Detection of RNA Hybridization by Pyrene‐Labeled Probes

Abstract

Powerful pyrene probes: Two kinds of pyrene-labeled oligonucleotides (HNA- and RNA-skeleton probes) were explored. The enhanced fluorescence intensity in the monomer region and the disappearance of aggregate/excimer emission in duplexes has been successfully used to detect the hybridization of oligonucleotides. By covalently attaching pyrene chromophores with different linkers onto altritol nucleotides or ribonucleotides, and by varying the number of these pyrene modified altritol nucleotides and ribonucleotides in HNA (hexitol nucleic acid) and RNA, respectively, we have explored the general applicability of pyrene absorbance and especially fluorescence as a probe to monitor RNA hybridization. The results reveal that the backbone of the probes, the number of pyrene units attached and the nature of the tether can all substantially affect the absorbance and fluorescence properties of the probes both in single strand and double strand form. Moreover, the strength of hybridization is also affected. The disappearance of pyrene aggregate/excimer emission and simultaneous increase in monomer emission intensity of the multipyrene-labeled probes has been successfully used to monitor the hybridization of oligonucleotides, including a hairpin structure. Differences in optical response between the HNA- and RNA-skeleton probes upon hybridization indicate that the interaction of pyrene with the nucleobases in both types of duplexes is different.

2‐(1‐Ethynylpyrene)‐Adenosine as a Folding Probe for RNA—Pyrene in or out

“…In pyrene and ethynylpyrene, the observed blue shifts have generally been attributed to exposure of the dye to a more polar environment. [20,30,[53][54][55] The effect of duplex melting on the absorption of the doubly pyrene-modified compound (Figure 4) is slightly different than that of the singly modified duplexes. Again, a blue shift can be seen, yet, unlike before, this blue shift does not affect the whole spectrum equally.…”

“…They have proven useful in the context of recognition and as molecular probes, [6][7][8][9][10][11][12][13] electron injection, [14,15] bioengineering, [16,17] structure stabilisation [18] and structure determination [19][20][21][22][23] as well as nucleobase photophysics. [24,25] Their relatively high quantum efficiency, sensitivity to environmental factors [26,27] and capability to form excimers have opened many possibilities in the field of DNA and RNA hybridisation studies.…”

Labeling-free fluorescent detection of DNA hybridization through FRET from pyrene excimer to DNA intercalator SYBR green I

“…1. The two fluorescence bands in the wavelength ranges of 360-410 nm and 420-600 nm are attributed to the pyrene monomer emission and excimer emission, respectively (Wang et al, 2009). It should be noted that the excimer emission band of the polymer PFP overlaps the absorption band of SG very well.…”

Nucleic Acids with a Six‐Membered ‘Carbohydrate’ Mimic in the Backbone

“…been compared [77]. Significant changes of fluorescence intensity upon hybridization are observed for multichromophoric probes.…”