2'-Pyrene modified oligonucleotide provides a highly sensitive fluorescent probe of RNA

Abstract

Oligonucleotide 9mers containing 2'-O-(1-pyrenylmethyl)uridine [U(pyr)] at the center position were synthesized by using a protected U(pyr) phosphoramidite. The UV melting behaviors indicate that the pyrene-modified oligonucleotides can bind to both their complementary DNA and RNA in aqueous solution. When compared with the unmodified oligonucleotides, the pyrene-modified oligonucleotides showed higher affinity for DNA while exhibiting lower affinity for RNA. The pyrene-modified oligonucleotides in diluted solution exhibited fluorescence typical of pyrene monomer emission [lambdamax 378 (band I) and 391 nm (band III)]. When these oligomers bound to DNA, the fluorescence intensity ratio of band III/band I was increased. With this fluorescence change, a new broad emission (lambdamax 450 nm) due to exciplex between the pyrene and an adjacent nucleobase appeared. In contrast, addition of RNA to the pyrene oligonucleotides resulted in enhancement of the pyrene monomer emission with decrease in the fluorescence band ratio. The extent of the emission enhancement was found to be highly dependent on the nucleobase adjacent to the U(pyr) in the pyrene oligomers. The pyrene oligonucleotide containing dC at the 3'-site of the modification showed remarkable increase (approximately 250 times) in fluorescence (375 nm) upon binding to complementary RNA. The present findings would open the way to the design of a highly sensitive fluorescent probe of RNA.

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

“…This would account for both tertiary structure destabilization and the fluorescence enhancement induced by ligands. Similar behavior of pyrene tags has been seen in other nucleic acid systems (41,42). The weaker protein affinity for the py-U1082 RNA has actually been an advantage for measuring the large enhancement of protein binding in the presence of thiostrepton, and in any case affinities for unmodified RNAs can be obtained from competition experiments.…”

Detection of RNA Hybridization by Pyrene‐Labeled Probes

“…This was attributed to the difference in environment for the pyrene probes appended to the sugar unit of pyrene-RNA and pyrene-DNA duplexes. [3][4][5] Yet for some other pyrenelabeled DNA oligonucleotides (ONs), the fluorescence emission was reported to increase rather than decrease upon hybridization. [6][7] Although pyrene has been used as a fluorescent probe to monitor the hybridization or tertiary folding of nucleic acids, [8] pyrene-oligonucleotide probes based on monomer emission intensity are usually disturbed by fluorescence quenching through an electron-migration process between excited pyrene and nucleotide bases.…”

“…[22][23][24] Results and Discussion Design and synthesis of pyrene-labeled probes Several ONs containing 2'-O-(pyren-1-ylmethyl)uridine [25] were prepared and used as fluorescent probes of DNA and RNA in hybridization assays. [3][4][5][25][26] Here we report on the synthesis of ONs containing pyrenyl residue attached to 2'-O-position of adenosine with different linkers. Moreover the adenine derivative of 3-O-(pyren-1-ylmethyl)-2-deoxy-d-altro-hexitol was also incorporated into hexitol ONs.…”

Influence of C-5 halogenation of uridines on hairpin versus duplex RNA folding

“…Finally, in addition to halogenation of uridines and cytosines for X-ray crystallography, other modifications are commonly used in structural studies of nucleic acids: ribose modification for EPR spectroscopy (Macosko et al 1999), 29-fluoro-nucleotides (Kreutz et al 2005(Kreutz et al , 2006 or 5-fluoropyrimidines (Marshall and Smith 1977;Rastinejad et al 1995) for NMR spectroscopy, introduction of various fluorescent bases for fluorescence studies (Qin and Pyle 1999;Yamana et al 1999;Ben Gaied et al 2005), or, more recently, incorporation of selenium for crystal structure determination (Wilds et al 2002;Hobartner and Micura 2004). Our results highlight the importance of checking the possible influence on RNA folding of even subtle nucleic acids modifications.…”