A two unit antisense RNA cassette test system for silencing of target genes
Hilde M. Engdahl
1
Tord . H. Hjalt
0
1
E. Gerhart H. Wagner
1
0
Department of Microbiology, Uppsala University, Biomedical Center
, Box 581, S-75123 Uppsala,
Sweden
1
Department of Microbiology, Swedish University of Agricultural Sciences, Genetic Center
, Box 7025, Genetikvgen 1, S-75007 Uppsala,
Sweden
This communication describes a two unit antisense RNA cassette system for use in gene silencing. Cassettes consist of a recognition unit and an inhibitory unit which are transcribed into a single RNA that carries sequences of non-contiguous complementarity to the chosen target RNA. The recognition unit is designed as a stem-loop for rapid formation of longlived binding intermediates with target sequences and resembles the major stem-loop of a naturally occurring antisense RNA, CopA. The inhibitory unit consists of either a sequence complementary to a ribosome binding site or of a hairpin ribozyme targeted at a site within the chosen mRNA. The contributions of the individual units to inhibition was assessed using the lacI gene as a target. All possible combinations of recognition and inhibitory units were tested in either orientation. In general, inhibition of lacI expression was relatively low. Fifty per cent inhibition was obtained with the most effective of the constructs, carrying the recognition stem-loop in the antisense orientation and the inhibitory unit with an anti-RBS sequence. Several experiments were performed to assess activities of the RNAs in vitro and in vivo: antisense RNA binding assays, cleavage assays, secondary structure analysis as well as Northern blotting and primer extension analysis of antisense and target RNAs. The problems associated with this antisense RNA approach as well as its potential are discussed with respect to possible optimization strategies.
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Antisense strategies for gene silencing have attracted much
attention in recent years (1,2). The underlying concept is simple
yet (in principle) effective: antisense nucleic acids (NA) base pair
with a target RNA resulting in inactivation. Target RNA recognition
by antisense RNA or DNA can be considered a hybridization
reaction, although this view is sometimes clearly misleading (see
below). Since the target is bound through sequence
complementarity, this implies that an appropriate choice of antisense NA should
ensure high specificity. Inactivation of the targeted RNA can
occur via different pathways, dependent on the nature of the
antisense NA (either modified or unmodified DNA or RNA) and
on the properties of the biological system in which inhibition is to
occur, i.e. the sum of the metabolic activities that mediate inhibition.
Major differences between exogenous [antisense
oligo(deoxy)ribonucleotides; AONs] and endogenous antisense RNA strategies
lie in: (i) delivery of the inhibitor; (ii) presumed mode of binding
to the target RNA; (iii) details of the inhibitory reaction. AONs
are generally applied extracellularly and taken up, whereas antisense
RNAs are most often transcribed intracellularly, after transient or
stable introduction of an appropriate antisense gene (1,3). AONs
are designed to contain a low degree of secondary structure in
order to permit efficient hybridization, whereas antisense RNAs
are often larger and structurally more complex. The latter implies
that binding reactions, unlike the AON case, occur between
folded RNAs and therefore may follow different rules. Antisense
DNA approaches often rely on endogenous RNase H activity to
inactivate the target RNA. Numerous modifications of AONs
have been introduced, resulting in metabolically more stable
compounds and those with altered stereochemistry (see for
example 4). In contrast, antisense RNA-mediated silencing can
occur by duplex-dependent blockage of a ribosome binding site
(RBS) within an mRNA, duplex-dependent facilitated mRNA
decay, antisense RNA-induced premature termination of
transcription and cleavage of the mRNA by an antisense RNA with
ribozyme activity. Inhibition of gene expression by introduced
antisense RNA genes has been achieved in, for example, bacteria
(5), plants (6) and mammalian cells (7).
Numerous naturally occurring antisense RNA systems have
been found in bacterial accessory DNA elements, such as phages,
plasmids and transposons (for a review see 8). These systems are
useful sources of information regarding binding rate, specificity
and mechanisms of target RNA inactivation. The copy number
control circuit of bacterial plasmid R1 with its key elements CopA
(antisense RNA) and CopT (target) has been studied extensively.
Its efficiency, both in terms of binding rate and specificity (912),
prompted us to design antisense RNA cassettes that incorporate
favourable characteristics of this system. In particular, it is known
that stemloop II (SL II) of CopA suffices for kissing complex
formation with the target RNA and that formation of this transient,
but long-lived, intermediate is rate limiting for pairing (11).
The basic features (...truncated)