Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments

Abstract

DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein–DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer–template DNA constructs labeled with Cy3/Cy5 donor–acceptor Förster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked ‘externally’ to the bases with flexible tethers, direct ‘internal’ (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion ‘background’ signals, thereby simplifying interpretations of both smFRET and smFLD experiments, while the decreased effects of the internal probes on local thermodynamic stability also make fluctuations sensed by these probes more representative of the unperturbed DNA structure. We suggest that internal probe labeling may be useful in studies of many DNA–protein interaction systems.

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model

“…Cy dyes are one important class of such modifications. The dyes have sharp absorption bands, high extinction coefficients, excellent resistance to photobleaching and make DNA oligomers highly fluorescent, so that even single molecules can be observed [1,2]. Their chemical structure is built from two indole rings that are connected by a polymethine chain (Fig.…”

MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements

“…The sequence and modification of miR-590-5p for FRET analysis are difficult to design. Typical FRET pairs for RNA such as Cy3-Cy5 [37] were not suitable for our study, as the wavelength of Cy3 emission was too short to excite Cy5 by image flow cytometry (Fig. 6A).…”

“…6A). Cy3 and Cy5 are problematic for use with miRNAs [37]. Another typical FRET pair FAM-TAMRA has been commonly used in qPCR [48].…”

“…Together, these data not only indicate the specific interaction of miR-590-5p and anti-miR-590-5p but also provide real-time images of RNA complementation. Another study shows that unrelated miRNA (the core region is not complementary) will not increase the FRET signal [37]. The number of FRET-fluorescence spots was not significantly altered by the dose or duration of miR-mock-Fam-F treatment ( Fig.…”

AA-Dutp-Cy3 a Novel Fluorescent Labeling Agent for Nucleotides

“…The reactive groups allow the dyes to be chemically linked to either aminoallyl-modified nucleic acids or directly to proteins and peptides. The dyes are suitable for a wide variety of biological applications including comparative genomic hybridization and expression array profiling, as commonly employed in transcriptomics [2]. They are also suitable for labeling proteins and nucleic acids for a variety of other applications relating to proteomics and genomics [3].…”