1-Phenylethynylpyrene (PEPy) as a novel blue-emitting dye for qPCR assay

Abstract

An alkyl azide derivative of 1-phenylethynylpyrene (PEPy) dye was prepared and used in the functionalization of oligonucleotides via click chemistry. Spectral and photo-physical properties of the PEPy-modified oligonucleotides as a single strand, and in perfect or mismatched duplexes, have been studied. A series of PEPy-Dabcyl fluorogenic TaqMan probes were synthesized and tested in qPCR. PEPy proved to be a superior substitute for AMCA as a short wavelength fluorescent dye for qPCR probes. PEPy probes were shown to significantly reduce Cq (a fractional PCR cycle used for quantification) vs. AMCA labeled probes, thus improving on the reliability of detection. Moreover, a larger increase of fluorescence during amplification was observed in the case of PEPy probes that makes this dye very suitable for an end-point PCR technique. This study broadens the panel of fluorescent dyes suitable for the use in probes for quantitative real-time PCR.

Excimer-FRET Cascade in Dual DNA Probes: Open Access to Large Stokes Shift, Enhanced Acceptor Light up, and Robust RNA Sensing

“…Pyrene derivatives are capable of interacting with dsDNA either by intercalation between base pairs or by minor-groove binding. − These interactions are accompanied by strong suppression of emission due to photoinduced energy transfer (PET), especially in proximity to G-rich sequences. , On the contrary, dsDNA reduces the photoisomerization speed of covalently bound Cy3 dye, which basically leads to the increase of fluorescence quantum yield (Φ f ) in comparison to the free dye . In this respect, we optimized the structure of the probes and simultaneously studied FRET properties of the pyrene donor and sCy3 acceptor.…”

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

“…A large diversity of the pyrene-modified nucleotide monomers obtained through presynthetic [ 69 , 70 , 71 , 72 , 73 , 74 , 75 ] or postsynthetic [ 76 , 77 , 78 , 79 , 80 , 81 ] conjugation of pyrene derivatives with modified nucleotides or oligonucleotides via Cu(I)-catalyzed azide alkyne Huisgen 1,3-dipolar cycloaddition (CuAAC) has been reported over the past several years.…”

Cy5/BHQ dye–quencher pairs in fluorogenic qPCR probes: effects of charge and hydrophobicity

“…11 Oligonucleotide probes were purified by denaturing PAGE followed by RP-HPLC. Oligonucleotides were cleaved from the support and deprotected using conc.…”

“…Unpuried or puried 5 0 -alkyne oligonucleotides were conjugated with Cy5 or sCy5 azides as described previously for 1-PEPy dye. 11 Oligonucleotide probes were puried by denaturing PAGE followed by RP-HPLC. Conditions of PAGE, HPLC purications and analysis were the same as before.…”