Journal
BIOCONJUGATE CHEMISTRY
Volume 18, Issue 6, Pages 2159-2168Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bc700233w
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Funding
- NCI NIH HHS [R21 CA099246, R33 CA099246, CA099246] Funding Source: Medline
- NHGRI NIH HHS [R01 HG001499, HG001499] Funding Source: Medline
- NIBIB NIH HHS [R01 EB006639-03, R01 EB006639] Funding Source: Medline
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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 MPc-oligonucleotide conjugates as primers for PCR (polymerase chain reaction) amplifications with the products sorted via electrophoresis and detected using near-IR fluorescence (lambda(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.
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