Determination of potent antisense oligonucleotides In Vitro by semiempirical rules

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 seq...

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Bibliographic Details
Published in:Journal of bioscience and bioengineering 2007-03, Vol.103 (3), p.270-277
Main Authors: Yanagihara, Naoki, Tadakuma, Hisashi, Ishihama, Yo, Okabe, Kohki, Funatsu, Takashi
Format: Article
Language:English
Subjects:
antisense oligodeoxynucleotides
ARN
Base Sequence
Binding Sites - genetics
Biological and medical sciences
Biotechnology
FLUORESCENCE
fluorescence microscopy
FLUORESCENCIA
Fundamental and applied biological sciences. Psychology
Genes, fos
Green Fluorescent Proteins - genetics
In Vitro Techniques
Microscopy, Fluorescence
Nucleic Acid Conformation
Nucleic Acid Hybridization
NUCLEOTIDE
NUCLEOTIDE SEQUENCE
NUCLEOTIDES
NUCLEOTIDOS
Oligodeoxyribonucleotides, Antisense - analysis
Oligodeoxyribonucleotides, Antisense - genetics
PCR
Recombinant Proteins - genetics
RNA
RNA, Messenger - chemistry
RNA, Messenger - genetics
SECUENCIA NUCLEOTIDICA
Sequence Analysis, DNA - methods
SEQUENCE NUCLEOTIDIQUE
Thermodynamics
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Summary: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.
ISSN:1389-1723
1347-4421
DOI:10.1263/jbb.103.270