Review

Bioana­lysis of siRNA and oligonucleotide

therapeutics in biological fluids and tissues

This article summarizes bioanalytical avenues for the determination of siRNA and oligonucleotide therapeutics,
with an emphasis on hybridization methods. Aspects of the chemistry and delivery of investigational oligonucleotide
therapeutics are considered. The nature of the oligonucleotide under investigation will dictate the best analytical
course of action; each method has its advantages and disadvantages, depending upon the oligonucleotide test article
and the anticipated toxicokinetic and pharmacokinetic study parameters. Stringent method development and specific
validation criteria are essential to attain the best quality results in support of a regulatory filing.

Oligonucleotide (OGN) biopharmaceuticals are
currently being investigated at preclinical and
clinical stages for various disease indications. To
date, fomivirsen, an antisense oligonucleotide
(ASO) phosphorothioate (PS) targeted at cyto-
megalovirus retinitis, and pegaptanib, an anti-
angiogenic pegylated aptamer for the treatment
of neovascular age-related macular degeneration,
have been approved by the US FDA in 1998 and
2004, respectively [301,302] .
Investigational nucleic acid-based therapeu-
tics occupy an increasingly important space
in the biopharmaceutical drug-discovery and
-development landscape. It is expected that
more OGN drugs will reach the market, con-
sidering the relative potency of later generation
compounds, such as siRNA [1] , the capacity
for controlled manufacturing of OGN and
the appealing prospect of simple and effective
drug design.
Oligonucleotide drug candidates require
robust bioanalytical assays for their determina-
tion in increasingly complex biological matrices,
such as skin, colon or brain tissue. In addition,
sensitive assays are required for the lower thera-
peutic doses that more effective drug regimens
and targeted delivery will bring about. The
bioanalytical method platform is also carefully
selected based on the structure and function of
the therapeutic OGN. Numerous classes of
therapeutic OGN compounds exist, including:
n siRNA [2,3]
n ASO OGNs [4]
n Aptamers [5]
n Immunomodulatory OGNs (IMOs) [6,7]
n miRNA-blocking OGNs [8] Guy A Tremblay1 &
n RNA decoys [9] Philip R Oldfield1†
†
Author for correspondence
n Splice switching/exon skipping OGNs [10] 1
Immunochemistry, Charles
n Ribozymes and DNA enzymes [11] River Preclinical & Clinical
Services, 22022
Current investigational therapeutic OGNs are Transcanadienne, Senneville,
typically either siRNA, ASO, IMOs or aptamers. QC H9X 3R3, Canada,
ASOs are complementary to a targeted, disease- Tel.: +1 514 630 8263
associated RNA molecule, generally a mRNA. Fax: +1 514 630 8230
The classical ASO mechanism is based on trig- E-mail: philip.oldfield@crl.com
gering RNase H cleavage of the ASO–RNA tar-
get duplex [4] . ASOs pioneered the therapeutic
Antisense
OGNs, and a number of OGN chemistries were
Complement strand of a
developed for ASO that have been adapted for targeted nucleic acid,
use with other OGNs. typically mRNA
Toll-like receptors (TLRs) are pattern­
recognition receptors and recognize molecules Aptamer
common to bacteria and viruses. TLR9 is
Oligonucleotide ligand obtained
expressed in two types of cells of the immune by in vitro evolution
system: plasmacytoid dendritic cells and B cells.
IMOs containing unmethylated CpG motifs
siRNA
mimic nonmammalian DNA and therefore bind
19–25-nucleotide long
to the TLR9, thereby inducing proinflammatory double-stranded RNA
cytokines and Th1-type immune responses [6,7] . molecules involved in the
They are agonists of the innate immune system. RNAi pathway
Aptamers are OGNs that have been selected via
in vitro molecular evolution techniques [5] . They Therapeutic oligonucleotide
are ligands for specific molecular targets, mostly A class of oligonucleotides
proteins of therapeutic relevance. Aptamers are consisting of approximately
typically selected using an iterative molecu- 10–40 DNA, RNA or modified
nucleic acid monomers used for
lar evolution technique known as Systematic
therapeutic applications
Evolution of Ligands by Exponential enrich-
ment (SELEX) [12] . They are habitually designed
to work at the extracellular level.
Recent advances with RNAi and siRNA syn-
thetic compounds have fueled interest in thera-
peutic OGNs in recent years. Andrew Z Fire and

