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Determination of potent antisense oligonucleotides in vitro by semiempirical rules
Naoki Yanagihara1, Hisashi Tadakuma, Yo Ishihama
1Major in Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Abstract:
The selection of effective antisense target sites on a given mRNA molecule is a major problem in the detection of target mRNA in oligonucleotide arrays. In general, antisense oligodeoxynucleotides (asODNs) of about 10-20 nucleotides (nt) in length are used. However, the demand for predicting the sequence of potent asODNs much longer than those mentioned above has been increasing. Here, we prepared 40-nt asODNs directed against fluorescence-labeled green fluorescent protein (GFP) mRNA and quantified their hybridization efficiencies by fluorescence microscopy. We found that the hybridization efficiency depended on the TC content or the minimum free energy of the asODNs. On the basis of these findings, a semiempirical parameter called accessibility score was introduced to predict the potency of asODNs. The results of this study aided in the development of an effective two-step procedure for determining mRNA accessibility, namely, the computer-aided selection of asODN binding sites using an accessibility score followed by an experimental procedure for measuring the hybridization efficiencies between the selected asODNs and the target mRNA by fluorescence microscopy.
