Fluorescence Detection of Specific RNA Sequences Using 2′-Pyrene-Modified Oligoribonucleotides

Uridine 2′‐Carbamates: Facile Tools for Oligonucleotide 2′‐Functionalization

“…This gives evidence that the excited pyrene 2′‐carbamate is effectively quenched in single‐stranded oligodeoxyribonucleotides and in DNA‐DNA duplexes, but not in DNA‐RNA duplexes. Similar examples of fluorescence increases upon hybridization with a complementary RNA are demonstrated for probes containing 2′‐attached pyrene (Yamana et al, ).…”

“…As expected, a single modification close to the 5′‐terminal position of the oligonucleotide caused less destabilization than one in the middle. A number of studies of pyrene attached to the 2′‐position of individual nucleosides within duplexes have been published recently (Silverman and Cech, ; Yamana et al, ). Pyrene as well as other planar polyaromatic hydrocarbons often show a greater stabilizing effect in DNA‐DNA duplexes than in DNA‐RNA duplexes (Yamana et al, ).…”

“…A number of studies of pyrene attached to the 2′‐position of individual nucleosides within duplexes have been published recently (Silverman and Cech, ; Yamana et al, ). Pyrene as well as other planar polyaromatic hydrocarbons often show a greater stabilizing effect in DNA‐DNA duplexes than in DNA‐RNA duplexes (Yamana et al, ). The pyrene 2′‐carbamate modification is not an exception, giving a clear stabilizing effect of pyrene superimposed on the destabilizing influence of the 2′‐carbamate group, while even the spermine carbamate, which should carry three positive charges under physiological pH, is only negligibly stabilizing in just one most favorable situation.…”

Microenvironmental Effect of 2′-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA

“…In previous reports, A-form helix structure of RNA duplex was essential for a 2′-pyrene modified oligonucleotide probe for efficient sensing of target RNAs. ,, In order to display the overall structure of the duplexes and the local microenvironments around pyrene moiety, we measured circular dichroism (CD) spectra of all the duplexes between ONs and the complementary RNA, as well as RNA/RNA and DNA/RNA duplexes (Figure ). The CD spectra of ON-OH/RNA duplex and ON-OMe/RNA duplex were very close to that of the unmodified RNA duplex (antiRNA/RNA) with a large positive peak at 260 nm and a large negative peak at 210 nm, a typical CD spectrum of A-form double helix structure.…”

“…Fluorescent oligonucleotide probes with pyrene modification at different positions of nucleotides (such as nucleobases, sugar moieties, and phosphate) have been widely investigated. − ,− Oligonucleotide probes with 2′-pyrene modification can sensitively detect local mismatched nucleotides. − Yamana, Hrdlicka et al repectively designed a 2′- O -(1-pyrenylmethyl)­uridine modified RNA oligonucleotide probe, which showed a large hybridization-induced increase in fluorescence intensity upon binding to a complementary RNA target. However, full substitution of ribonucleotides with deoxynucleotides of the probe caused the loss of pyrene fluorescence enhancement when binding to the same RNA target. ,,, By model building and energy minimization, Yamana and co-workers indicated that the pyrene moiety in RNA oligonucleotides was located in the minor groove of RNA/RNA double helix, whereas the pyrene moiety in DNA oligonucleotides was intercalated into the DNA/DNA double helix. , The pyrene intercalated in DNA double helix is quenched by neighboring nucleobases via photoinduced electron transfer (PET) from the excited pyrene to nucleobases, but the pyrene located outside of the RNA double helix is free from the π-stacking interaction with nearby nucleobases and emits stronger fluorescence . Ostergaard and Hrdlicka proposed that pyrene moiety was more likely to intercalate into B-form duplex than into A-form duplex .…”