10.4155/BIO.09.66 © 2009 Future Science Ltd Bioanalysis (2009) 1(3), 595–609 ISSN 1757-6180 595
Review | Tremblay & Oldfield
Pharmacokinetics
Describes the relationship
between mechanisms of drug
absorption, distribution and
elimination over time
596
Craig C Mello were awarded the 2006 Nobel
Prize for their discovery of RNAi gene silenc-
ing by double-stranded RNA [303] . RNAi is a
natural mechanism of RNA inhibition medi-
ated by small, double-stranded RNA molecules
of 19–25 nucleotides. The RNAi field expanded
fast within academia, where siRNA molecules
were designed to knock-down the expression of a
selected gene in cell culture. It has been reported
that a proper selection of the target sequence may
typically lead to a knockdown of approximately
75% of a given mRNA and its protein product
thereof [1] .
Synthetic siRNAs have been demonstrated to
target genes in vivo for multiple diseases, includ-
ing ovarian [13] and bone cancer [14] , hypercholes-
terolemia [15] , liver cirrhosis [16] , respiratory syn-
cytial virus [17] , hepatitis B virus [18] and human
papillomavirus [19] .
Delivery and modification are important
areas of focus for OGN drug development, since
they can improve upon aspects of tissue-specific
targeting, cell entry, stability and potency.
Improved cell-specific targeting and transfec-
tion efficiency will help to lower the effective
dose. For those OGNs that act at the extracel-
lular level, robust modification chemistries that
enhance the half-life and solubility of the OGN
in plasma will be necessary in order to prevent
degradation by circulating nucleases.
Quantitative, highly specific and sensitive
bioanalytical methods are required to deter-
mine the toxicokinetic (TK)/pharmacokinetic
(PK) parameters and exposure–response in
order to choose the right dosage regimens of
therapeutic OGNs in support of their preclini-
cal and clinical development. The methods
will be used to measure OGN concentrations
in plasma, urine, bile, feces and solid tissues,
typically kidney, liver, brain and spleen; but
other target tissues, such as skin and vitreous
humor, have also been investigated.
Chemistry of investigational
OGN therapeutics
Several modifications to investigational thera-
peutic OGNs have been introduced over the
years. They can involve the phosphodiester
linkage group, the ribose sugar moiety or the
nucleotide bases. Termini can be modified as
well; for example, additions to the 5´ or 3´ ends
of OGNs with polyethylene glycol (PEG) for
aptamers [20] , cholesterol for siRNA [21] or
peptides for exon skipping OGNs [22] have
been developed.
Bioanalysis (2009) 1(3)
For therapeutic OGNs to be part of clini-
cal trials, they are required to be robust, safe
and effective. F igure 1 depicts native RNA
(Figure 1A) and modification chemistries used
with investigational therapeutic OGNs.
The approved ASO therapeutic fomiversen has
a phosphorothioate (PS) oligodeoxynucleotide
(ODN) chemistry. PS ODNs are the first-genera-
tion ASOs intended to increase the nuclease resis-
tance and cellular uptake of the phosphodiester
backbone in vivo [23] . The PS modification, where
a nonbridging oxygen in the phosphodiester link
is substituted with sulfur (Figure 1B) , imparts
considerable stability to PS ODNs in vivo. The
PS group also confers binding affinity towards
proteins [24] , which may help to protect the OGN
from circulating nucleases [25] .
The pegaptanib aptamer, the other approved
therapeutic OGN, is pegylated at the 5´ end and
has an inverted 3´-3´deoxythymidine residue at
the 3´ end. Pegaptanib is 2´-O-methylated (2´O-
Me) on purines and 2´-fluorine-modified (2´F)
on pyrimidines [26] .
The 2´-F-modified (Figure 1C) nucleic acid
derivatives adopt the A-form typically found in
RNA, bind targets with high affinity and are
more resistant to nucleases in vivo [27] . The 2´F
is a useful modification for aptamers, since the
2´F residues can be incorporated by RNA poly-
merases used with in vitro molecular evolution
techniques [28] . Of note, the RNAi pathway is
tolerant to 2´F derivatives, which also makes
the modification useful for siRNA therapeutic
applications [29] .
The 2´O-Me RNAs (Figure 1D) are second-
generation ASO modifications and confer con-
siderable protection from nucleases while having
similar hydrogen bonding properties to RNA–
RNA hybrids [23] . An interesting property of 2´O-
Me is related to their propensity to abrogate the
inherent immunostimulatory characteristics of
OGNs when added selectively to guanosine or
uridine residues of a siRNA [30] , considering the
observation that the siRNA under investigation
may function via an unspecific immune-stimu-
latory mechanism [31] . Interestingly, the 5-meth-
ylcytosine (5mC) nucleobase modification, natu-
rally found in CpG motifs, has also been shown
to reduce immunogenicity for a PS ODN [32] .
Typically used in combination with
PS , 2´- O -me t hox y e t hy l ( 2´MOE ;
F igure  1E )-modified ODNs are also second-
generation ASO modifications  [33] . In addition
to supporting RNase H cleavage [34] , OGNs with
the 2´MOE modification have increased affinity
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 Bioana­lysis of siRNA & oligonucleotide therapeutics in biological fluids and tissues | Review
A. RNA B. Phosphorothiate oligodeoxynucleotide

