Molecular Mechanisms of Antisense Oligonucleotides
Stanley T. Crooke
In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry
and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has
focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I
believe that the art of excellent science is to frame large important questions that are perhaps not immediately
answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that
begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step
research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new
things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have
had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides,
manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular
pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of
scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of
the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is
to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.
Keywords: RNase H, oligonucleotide, antisense, mechanisms, RNA, DNA
Introduction
T
hroughout my career, my primary research interests
have focused on understanding the molecular mecha-
nisms by which drugs work, molecular pharmacology. Early
in my career, I focused on the molecular mechanisms of an-
tineoplastic drugs such as bleomycin, cisplatinum, and an-
thracyclines [1–3]. I then focused on more traditional receptor
biological questions with a particular emphasis on leukotriene
receptor signaling pathways and other G-protein-coupled re-
ceptors [4]. In 1987 when I became interested in the notion of
antisense technology, I returned to my roots in RNA bio-
chemistry and began work to understand how oligonucleo-
tides behave in biological systems. Since 1989, my research
has focused primarily on this topic, although I have been in-
volved in most areas of research in antisense technology.
I believe that the art of excellent science is to frame large
important questions that are perhaps not immediately an-
swerable with existing knowledge and methods, and then
conceive a long-term (multiyear) research strategy that be-
gins by answering the most pressing answerable questions on
the path to the long-term goals. Then, a step-by-step research
pathway that will address the strategic questions posed must
be implemented, adjusting the plan as new things are learned.
This is the approach we have taken at Ionis Pharmaceuticals
(formerly, Isis Pharmaceuticals). Obviously, to create anti-
sense technology, we have had to address a wide array of
strategic questions, for example, the medicinal chemistry of
oligonucleotides, manufacturing and analytical methods,
pharmacokinetics and toxicology, as well as questions about
the molecular pharmacology of antisense oligonucleotides
(ASOs). Each of these endeavors has consumed nearly three
decades of scientific effort, is still very much a work-in-
progress, and has resulted in hundreds of publications. That
progress has also been chronicled in a number of books that I
have edited [5–8]. (Although it is clearly time to edit a new
volume on antisense technology.) As a recipient of the
Lifetime Achievement Award 2016 granted by the Oligo-
nucleotide Therapeutic Society, in this note, my goal is to
summarize the contributions of my group to the efforts to
understand the molecular mechanisms of ASOs.
Phases of Antisense Drug Action
The molecular events that result in the activities of ASOs can
be divided into three phases: prehybridization, hybridization,
Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, California.
ª Stanley T. Crooke, 2017; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative
Commons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use,
distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
NUCLEIC ACID THERAPEUTICS
Volume 27, Number 2, 2017
Mary Ann Liebert, Inc.
DOI: 10.1089/nat.2016.0656
70
and posthybridization. In my laboratory, we have attempted to
address all three phases. In the prehybridization phase, an ASO
must enter the cell, distribute within the cell, and achieve ef-
fective concentrations at the target RNA site(s). It must then
sort through the cellular nucleic acid sequence space to hy-
bridize to its cognate site. The concentrations of the cognate
site are extremely low relative to total cellular RNA and cer-
tainly relative to conditions used in in vitro hybridization as-
says. Hybridization to the cognate site in the cell is a much more
complex process than in a test tube and must involve interac-
tions with proteins, such as Ago2, or other cellular components
that facilitate hybridization [9]. Once the ASO is bound to its
cognate site, depending on the chemical design of the ASO, a
variety of events may be induced that alter the target RNA to
achieve the desired pharmacological outcome [10].
One of the more interesting features of the molecular phar-
macology of ASOs is that the kinetics are remarkably slow. We
have characterized the kinetics of the major steps in the mo-
lecular pharmacology of RNase H1 activating ASOs [11]
showing that the onset of action occurs about two hours after
transfection, with 60, 20, and 40 min required for intracellular
distribution, RNA sequence searching and hybridization to the
cognate site and RNase H1 recruitment and cleavage, respec-
tively. The recruitment of RNase H1 and subsequent RNase
H1-mediated cleavage of the target RNA increase the degra-
dation rate of the target RNA by 2- to 4-fold compared with the
intrinsic rate of cellular RNA degradation (2.0 or 2.4 kb/deg-
radation rate in the cytoplasm or nucleus, respectively) (Fig. 1).
