Review
Morpholino antisense oligomers: the case for an RNase H-independent structural type

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Abstract

RNase H-competent phosphorothioates (S-DNAs) have dominated the antisense field in large part because they offer reasonable resistance to nucleases, they afford good efficacy in cell-free test systems, they can be targeted against sites throughout the RNA transcript of a gene, and they are widely available from commercial sources at modest prices. However, these merits are counterbalanced by significant limitations, including: degradation by nucleases, poor in-cell targeting predictability, low sequence specificity, and a variety of non-antisense activities. In cell-free and cultured-cell systems where one wishes to block the translation of a messenger RNA coding for a normal protein, RNase H-independent morpholino antisense oligos provide complete resistance to nucleases, generally good targeting predictability, generally high in-cell efficacy, excellent sequence specificity, and very preliminary results suggest they may exhibit little non-antisense activity.

Section snippets

RNase H cleavage: origins of the broad acceptance of S-DNAs

A key requirement for effective antisense oligos is that they remain intact for many hours in the extracellular medium and within cells. The methylphosphonate-linked DNA analogs developed by Miller and Ts’o in the late 1970s constituted a major advance in the emerging antisense field by providing the first antisense type having good stability in biological systems [1]. However, concerns subsequently developed that methylphosphonates might be inadequate for many antisense applications,

Limitations of S-DNAs

With continued study of S-DNAs it is now widely recognized that their good efficacy and targeting versatility are counterbalanced by a variety of disadvantages.

Efficacy

A key property of the RNase H-competent S-DNAs which led to their broad adoption by the antisense community was their greatly increased efficacy (likely a consequence of their RNase H competency) relative to methylphosphonates. However, since then at least two RNase H-independent types (PNAs [16] and morpholinos [17] shown in Fig. 1) have been developed which often match or exceed the efficacy of S-DNAs in a cell-free translation system when said oligos are targeted to sequences in the region

Predictable targeting

A problem which has plagued antisense research with S-DNAs is the difficulty of predicting which antisense sequences will be effective in cells. As a consequence, multiple S-DNAs may need to be prepared and empirically tested in order to identify an oligo with good in-cell activity [23], [24]. Further, when one does find an effective oligo through such an empirical search it is not unusual to find that it is targeted in a region of the RNA, such as the 3′ untranslated region [25], which one

Positive test system

A long standing limitation in antisense research has been that the available test systems rely on down-regulation. This includes such a crude measure as inhibition of cell growth, as well as assays for inhibition of the synthesis or activity of a particular protein, and assays for degradation of a particular RNA (useful only with RNase H-competent oligos). The difficulty in these negative test systems is that a variety of non-antisense effects can also lead to down-regulation or what appears to

Delivery into cultured cells

In the 1980s a number of antisense experiments with cultured cells suggested that antisense oligos readily enter cultured animal cells and have good access to their targeted RNAs therein. These early results led scientists in the antisense field to believe that antisense oligos, and particularly non-ionic types, could readily diffuse across cell membranes. However, by the early 1990s reality reared its ugly head in the form of a number of careful experiments whose results indicated that neither

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