O O

O- P O base O- P O base
O O
O S

O OH O

C. 2´-fluoro RNA D. 2´-O-methyl RNA

O O

O- P O base O- P O base
O O
O O

O F O O
CH3
E. 2´-O-methoxy ethyl F. Locked nucleic acid

O O

O -
P O base O- P O base
O O
O O

O O CH3 O O
O

G. Morpholino oligomer

O
H3C
N P O base
H3C O
O

Figure 1. Chemical modifications developed for investigational therapeutic oligonucleotides.

towards target RNA and increased nuclease sta-
bility relative to unmodified phosphoramidate
(PN) ODNs [35] .
One example of a second-generation molecule
modified with PS and 2´MOE in clinical devel-
opment is OGX-011, an ASO designed to block
the production of clusterin, a cell-survival pro-
tein that is overproduced in several cancer indi-
cations [36] . OGX-011 is complementary to the
mRNA translation initiation site and has been
shown to inhibit clusterin expression in in vitro
and in vivo laboratory models [37] .

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Review | Tremblay & Oldfield
Hybridization assay
Is a ligand-binding assay used for
the determination of the
oligonucleotide therapeutic
598
Also currently in clinical trials, EZN-2968
is a synthetic antisense (AS) ODN-targeting
hypoxia-inducible factor (HIF), with potential
antineoplastic activity resulting in inhibition
of angiogenesis, the inhibition of tumor cell
proliferation and apoptosis [38] . EZN-2968 is
a mixed polymer consisting of a PS core with
locked nucleic acid (LNA) components at the
5´ and 3´ ends.
Along with protection from degradation in vivo,
the LNA modification (Figure 1F) keeps the ribose
moiety in a C3´-endo conformation and locks the
OGN in the A-form. This results in improved
nucleobase stacking interactions, as well as higher
affinity and specificity [39] . Designing probes with
LNA nucleotides will also increase hybridiza-
tion kinetics and impart stability to duplexes by
effectively increasing the melting temperature.
In morpholino OGNs, the ribose sugar moiety
is replaced with morpholine rings and the anionic
phosphodiester linkage is replaced with non-
ionic phosphorodiamidate groups (Figure 1G) .
The morpholino oligomers are often used as an
ASO technology to block translation or inter-
fere with RNA processing, including splicing and
miRNA maturation [40,41] . They differ from tra-
ditional AS ODNs in that they function by ste-
ric hindrance of the target sequence rather than
ASO-mediated RNase H degradation [42] . One
example is AVI-4658, an exon-skipping OGN in
clinical development, designed to skip exon 51
of the dystrophin gene, allowing for restoration
of the reading frame in the mRNA sequence in
patients with Duchenne muscular dystrophy [43] .
Delivery systems
Oligonucleotides can be formulated to increase
their half-life in vivo and to modulate delivery to
specific organs and tissues. Delivery vehicles will
also help the intrinsically negatively charged and
bulky OGN to cross the cell membrane. Thus,
although a number of OGN compounds, includ-
ing siRNA, have been shown to be active ‘naked’,
or unformulated, further OGN drugs will benefit
from safe and effective delivery systems [44] .
Liposomes and lipid-like particles can be
charged with pharmaceuticals in their aqueous
center, protected from the extracellular environ-
ment by a spherical lipid bilayer [45] . Liposomes
are divided into three classes: multilamellar
vesicles, small unilamellar vesicles and large
unilamellar vesicles. The latter class is preferred
for OGN drugs because of its ability to achieve
favorable drug–lipid ratios and more predictable
drug-release kinetics [23] .
Bioanalysis (2009) 1(3)
Several lipid-based technologies for nucleic
acid delivery exist. Recently, stable nucleic acid–
lipid particle formulations have been used for a
number of disease models in vivo. Zimmermann
