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Characterization of fluorescein–oligonucleotide conjugates and measurement of local electrostatic potential
Author(s) -
Sjöback Robert,
Nygren Jan,
Kubista Mikael
Publication year - 1998
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/(sici)1097-0282(199812)46:7<445::aid-bip2>3.0.co;2-5
Subject(s) - fluorescein , chemistry , oligonucleotide , nucleic acid , fluorescence , moiety , conjugate , conjugated system , quantum yield , photochemistry , analytical chemistry (journal) , chromatography , stereochemistry , organic chemistry , dna , biochemistry , mathematical analysis , physics , mathematics , quantum mechanics , polymer
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 pK a 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: ε 494 = 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