Bispyrene-Conjugated 2′-O-Methyloligonucleotide as a Highly Specific RNA-Recognition Probe

Abstract

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...

Biosensors Based on Biological Nanostructures

“…This was the idea of Yang et al in the construction of sensors for cations (Yang, Lin et al, 2001;Liao, Chen et al, 2002) when constructing a sensor for anions. Furthermore, since pyrene is a fluorophore that can easily form excimers, it has been used for the construction of sensors for DNA detection, as described by Mahara et al (Mahara, Iwase et al, 2002) as it needs the formation of pyreneconjugated oligonucleotides where excimers can occur or in sensor for protein-protein recognition, as described by Sahoo et al (Sahoo, Narayanaswami et al, 2000) Martins et al (Martins et al, 2011 in press) showed that peptide nanostructures containing pyrene derivatives sorbed onto them can sense distinct environments, with effect on fluorescence lifetimes, even when excimer are not formed. According to several authors, the challenge of developing these types of sensors is in the proposal of an all-serving-sensor.…”

Homogeneous DNA-detection Based on the Non-enzymatic Reactions Promoted by Target DNA

“…Mahara et al developed bispyrene probes that respond to RNA by excimer formation. 19 Kashida et al inserted two pyrene into ODNs with one or two intervening nucleotides to make the probes for deletion polymorphisms. 20 The ODN conjugate bearing two pyrenes on both of its terminus was synthesized by Fujimoto et al, as the MB based on excimer/monomer transformation.…”

Transcriptome analysis device based on liquid phase detection by fluorescently labeled nucleic acid probes

“…In order to overcome such disadvantages, our group has been proposing the direct detection of mRNA in the liquid phase (Mahara et al 2002). The detection technique has great advantages over the one in the solid phase in terms of the total process time.…”