et al. were the first to demonstrate sequence-spe-
cific RNAi in nonhuman primates using a stable
nucleic acid–lipid particle formulation [46] . A
single injection of siRNA resulted in a maximal
silencing of more than 90% of the ApoB mRNA
expression in the liver 48 h after administration.
In addition, silencing persisted for 11 days at the
highest administered dose [46] .
Another important class of in vivo drug-
delivery system is comprised of positively
charged peptides and polymers, formulated
with negatively charged nucleic acids, result-
ing in stable nanoparticles. PEG can be used to
stabilize the nanoparticles and prevent aggrega-
tion [23] . Polyethyleneimine (PEI) is perhaps the
most widely used cationic polymer for in vivo
nucleic acid drug delivery. PEI can be either
branched or linear. There has been some con-
cern regarding the toxicity of PEI; however, dif-
ferent chemistries are implemented to minimize
those effects [47,48] .
Yet another cationic polymer, cyclodextrin, is
a circular polysaccharide that has been used for
siRNA-mediated gene knockdown. Used in con-
junction with the transferrin protein for target-
ing cancer cells, Heidel et al. delivered a siRNA
payload to target the M2 subunit of ribonucleo-
tide reductase, making it the first nonhuman
primate study on targeted, systemic delivery of
siRNA [49] .
The ideal bioanalytical method will take into
consideration the delivery vehicle and formula-
tion specifics of an investigational OGN. The
use of mild nonionic detergents that disrupt the
lipid bilayer, such as Triton® X-100, alone or in
combination with thermal denaturation will
enable solubilization of most lipids and poly-
mers, and release the nucleic acids for hybrid-
ization without the further need for purification
of the OGN for downstream quantification with
hybridization assays.
Comparison of recovery of formulated ver-
sus unformulated quality control (QC) sam-
ples will show whether the OGN is completely
released from the delivery vehicle in the course
of the bioanalytical method development. In
addition, the routine use of formulated QC
samples determined from an unformulated
standard curve will ascertain whether release of
the test article is representative of any generated
study samples.
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 Bioana­lysis of siRNA & oligonucleotide therapeutics in biological fluids & tissues
Administration & PK
Oligonucleotides have been administered by
various routes in support of preclinical and
clinical studies, including ocular [50] , paren-
teral intravenous injection [35] , intravenous
infusion  [51] , topical [52] , subcutaneous injec-
tion [53] , intramuscular injection [54] , lung inha-
lation [55] , intranasal inhalation [56] , intracere-
bral  [57] and even oral routes  [58] . The choice
of drug regimen is determined by indication,
intended clinical route of administration, con-
siderations for systemic exposure and character-
ization of toxicity. Parenteral administration is
clinically the most common way to administer
OGNs and is typically the route of choice for
most oncology indications. Administration of
OGNs via the inhalation route is also an ideal
way to locally administer therapeutics to the
upper airway and lungs.
Accumulation of the OGN test article is
found primarily in kidney and liver tissues fol-
lowing systemic administration [23] . Potential
toxicological effects exist as a result of comple-
ment activation and stimulation of the immune
system (which is desirable for IMO OGNs).
Additional clinical pathology parameters related
to the kidney and liver, histological pathology
staining, immunology assessments and liver
enzymes can be included in the study design
to characterize these effects. The systemic tox-
icity can be minimized by local administration
