Rational design and rapid screening of antisense oligonucleotides for prokaryotic gene modulation

Antisense oligodeoxynucleotides (oligos) are widely used for functional studies of both prokaryotic and eukaryotic genes. However, the identification of effective target sites is a major issue in antisense applications. Here, we study a number of thermodynamic and structural parameters that may affect the potency of antisense inhibition. We develop a cell-free assay for rapid oligo screening. This assay is used for measuring the expression of Escherichia coli lacZ, the antisense target for experimental testing and validation. Based on a training set of 18 oligos, we found that structural accessibility predicted by local folding of the target mRNA is the most important predictor for antisense activity. This finding was further confirmed by a direct validation study. In this study, a set of 10 oligos was designed to target accessible sites, and another set of 10 oligos was selected to target inaccessible sites. Seven of the 10 oligos for accessible sites were found to be effective (>50% inhibition), but none of the oligos for inaccessible sites was effective. The difference in the antisense activity between the two sets of oligos was statistically significant. We also found that the predictability of antisense activity by target accessibility was greatly improved for oligos targeted to the regions upstream of the end of the active domain for β-galactosidase, the protein encoded by lacZ. The combination of the structure-based antisense design and extension of the lacZ assay to include gene fusions will be applicable to high-throughput gene functional screening, and to the identification of new drug targets in pathogenic microbes. Design tools are available through the Sfold Web server at .

[1]  H. Allawi,et al.  Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase. , 2001, RNA.

[2]  N. Dean,et al.  Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters. , 1994, The Journal of biological chemistry.

[3]  S. Freier,et al.  Effects of RNA secondary structure on cellular antisense activity. , 2000, Nucleic acids research.

[4]  Ye Ding,et al.  Sfold web server for statistical folding and rational design of nucleic acids , 2004, Nucleic Acids Res..

[5]  E. Southern,et al.  Determining the influence of structure on hybridization using oligonucleotide arrays , 1999, Nature Biotechnology.

[6]  G. Trainor,et al.  Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries. , 1996, Nucleic acids research.

[7]  S. Withers,et al.  Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli. , 1992, The Journal of biological chemistry.

[8]  A D Tsodikov,et al.  Thermodynamic calculations and statistical correlations for oligo-probes design. , 2003, Nucleic acids research.

[9]  Yedy Israel,et al.  Tetranucleotide GGGA Motif in Primary RNA Transcripts , 1998, The Journal of Biological Chemistry.

[10]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[11]  E. Southern,et al.  Antisense oligonucleotides selected by hybridisation to scanning arrays are effective reagents in vivo. , 2001, Nucleic acids research.

[12]  D. Cunningham,et al.  Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma. , 2000, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[13]  N. Sugimoto,et al.  Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. , 1995, Biochemistry.

[14]  D. Ecker,et al.  Implication of RNA structure on antisense oligonucleotide hybridization kinetics. , 1992, Biochemistry.

[15]  B. Matthews,et al.  Three-dimensional structure of beta-galactosidase from E. coli. , 1994, Nature.

[16]  P. Zamecnik,et al.  Targeting the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase complex as a therapeutic strategy against tuberculosis: Proof of principle by using antisense technology , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Ye Ding.  Statistical and Bayesian approaches to RNA secondary structure prediction. , 2006, RNA.

[18]  C. Marwick,et al.  First "antisense" drug will treat CMV retinitis. , 1998, JAMA.

[19]  Juliano Timm,et al.  Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series , 1994, Journal of bacteriology.

[20]  R. E. Huber,et al.  Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (Escherichia coli). , 1990, Archives of biochemistry and biophysics.

[21]  E. Southern,et al.  Selecting effective antisense reagents on combinatorial oligonucleotide arrays , 1997, Nature Biotechnology.

[22]  Fluorescence-based detection of lacZ reporter gene expression in intact and viable bacteria including Mycobacterium species. , 1999, FEMS microbiology letters.

[23]  S. Freier,et al.  Antisense oligonucleotides inhibit intercellular adhesion molecule 1 expression by two distinct mechanisms. , 1991, The Journal of biological chemistry.

