Phthalocyanine Dimerization-Based Molecular Beacons Using Near-IR Fluorescence

Abstract

Herein we demonstrate the use of a novel dimerization-based molecular beacon (MB) probe consisting of two metallo-phthalocyanine (Pc) fluorophores that use near-IR fluorescence, appropriate for highly specific and sensitive in-vivo and/or in-vitro DNA/RNA detection. Pc's possess a propensity to form non-fluorescent H-dimers that is utilized as the molecular "off" switch in the closed MB conformation. The "on" switch, which is generated when the solution target binds to the loop of the MB forming the open form, also provides two fluorophores for transduction resulting in a doubling of the extinction coefficient and improving the resulting fluorescence yield compared to a classical single-fluorophore/quencher MB system. In addition, the Pc-based MBs possess high thermal, photo and chemical stabilities that are essential for many highly sensitive applications, such as molecular imaging. The dimer-based MBs were obtained using a simple singlestep synthesis procedure and demonstrated excellent quenching efficiencies (98%) as well as a high signal-to-background ratio (~60) exceeding the performance characteristics of many conventionallyavailable MB probes.Since their 1996 inception by Tyagi and Kramer, 1 molecular beacons (MBs) have become powerful tools for a variety of applications in DNA/RNA detection (e.g. real-time PCR, SNP detection, in vivo cellular imaging, cancer imaging etc.). MB-based detection systems are separation-free and highly specific however, their sensitivity (defined as the ratio of the fluorescence in the open versus closed form; signal-to-background, SB) is limited due to incomplete quenching of the closed form while signal in an open form is confined to one fluorophore. Various approaches for SB enhancement have been pursued including introduction of multiple fluorophores and/or quenchers, 2 eximer emission, 3, 4 FRET, 5 conjugated-polymers, 6 electrochemiluminescence 7 or quantum-dots. 8 The quest for more sensitive probes is still necessary along with the need to increase the MB reporting fluorophore's photostability to prolong observation times in imaging applications and conducting assays in the near-IR, where significant autofluorescence can be avoided. 9Water soluble metallo-phthalocyanines (Pc's) are near-IR fluorophores that possess high extinction coefficients, favorable quantum yields, narrow absorption/emission envelopes and display extraordinarily high photostabilities. 10, 11 These qualities make Pc's particularly attractive for MB applications along with other unique characteristics, such as: (i) Intrinsic propensity to form non-fluorescent H-type dimers that can be utilized as a molecular "off" switch for the closed MB form while two flourophores instead of one would be available for readout in the open form; (ii) diminished Pc dimerization when it is part of a relatively large oligonucleotide construct (e.g. MB hybridized to a complementary target as has been demonstrated by us), but the lack of fluorescence when bound to a relatively short DNA E-mail: chsope@lsu.edu. co...

Near-Infrared Dyes and Their Use in Medical Science

“…Pcs have a number of characteristic properties such as high thermal stability, unique photophysical properties, intense color, non-toxicity, and high phototoxicity upon irradiation with light, contributing to their effectiveness in different research areas. Their easily tunable photochemical and photophysical properties such as narrow absorbance and emission band and resistance to photobleaching, by changing the substitution pattern around the Pc aromatic core and/or the central metal atom, makes these compounds highly appropriate for applications such as PCR, single gene mutation detection, and resonance energy transfer-based assays (9).…”

“…They are specifically useful fluorescence on-off systems offering high target to background ratio due to Pcs high extinction coefficients and strong and narrow emission profiles. In the light of this information, Nesterova et al (9) have published another article in the Journal of American Chemical Society in 2009 as a follow up study (9). In this study, authors, for the first time, have designed, prepared, and evaluated the use of double-labeled dimerization-based molecular beacons A different perspective of Pc aggregation-based system (on/off probe) has been described in another study published in the Analyst in 2010.…”

Recent trends in molecular beacon design and applications

“…Water-soluble metallophthalocyanines are one class of metal complexes that were also introduced into the design of MBs by Nesterova et al [19]. They are near-IR PLS with unique photophysical characteristics, such as (1) an intrinsic propensity to form nonfluorescent H-type dimers and (2) diminished metallophthalocyanine dimerization when the Fig.…”

Zinc Phthalocyanine Labelled Polyethylene Glycol: Preparation, Characterization, Interaction with Bovine Serum Albumin and Near Infrared Fluorescence Imaging in Vivo

“…Based on the optical sensitivity of phthalocyanines, they were often applied as photodynamic therapy (PDT) agents to treat cancer or against bacteria [24][25][26][27][28][29]. Besides, zinc phthalocyanines were reported recently as optical probes either in vitro or in vivo due to the low absorbance of biosystems in the near infrared spectral window [30][31][32]. Nesterova et al studied phthalocyanine dimerizationds as near infrared fluorescence probes for in vivo and in vitro DNA/RNA detection [30].…”

“…Besides, zinc phthalocyanines were reported recently as optical probes either in vitro or in vivo due to the low absorbance of biosystems in the near infrared spectral window [30][31][32]. Nesterova et al studied phthalocyanine dimerizationds as near infrared fluorescence probes for in vivo and in vitro DNA/RNA detection [30]. Mantareva et al reported cationic zinc phthalocyanines as advanced fluorescent contrast agents for pigmented melanoma tumors [31].…”