of the material to the tissue of interest. Peak
concentration-related toxicity, often associated
with large intravenous bolus doses, can also be
minimized or avoided by administration via
intravenous infusion, where lower steady states
can be achieved and exposure controlled by
the duration of the infusion period. Infusion
is also ideal for OGNs with a short biologi-
cal half-life  [23] . A thorough overview of PK,
as well as routes and formulations for OGNs
can be found in Chapters 7 and 8 of Antisense
Drug Technology. Principles, Strategies and
Applications; toxicity-related aspects of thera-
peutic OGNs are also discussed in Chapter 13
of the book [23] .
Hybridization assays
„„General considerations
Hybridization assays (also known as hybridiza-
tion immunoassays or hybridization ELISAs) are
carried out in a microtiter plate format, such as
in 96-well plates, with an OGN analyte instead
of an antigen analyte, as is the case with a typical
ELISA. Hybridization assays typically involve
future science group
| Review
the hybridization of the OGN to a capture probe
(immobilization) and/or to a detection probe
(signaling), the detail of which is described in
the following sections.
Hybridization-based immunoassay methods,
in general, provide the best reported assay sen-
sitivity and throughput compared with other
bioanalytical methods for OGNs. Hybridization
assays require little or no sample clean-up and
are therefore less time consuming. They have
been used widely for the quantitative ana­lysis
of OGN in support of TK/PK evaluation and
are particularly useful for the terminal-phase PK
assessment [59,60] .
A number of parameters can be optimized to
increase the sensitivity of hybridization assays, if
and when samples contain very low amounts of
the OGN test article or if high metabolite pro-
files are expected. Optimization of the (capture
and/or detection) probe concentration is of para-
mount importance, as this will directly impact
the signal-to-noise ratio; the noise being derived
mainly from the plasma or tissue matrices found
in hybridization mixtures.
The range of the standard curve between the
lower limits of quantifications (LLOQ) versus
the upper limits of quantification (ULOQ)
can also be optimized. Customary calibration
curve ranges (ULOQ:LLOQ ratio) of 50:1 with
fluorimetric detection and 30:1 with colorimet-
ric detection are typically attained. The use of
nonlinear logistic regression ana­lysis with four
parameters (4-PL) or, more recently, with five
parameters (5-PL; reviewed in [61]), as opposed to
linear regression, is typical for generating the stan-
dard curve in ligand-binding assays as it helps the
range, the LLOQ and the concentration accuracy
of PK/TK samples.
Chemically modified probes, such as LNA,
can also be used to increase the hybridization
kinetics [62] .
Commercially available streptavidin clear
plates for colorimetric analysis or opaque black
plates for fluorescent detection coated with
streptavidin can be used. Primary amine-
conjugation plates for immobilization of 5´
amino-derived capture probes can also be
used for immobilization. The detection probe
can be labeled with digoxigenin for detection
with commercially available antidigoxigenin
conjugates, for example.
Signaling is produced via the reporter enzymes
typically used for ELISA, such as alkaline phos-
phatase or horseradish peroxidase (HRP), with
colorimetric or fluorimetric substrates. One
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Review | Tremblay & Oldfield
advantage of HRP is lower noise compared with
alkaline phosphatase, whose substrates may have
labile phosphate groups.
For the determination of OGN in plasma
samples with a hybridization assay, purifica-
tion of the OGN from biological samples is not
