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2 Phosphorylating the 5« Termini of Oligonucleotides 10.

To direct the activity of RNase H in vivo, methylphosphonate:phosphodiester chimaeric antisense oligonucleotides were synthesised to incorporate the sites demonstrating cleavage in vitro within the internal phosphodiester element of the chimaera (Table ). Methylphosphonate oligodeoxynucleotide derivatives are refractory to RNase H endonucleolysis. As little as a 6 base complementarity region of an unsubstituted phosphodiester oligomer to target is sufficient to elicit human RNase H1 activity (), therefore a central 8 base phosphodiester element was included in the design of the antisense compounds.

7 Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers 9.

The identification of sites demonstrating complex cleavage can be determined by the temporal monitoring of the development of the RNA fragmentation profile. For example, the secondary and tertiary structure of the transcript may be disrupted by oligonucleotide stand displacement or effectively null sites may become susceptible to secondary cleavage once unmasked by a primary cleavage event. Further complications may arise with the potential for the multiple turnover of oligonucleotides in combinatorial assays. This has been implicated at sites of high affinity and hybridisation rate where the library concentration is theoretically limiting (). The identification of target sites that are susceptible to such anomalous cleavage should help to prevent the design and application of independent antisense compounds that demonstrate relatively little activity in vivo. Target sites demonstrating such a false positive reaction can be selected against when monitoring the evolution of the RNA fragmentation profile.

2 Oligonucleotide-directed Mutagenesis of Single-stranded DNA 13.

9 Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension 9.

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.

Antisense oligonucleotides (ODNs) are designed to hybridise to a specific mRNA sequence in order to prevent translation (). Antisense reagents can be readily synthesised, are sequence specific and have the potential to down-regulate the expression of any mRNA of known sequence. They are useful for determining protein function and interactions (–). The potential of antisense for therapy has been developed using animal models and selective clinical trials (,). Therapeutic agents have now been approved ().

DNA strand breaks using a random oligonucleotide-primed synthesis ..

3 Purification of Radiolabeled Oligonucleotides by Precipitation with Ethanol 10.

For example if 1/10 of the RNA prep was used to make cDNA and 1/100 of the cDNA was used in each qPCR then spike the sample with 107 copies of the synthetic RNA oligo.

The ability of the in vitro assay to predict oligonucleotide efficacy was superior to either computationally based RNA structural predictions, ΔG calculations and in vivo trial and error methodologies. Such results are illustrative of the potential of the coparallel competitive screening of the in vitro selection methodology to predict effective antisense oligonucleotide sequences.

4 Purification of Radiolabeled Oligonucleotides by Precipitation with Cetylpyridinium Bromide 10.
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Summary: random oligonucleotide-primed synthesis (ROPS) assay [13]

The DIG System is an effective system for the labeling and detection of DNA, RNA, and oligonucleotides. The protocols for labeling with digoxigenin (Figure 1) and subsequent detection are based on well established, widely used methods. DNA, RNA, and oligonucleotide probes are labeled according to the methods (usually enzymatic) used for preparing radioactive probes. Hybridization of digoxigenin-labeled probes (e.g., to target DNA or RNA on a Southern or Northern blot) is also carried out according to standard protocols, except that a special blocking reagent is used to eliminate background. The signal on the nucleic acid blot is detected according to the methods developed for western blots. The incorporation and spacing of digoxigenin in DNA, RNA, and oligonucleotides can be varied by using different labeling protocols.

Random Oligonucleotide–Primed Synthesis; ..

Fluorescently tagged chimaeric methylphosphonodiester:phosphodiester ODNs were synthesised as previously described (). The 20mer ODNs were composed of 6MP:8PO:5MP internucleoside linkages. Oligonucleotides were synthesised from phosphoramidites and methylphosphonamidites (Cambio, Cambridge, UK) using the slow 1 µmol cycle (version 1.23) on an Applied Biosystems 381A DNA synthesiser. A linker group [5′-amino-modifier C6-TFA (Glen Research)] was included as the last cycle on the synthesiser. Base deprotection was achieved as described by Hogrefe et al. (,) extended over 72 h. Fluorescent tagging was achieved using 5(6)-carboxyfluorescein N-hydroxysuccinimide ester (Molecular Probes Inc., Cambridge, UK) ().

Random oligonucleotide-primed synthesis

Random combinatorial oligonucleotide libraries of 10 () or 11 nt () have been used to identify sites accessible to heteroduplex formation, from which the sequences of 20 nt antisense oligonucleotide compounds have been extrapolated. However, optimisation of antisense oligonucleotide efficacy is not only dependent upon the target site, but is also dependent upon oligonucleotide length. We have taken an experimental approach that will permit the in vitro identification of accessible sites upon any mRNA transcripts that are amenable to the optimal hybridisation of antisense oligonucleotides, with respect to oligonucleotide length and efficacy.

mRNA Using Random Oligonucleotide Primers 9

Chapter 9Preparation of Radiolabeled DNA and RNA Probes 9.11 Random Priming: Radiolabeling of Purified DNA Fragments by Extension of Random Oligonucleotides 9.

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