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.

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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Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask.