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.

The design, synthesis, and properties of a new pyrene excimer-forming probe of DNA have been described. 2,2-(Aminomethyl)propanediol was converted by the reaction with 1-pyrenebutylic acid to bis-pyrene-modified propanediol as a fluorescent non-nucleosidic linker. The bis-pyrene-modified linker can be incorporated via phosphoramidite chemistry into the 5'-terminal or internal positions of oligonucleotides (ODNs). The terminally modified ODNs showed almost similar affinity for complementary DNA when compared with the corresponding unmodified ODNs. The duplexes containing the bis-pyrene in the main chain exhibited higher melting temperatures relative to the corresponding duplexes containing propanediol linker at the same position. The UV and CD spectral studies indicate that the stacking interactions between the pyrene and DNA bases occur in the internally modified duplex and do not in the terminally modified duplex. The bis-pyrene modified linker itself displays excimer (E at 480 nm) and monomer (M at 380 nm) emission in a quantum yield (QY) of 0.17 and the E/M intensity ratio of 15. Incorporation of this linker into the terminal or internal positions of ODNs reduced the QY (0.003-0.009) and the E/M ratio (0.3-0.8). While small changes in the QY and E/M ratio was obtained in binding of the internally labeled ODNs to DNA, up to 27-fold increase in the QY and 17-fold increase in the E/M ratio was observed upon hybridization of the terminally labeled ODNs with DNA. The excimer and monomer fluorescence changes were found to be sensitive to a mismatch base present in the target DNA. The bis-pyrene-modified ODNs thus provide a sequence-sepcific fluorescent probe of DNA.

A helical pyrene-array was formed by incorporation of multiple pyrenes into the sugar residues of duplex RNA leading to strong excimer fluorescence.

We describe a new strategy for multipyrene modification of RNA sequences to form unique structures of pyrene aromatic arrays, the "pyrene-zipper array", on duplex RNA that exhibits remarkably strong excimer fluorescence.

In designing antisense molecules, it is crucial to detect the regions of the target RNA where the antisense molecule can hybridize. [1] To date, several studies have focused on the detection of these regions. The studies include theoretical calculations, [2] enzymatic digestion, [3] and DNA arrays, [4] and all of them have achieved a partial success. However, none of these methods have the potential to be applied to cells in which various endogenous molecules interact with RNA in a variety of manners.Solution-based fluorescence methods should be suitable to deduce the RNA structure in vitro, because these methods primarily function without disturbing the RNA structure. One of the most useful fluorescence methods is based on fluorescence resonance energy transfer (FRET), which provides information about the distance between two chromophores. A three-dimensional structure of a ribozyme was revealed by such methods. [5] A molecular beacon, which is based on FRET, was applied to the detection of RNA in living cells. [6] In another method, various types of pyrene-conjugated oligonucleotides have been used for the detection of both RNA and DNA sequences. [7] The methods were based on excimer formation upon the formation of hybrid molecules and could not discriminate RNA from DNA. As a final goal, we intend to elucidate the detailed structure of RNA in living cells, and have developed a novel bispyrene-conjugated 2'-O-methyloligonucleotide probe (OMUpy2; Figure 1). The probe is also expected to be feasible for use in analysis of intracellular RNA traffic and consequently to evaluate RNA functions.OMUpy2 was synthesized and purified according to the reported procedure with slight modifications. [8] To evaluate whether OMUpy2 can detect single-stranded regions of native-folded RNA molecules, we used E. coli 5S-rRNA, which contains 120 nucleotides (nt). 5S-rRNA is a component of the 50S ribosome subunit and the secondary structure has been reported ( Figure 2). [9] We chose the regions for probing with OMUpy2 as follows; OMUpy2-I was complementary to region I (38±47 nt), and OMUpy2-II and III were complementary to regions II (45±56 nt) and III (106±115 nt), respectively.The fluorescence characteristics of OMUpy2 in the absence and presence of the equimolar solution of its complementary oligodeoxyribonucleotide (cODN) or oligoribonucleotide (cORN) are shown in Figure 3. Compared with 1-pyrenylmethanol, the structured emission caused by the pyrene of the OMUpy2-I around 380 nm was largely quenched, and a weak, broad, and structureless emission around 480 nm was observed. It is likely that the monomeric pyrene emission was quenched by the intercalation of the pyrene between nucleobases. [8,10] On the other hand, when the OMUpy2-I was hybridized with cORN, the broad structureless fluorescence band around 480 nm was increased by 43.7-fold compared with the corresponding single-stranded OMUpy2-I, and the structured emission around 380 nm was substantially unchanged. The mechanism of the increment in fluorescence intensity...

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RNA oligomers having multiple (2 to 4) pyrenylmethyl substituents at the 2'-O-sugar residues were synthesized. UV-melting studies showed that the pyrene-modified RNAs could form duplexes with complementary RNA sequences without loss of thermal stability. Absorption, fluorescence, and circular dichroism (CD) spectra revealed that the incorporated pyrenes projected toward the outside of A-form RNA duplexes and assembled in helical aromatic arrays along the minor grooves of the RNA duplexes. Results of computer simulations agreed with the assembled structures of the pyrenes. The helical pyrene arrays exhibited remarkably strong excimer fluorescence, which was dependent on the sequence contexts of RNA duplexes.

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