them is increased from 6.43 for free fluorescein to about 6.90 for fluorescein attached to both single‐ and double‐stranded oligonucleotides of at least 12 bases/base pairs. This difference reflects the local electrostatic potential around the nucleic acid, which is calculated to −28 mV. The molar absorptivities and spectral responses of the conjugated fluorescein protolytic species are also determined, from which the concentrations of fluorescein–oligonucleotide conjugates can be calculated by assuming: ε494 = 62000/[1 + 10−(pH−6.90)] + 12000/[1 + 10(pH−6.90)] (M−1 cm−1). The fluorescence quantum yield of the conjugates depends, in a complex way, on temperature, environment and oligonucleotide length, sequence and conformation, and must be determined for each experimental situation. © 1998 John Wiley & Sons, Inc. Biopoly 46: 445–453, 1998

Trans-Scleral Permeability of Oregon Green 488®

“…Fluorescein has been used to conjugate many ocular drugs, such as antibiotics, dexamethasone, methotrexate, dextran, oligonucleotide, and ovalbumin, in order to investigate the trans-scleral drug pharmacokinetics for posterior segment disorders. [13][14][15] However, fluorescein is easily photobleachable 3 and pH-sensitive, 4 which can make it difficult to combine with some drugs and unstable.…”

“…Specifically, the permeation of labeled drug through donor sclera can be measured accurately via fluorometry, labeled drug concentration in the vitreous can be measured via ocular fluorophotometry, and the presence of drug within the sclera can be detected with fluorescein microscopy. 1,2 Even though NaF has proven useful in drug-delivery research, it is limited by photobleaching 3 and pH sensitivity, 4 and thus, more stable, alternative fluorescent agents would be beneficial.…”

Ordered Nano-Laminated Films and Fluorescence Dynamic Characterization of Tb-Bisphthalocyanine

“…The pulsed Nd:YAG laser described earlier in section 3 has been used as light source and the same interference filter (Ko et al 2006). This means that the intensity of the fluorescent emission would decrease when temperature increases; however, the intensity profile would not change (Ko et al 2006, Sjoback et al 1998). …”

Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

“…These limitations have often been overcome by the use of fluorescein, which is an extrinsic label with a strong emission signal far removed from the ultraviolet range. 35 As an intrinsic DNA fluorescence alternative, we chose 6MI since it is a relatively bright fluorophore in ssDNA (quantum yield ~ 0.3) 55 and its emission peak is red-shifted from that of the tryptophan signal. 43 In order to achieve strong signals, we used short oligonucleotides to maximize the proportion of fluorophore in the ssDNA.…”

“…[34][35][36][37][38]40,69,71,74,80,81 In particular, the emission signal from fluorescein is in the visible wavelengths and this reporter provides strong signals in the absence of a RecA protein tryptophan background. However, an extrinsic fluorophore's signal reflects the microenvironment of the dye rather than that of the DNA.…”