GFP-like Fluorophores as DNA Labels for Studying DNA–Protein Interactions

Abstract

GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

“…Remarkably, metal nanoclusters can detect natural nucleic acids, such as cancer related mRNAs, directly in living cells [31]. Finally, the use of so-called environmentally sensitive modified nucleosides and nucleotides also offers exciting opportunities for the specific detection of nucleic acids [32][33][34][35][36]. The distinguishing feature of these is enhanced fluorescence upon a change in the local microenvironment.…”

“…They were able to achieve this owing to the environmentally sensitive intramolecular charge transfer (ICT) between the nucleobase and naphthalene moieties [33]. Other successful examples of environmentally sensitive modified nucleosides include light-up probes for the detection of triplex formation and polarity sensitive probes [34][35][36]. One particularly useful mechanism involves introducing intramolecular conformational changes at the bond between the attached modifications and nucleobase [34,35].…”

Synthesis and properties of 4-(diarylmethylene)imidazolinone-conjugated fluorescent nucleic acids

“…One of the most standard methods for labeling nucleic acids involves the attachment of a fluorescent dye to the C5-position of pyrimidine nucleosides through alkynyl linkage, because this enables predictive positioning of the fluorophores in the major groove of duplexes without the loss of Watson-Crick base-paring properties. [9][10][11][12][13][14][15] The fluorescent properties of these nucleosides are mainly dependent on the fluorophores chosen; thus, the specific type of fluorophore that is loaded onto the nucleoside is an essential consideration to best detect the local environmental change of nucleic acids.…”

Design and synthesis of 7-naphthyl-8-aza-7-deaza-2′-deoxyadenosines as environmentally sensitive fluorescent nucleosides

“…Fluorescent pyrimidine nucleosides containing GFP-like fluorophore at the 5-position have also been reported as an ESF nucleoside for studying DNA-protein interactions. 7 In our continuous efforts to develop practically useful ESF nucleosides, 8 we recently reported that 7-naphthylethynylated 8-aza-7-deaza-2 0 -deoxyadenosine cna A exhibited dual fluorescence emission arising from an intramolecular charge-transfer (ICT) state and a locally excited (LE) state depending upon the coplanarity between naphthalene ring and 8-aza-7-deazaadenine skeleton through ethynyl linker (Fig. 1 discriminate perfectly matched thymine on the complementary strand by a significant shift of emission wavelength to shorter wavelength via probing the differences in microenvironment caused by a structural change.…”