Prehybridization Events
One of our long-term goals has been to understand the events
that take place before hybridization at the target sequence in
RNA by the ASO. Clearly to hybridize to the ‘‘receptor’’ se-
quence, effective concentrations in the region, in which the
target RNA resides, must be achieved. We began by cataloging
the major intracellular proteins that bind phosphorothioate
(PS) ASOs using an affinity capture method to identify the
proteins in cellular homogenates involved in those interactions
[12]. Somewhat surprisingly, only 58 proteins were identified
that bind PS ASOs. The affinity capture method we have used
would not be expected to identify proteins with low affinity for
PS ASOs or proteins that are present in lo w concentrations.
Nor is it likely that the method would identify proteins that
bind only when complexed with other proteins or other
cellular components. However, since we are primarily in-
terested in bulk movements of PS ASOs in cells, this catalog
of proteins likely constitutes most of the abundant proteins
of interest and is certainly a good starting point.
Not surprisingly, many of the proteins that bind PS ASOs
contain nucleic acid-binding domains or are chaperone pro-
teins. However, a number of proteins that might not have been
expected to bind PS ASOs were identified, for example, an-
nexin A2 [13]. Then, by reducing and overexpressing various
proteins, we identified several proteins that either reduce or
enhance the potency of PS ASOs. We have characterized the
mechanisms accounting for the effects on antisense activity of
a number of these proteins. Some, through various mecha-
nisms, inhibit ASO activity, for example, by competing with
RNase H1 for binding to the RNA-ASO heteroduplex [14].
Others increase ASO activity by various mechanisms, for ex-
ample, alteration of subcellular localization [15]. Other pro-
teins affect ASO cellular toxicities, for example, members of
the drosophila behavior human splicing (DBHS) family in-
cluding, PSF and P54nrb [16]. Still others, such as HSP90 [17],
have effects for which the mechanisms remains to be defined.
Next, we identified the major sites of subcellular accu-
mulation and characterized the kinetics, with which these
sites accumulate PS ASOs after transfection, free uptake, and
electroporation [12,13,15,18,19]. By coupling this informa-
tion with the time course of drug action of PS ASOs, we were
able to associate various sites of accumulation with the onset
of activity, peak effects, and duration of effects of PS ASOs.
Reduction of specific proteins that caused changes in sub-
cellular localization of PS ASOs then supported the identi-
fication of subcellular sites, at which PS ASOs are active, or
inactive. For example, paraspeckles, PS bodies, P bodies, and
lysosomes are all sites, in which these ASOs are inactive
[12,15]. Two particularly informative examples are PS bod-
ies and paraspeckles. PS bodies form in the nucleus when
TCP1b binds PS ASOs. Reducing TCP1b resulted in reduc-
tion of PS bodies and PS ASO activity. Similarly, we showed
that PS ASOs can displace a long noncoding RNA, NEAT1,
and form functional paraspeckles in the nucleus, and these
paraspeckle proteins inhibit PS ASO activities [18]. Thus, we
have a full catalog of intracellular sites, in which ASOs ac-
cumulate over time after transfection, free uptake, and elec-
troporation and the proteins responsible for accumulation of
PS ASOs at the various sites (Fig. 2).
We then applied the same methodology to characterize the
major interactions of PS ASOs at the cell surface [12]. Al-
though much of this work is still in the process of being
published, we believe that the main cell surface interactions
of PS ASOs are once again with proteins. We have identified
new cell surface proteins that bind PS ASOs and confirmed
the interactions of a number of proteins previously reported
to bind to these agents [12,20,21]. We have also identified
key membrane lipids that appear to affect, although indi-
rectly, interactions with cell surface proteins (S. Wang et al.,
unpublished data) and shown that the decision to enter pro-
ductive or nonproductive subcellular distribution pathways is
likely made at the cell surface. Finally, we have begun to
characterize the processes, by which PS ASOs distribute in-
tracellularly, for example, we have shown that PS ASOs are
released mainly from late endosomes and this leads to ASO
activity (S. Wang et al., unpublished data).
Thus, progress over the last few years provides a detailed
road map of where in the cell ASOs accumulate, how they get
to those sites, and the sites that appear to be associated with
ASO activity. We know that proteins determine the fate of PS
ASOs at the cell surface and within the cell. We have a solid
understanding of the kinetics of PS ASO distribution in cells
and opportunities to enhance ASO performance by using this
information in the design of new PS ASOs that more effectively
take advantage of proteins that contribute to ASO activities.
