The fluorescence resonance energy transfer (FRET) gate: A time-resolved study

Abstract

The two-step energy-transfer process in a self-assembled complex comprising a cationic conjugated polymer (CCP) and a dsDNA is investigated by using pump-dump-emission spectroscopy and time-correlated single-photon counting; energy is transferred from the CCP to an ethidium bromide (EB) molecule intercalated into the dsDNA through a fluorescein molecule linked to one terminus of the DNA. Time-dependent anisotropy measurements indicate that the inefficient direct energy transfer from the CCP to the intercalated EB results from the near orthogonality of their transition moments. These measurements also show that the transition moment of the fluorescein spans a range of angular distributions and lies between that of the CCP and EB. Consequently, the fluorescein acts as a fluorescence resonance energy-transfer gate to relay the excitation energy from the CCP to the EB. conjugated polymers ͉ DNA sensors ͉ time-resolved anisotropy ͉ ultrafast spectroscopy C ationic conjugated polymers (CCPs) have been demonstrated to have utility in DNA sequence detection (1-3). In the approach initially proposed by Gaylord et al. (1), the luminescent conjugated polymer serves as the light-harvesting entity with subsequent energy transfer to a fluorescein (FL) molecule attached to a peptide nucleic acid (PNA) oligomer comprised of a specific sequence of bases that defines the target. Fluorescence resonance energy transfer (FRET) from the CCP to the PNA-FL occurs by self-assembly of the CCP and the PNA in a double helix with a complementary single-stranded DNA target. In this approach, the electrostatic interaction between the CCP and the negatively charged PNA-FL͞DNA duplex brings the donor (CCP) and the acceptor (FL) in sufficiently close proximity to enable efficient FRET. When the base sequence on the DNA is not complementary to the base sequence on the FL-labeled PNA, the PNA-FL remains as a charge-neutral single-strand oligomer. At the dilute concentrations used, the typical distance between a neutral PNA-FL and a CCP is too large for efficient FRET. By using this FRET-based biosensor, specific targeted sequences of DNA were detected at concentrations as low as 10 pM (1).Wang et al. (2) reported an alternative DNA sequencedetection scheme (Scheme 1) based on intercalation of ethidium bromide (EB) within a dsDNA. Considering that the synthesis and purification of labeled DNA is more straightforward and that both single-stranded DNA and dsDNA have good solubility in buffered water (whereas with PNA, hydrophobic interactions are important), the EB intercalation approach is attractive. However, direct FRET from the CCP to EB was found to be inefficient despite the excellent spectral overlap between the donor emission and acceptor absorption. The absence of efficient FRET was attributed to the orthogonality of the transition dipoles of the CCP and EB in the self-assembled structure (see Scheme 1). When a FL molecule is linked to one terminus of the dsDNA as shown in Scheme 1B, the EB emission is significantly enhanced after excitation...

Cooperation of Escherichia coli Hfq hexamers in DsrA binding

“…6B). These phenomena clearly demonstrate dipolar-mediated energy transfer (FRET) between these two fluorophores (Xu et al 2005;Gilmore et al 2011). The intensity of SRB reached the lowest level when DsrA was titrated to ;67% of the Hfq hexamer molar concentration.…”

Energy Transfer Between a Cationic‐Conjugated Poly(fluorene‐co‐phenylene) and Thiazole Orange for DNA Hybridization Detection Involving G‐Rich Sequences

“…Figure 3(a) also shows that under similar conditions, the energy transfer efficiency is much lower with PFN/dsDNA/TO, most likely as a result of orientation mismatch between the transition moment of intercalated TO and the polymer backbone, as previously observed for the case of ethidium bromide. [21] At [RU] ¼ 2 Â 10 À6 M, the PFN-sensitized emission for ssDNA 1 / TO is approximately eight-fold more intense than that observed for PFN/ dsDNA/TO. The better response of PFN to ssDNA 1 /TO could be mainly attributed to the improved orientation between PFN and ssDNA 1 /TO, which is due to the random orientation of TO associated with the flexible ssDNA 1 .…”

“…[19] However, a recent FRET study between a CCP and ethidium bromide intercalated into dsDNA showed poor efficiencies. [20,21] Time-dependent anisotropy measurements revealed that the low FRET efficiency was due to a non-optimal (orthogonal) orientation between the transition moment of the optically active polymer backbone and that of ethidium bromide within the dsDNA structure. [22] We anticipated that TO dyes associated with ssDNA would have more geometric freedom to conform to the CCP structure, when compared to dyes intercalated within the more rigid dsDNA.…”

A New FRET‐Based Sensitive DNA Sensor for Medical Diagnostics using PNA Probe and Water‐Soluble Blue Light Emitting Polymer

“…When this distance is close to Forster critical radius, the energy transfer occurs within femtoseconds [26]. On the other hand, the excited state decay of donor PFP occurs exponentially with an average lifetime of ≈90 ps (≈30 ps in presence of the acceptor) [27]. The electrostatic energy is, therefore, transferred from donor to acceptor before the former can emit its photoluminescence (PL).…”