Metallo-Phthalocyanine Near-IR Fluorophores: Oligonucleotide Conjugates and Their Applications in PCR Assays

Abstract

Water soluble, metallo-pthalocyanine (MPc) near-IR fluorophores were designed, synthesized, and evaluated as highly stable and sensitive reporters for fluorescence assays. Their conjugation to oligonucleotides was achieved via succinimidyl ester-amino coupling chemistry with the conditions for conjugation extensively examined and optimized. In addition, various conjugate purification and isolation techniques were evaluated as well. Results showed that under proper conditions and following purification using reverse-phase ion-pair chromatography, labeling efficiencies near 80% could be achieved using ZnPc (Zn phthalocyanine) as the labeling fluorophore. Absorption and fluorescence spectra accumulated for the conjugates indicated that the intrinsic fluorescence properties of the MPc's were not significantly altered by covalent attachment to oligonucleotides. As an example of the utility of MPc reporters, we used the MPcoligonucleotide conjugates as primers for PCR (polymerase chain reaction) amplifications with the products sorted via electrophoresis and detected using near-IR fluorescence (λ ex = 680 nm). The MPc dyes were found to be more chemically stable under typical thermal cycling conditions used for PCR compared to the carbocyanine-based near-IR reporter systems typically used and produced single and narrow bands in the electrophoretic traces, indicative of producing a single PCR product during amplification.

NIR dyes for bioimaging applications

“…Their asymmetrically-substituted phthalocyanines and their oligonucleotide conjugates have found immediate utility in PCR applications [18] and as molecular beacons [19]. …”

Pegylated phthalocyanines: synthesis and spectroscopic properties

“…However, due to Pcs’ intrinsic property of insolubility and high tendency for aggregation in aqueous solutions, research has centered on the synthesis of soluble Pcs and the tuning of their photophysical properties. 3,4 A general approach consists of attaching multiple hydrophilic or amphiphilic substituents to the periphery or the core of Pcs, such as carboxylates, 5 sulfonates, 6 phosphonates, 7 pyridinium ions, 8 hydroxyl groups, 9 peptides, 10 oligonucleotides, 11 and polyethylene glycol (PEG) 12 groups. In particular, the use of PEG as effective carriers 13–15 or covalently attached for drug delivery to target tissues, 16,17 is well documented.…”

Rationally designed molecular beacons for bioanalytical and biomedical applications

“…19 Water soluble metallo-phthalocyanines (MPcs) are near-infrared fluorophores that possess high extinction coefficients, favorable quantum yields, and narrow absorption/emission envelopes and which display extraordinarily high photostabilities. 73-74 These qualities of MPc dyes can provide dimer-based MB probes (DBMBs) with unique properties and high signal-to-background ratio. 75 …”