Hybridization
In principle, PS ASOs of any chemistry should not be
expected to hybridize to their cognate sequences in cells [9].
For most ASOs, this step remains very much a black box, as
despite years of effort, proteins or lipids that might facilitate
hybridization have not been identified. The single exception,
however, serves as an excellent model. Ago2 binds the guide
MOLECULAR MECHANISMS OF ANTISENSE OLIGONUCLEOTIDES 71
FIG. 1. Rates of steps in RNase H1 activating ASOs activity [11]. (A) After transfection, about 60 min is required for
intracellular distribution of ASOs. In both the nucleus and cytoplasm, about 20 min are required to screen nucleic acid
sequence and bind to the cognate site. Approximately 40 min are then required to recruit RNase H1 and achieve measurable
RNA target reduction. (B) The transcription and splicing rates for the wild-type SOD1 construct and a mutant (187) with
substantially reduced splicing. (C) An effective RNase H1 activating ASO approximately doubles the intrinsic rate of
cellular RNA degradation. (D) The concentration of RNAse H1 is rate limiting. Over-expression of Escherichia coli RNase
H1 again doubles the rate reduction induced by an effective RNase H1 ASO. ASO, antisense oligonucleotide.
(Continued/)
72
FIG. 1. (Continued).
73
strand of double-stranded siRNA and single-strand RNAi
molecules and ‘‘preorganizes’’ the ASO, thus, facilitating
binding to the cognate site in the target RNA [9]. Thanks to
the work of many laboratories, a great deal is understood
about how Ago2 binds oligoribonucleotides and facilitates
hybridization [9]. The research continues to identify other
factors (proteins, lipids) that may facilitate the hybridization
of other chemical classes of oligonucleotides. However, we
do understand factors that influence both potency and spec-
ificity of the potential hybridization events. We know that
RNA structure is a major determinant while only rarely do
proteins that are bound to RNA targets alter ASO activity
[22]. We know that the number of copies of target RNAs is
irrelevant as is the RNA half-life, except for rapidly cleared
RNAs such as cMyc [14,22,23]. We also know that non-
promiscuous repeats are present in many pre-mRNAs and
these make excellent sites for ASO activity [24]. As we make
additional progress in understanding prehybridization events,
we hope that we will identify the cellular factors that enhance
the hybridization of ASOs of various chemical compositions,
similar to the effects of Ago2 with RNA-like ASOs [9].
Posthybridization Mechanisms
Depending on the design of the ASO, after hybridization a
number of mechanisms can be exploited to degrade, disable,
or modify the target RNA to achieve the desired pharmaco-
logic effects. This has also been one of the significant re-
search interests of my laboratory [5,10].
RNase H1
Mammalian cells contain two RNase H enzymes, H1 and
H2, which cleave RNA only when it is a DNA-RNA het-
eroduplex [25]. As RNase H1 cleavage of target RNAs has
proven to be a robust and versatile mechanism, by which
DNA-like ASOs induce pharmacological effects, we have
invested substantially in understanding the RNase H enzymes
and how they participate in ASO activities. We showed that
although RNase H2 is much more abundant than RNase H1 in
mammalian cells, in intact cells, only RNase H1 participates
in ASO activities [25–27]. This is probably because RNase
H2 is tightly bound to chromatin while RNase H1 is present
in the nucleus, cytoplasm, and mitochondria. We character-
ized the structure and enzymological properties of the RNase
H1 [28–37] and evaluated how the enzyme is regulated. We
showed that RNase H1 participates with a binding partner, a
protein involved in a number of RNA metabolic activities,
P32, and that in some circumstances P32 appeared to en-
hance the specificity of cleavage of RNase H1 [32]. These
studies supported optimization of chimeric ASOs designed
to activate RNase H1. To confirm that RNase H1 is the
only nuclease responsible for the antisense activity of DNA-
like ASOs, we constructed hepatocytes-specific RNase H1
FIG. 2. Subcellular distribution of PS ASOs and cleavage of target RNA by RNase H1. PS ASO distribute within the cell
via interactions with specific proteins. Once the ASO has bound to its cognate site in the target RNA, RNase H1 (and P32) is
recruited to degrade the target RNA. Post-RNase H1 cleavage and the fragments are processed by normal cellular RNA
degradation pathways. PS, phosphorothioate.
74 CROOKE