[24]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[25]  D. Turner,et al.  Predicting oligonucleotide affinity to nucleic acid targets. , 1999, RNA.

[26]  P. Zamecnik,et al.  Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate/glutamine cell wall structure, and bacterial replication. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[27]  M. Zuker.  On finding all suboptimal foldings of an RNA molecule. , 1989, Science.

[28]  C. Burge,et al.  Widespread selection for local RNA secondary structure in coding regions of bacterial genes. , 2003, Genome research.

[29]  M. Stephenson,et al.  Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Bennett,et al.  Amelioration of Chronic and Spontaneous Intestinal Inflammation with an Antisense Oligonucleotide (ISIS 9125) to Intracellular Adhesion Molecule-1 in the HLA-B27/β2 Microglobulin Transgenic Rat Model , 2002, Journal of Pharmacology and Experimental Therapeutics.

[31]  R. E. Huber,et al.  Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis. , 1990, The Journal of biological chemistry.

[32]  M. Phillips,et al.  Basic principles of using antisense oligonucleotides in vivo. , 2000, Methods in enzymology.

[33]  C. Lawrence,et al.  A statistical sampling algorithm for RNA secondary structure prediction. , 2003, Nucleic acids research.

[34]  E. Southern,et al.  Selecting optimal antisense reagents. , 2000, Advanced drug delivery reviews.

[35]  G Sczakiel,et al.  A theoretical approach to select effective antisense oligodeoxyribonucleotides at high statistical probability. , 1999, Nucleic acids research.

[36]  Ye Ding,et al.  A Bayesian Statistical Algorithm for RNA Secondary Structure Prediction , 1999, Comput. Chem..

[37]  Sverdrup,et al.  Antisense oligonucleotides: a systematic high-throughput approach to target validation and gene function determination. , 1999, Drug discovery today.

[38]  V. Patzel,et al.  Theoretical and Experimental Selection Parameters for HBV-Directed Antisense RNA Are Related to Increased RNA-RNA Annealing , 1997, Biological chemistry.

[39]  F. Celada,et al.  An immunological study of complementary fragments of beta-galactosidase. , 1974, Biochemistry.

[40]  W. Jacobs,et al.  Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene. , 1992, Journal of general microbiology.

[41]  Lei Tang,et al.  Clinical Potential of Respirable Antisense Oligonucleotides (RASONs) in Asthma , 2003, American journal of pharmacogenomics : genomics-related research in drug development and clinical practice.

[42]  David H Mathews,et al.  Revolutions in RNA secondary structure prediction. , 2006, Journal of molecular biology.

[43]  A D Tsodikov,et al.  Thermodynamic criteria for high hit rate antisense oligonucleotide design. , 2003, Nucleic acids research.

[44]  C. Lawrence,et al.  Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond. , 2001, Nucleic acids research.

[45]  Randall R. Sakai,et al.  Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries , 1998, Nature Biotechnology.

[46]  S. P. Walton,et al.  Prediction of antisense oligonucleotide binding affinity to a structured RNA target. , 1999, Biotechnology and bioengineering.

[47]  D. Spiller,et al.  Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA. , 2001, Nucleic acids research.

[48]  C. Lawrence,et al.  Clustering of RNA secondary structures with application to messenger RNAs. , 2006, Journal of molecular biology.

[49]  C. Stein.  Two problems in antisense biotechnology: in vitro delivery and the design of antisense experiments. , 1999, Biochimica et biophysica acta.

[50]  Doriano Fabbro,et al.  Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase , 1996, Nature Medicine.

[51]  C. Lawrence,et al.  RNA secondary structure prediction by centroids in a Boltzmann weighted ensemble. , 2005, RNA.

[52]  A D Tsodikov,et al.  Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity. , 2000, Nucleic acids research.

[53]  D. Turner.  Faculty Opinions recommendation of RNA secondary structure prediction by centroids in a Boltzmann weighted ensemble. , 2005 .