necessary. If the assay involves a denaturation/
renaturation step for capture of the OGN test
article by a complementary probe, for example,
the fibrin and other blood components will also
denature when heated at 94°C for a few minutes.
However, since a relatively small amount of bio-
logical sample material (e.g., 40 µl) is required
in the hybridization mixture, the denatured
components of plasma will not interfere with
the hybridization per se or the assay in general
(Tremblay GA, Unpublished Data) .
For the determination of OGNs in tissues
with a hybridization assay, an initial step of
extraction can be carried out using liquid–liq-
uid extraction (LLE) with phenol and chloro-
form in order to remove the proteins and other
organic phase-soluble contaminants from the
tissues samples. This may be useful for OGNs
such as PS ODNs, well known to bind pro-
teins. Alternatively, a straightforward extrac-
tion procedure is performed on a small amount
of tissue sample (e.g., 50 µl of a 200 mg/ml
tissue homogenate) using proteinase K, soni-
cation and nonionic detergents for disruption
Label Label
Detection probe
Oligonucleotide
test article ‘Sandwich complex’
Capture probe
Biotin Biotin
Streptavidin- Streptavidin-
coated plate coated plate
Bioanalysis © Future Science Group (2009)
Figure 2. Schematic representation of the sandwich hybridization assay.
600 Bioanalysis (2009) 1(3)
of lipid bilayers, making the extraction much
easier than a LLE. With hydrophobic tissues,
such as the skin, the nonionic detergents can
be replaced with sodium dodecyl sulphate,
an ionic denaturing detergent that is compat-
ible with both hybridization and proteinase K
(Tremblay GA, Unpublished Data) .
„„Sandwich hybridization assay
The sandwich hybridization method is the sim-
plest version of a hybridization assay (Figure 2) .
In this dual hybridization method, a capture
probe is complementary to the first portion of
the OGN test article and modified to allow it to
be immobilized to a solid support, while a sec-
ond detection probe, modified for downstream
signaling, is complementary to the second por-
tion of the test article. The sandwich assay is
simple, straightforward and may be the method
of choice when dealing with complex or highly
modified OGN drugs.
Efler et al. used an amino-modified 5´ end
to immobilize the capture probe to a micro­titer
plate and a 3´-labeled biotin detection probe
for colorimetric detection using a substrate of
HRP coupled to streptavidin [63] . They obtained
very good sensitivity with a LLOQ of 10 pg/ml.
However the sensitivity is largely dependent
upon the sequence of the probes and the OGN
test article, and it is more typical to achieve
LLOQs of approximately 100 pg/ml with the
hybridization sandwich assay.
Apart from aptamers, which may be over 30
nucleotides long, therapeutic OGNs will often
range between 16 and 25 nucleotides. This
means that, for a 16-mer OGN drug, for exam-
ple, each detection and capture probe will only
be eight nucleotides long.
Locked nucleic acid probes can be used to
improve the sensitivity and kinetics of hybrid-
ization for the sandwich assay via thermal stabi-
lization of short hybrids. Improving the hybrid-
ization kinetics is useful if the capture probe
is prebound to the microtiter plate and also
for hybridization of the detection probe to the
immobilized OGN test article–capture probe
duplex [39] .
„„Hybridization–ligation assay
The hybridization–ligation assay [60] , also known
as the ligation-based hybridization assay, is a
specific and sensitive method for measuring an
OGN test article. The ligation assay requires that
the 3´-hydroxyl end group of the test article is free
and accessible for ligation to a phosphorylated
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 Bioana­lysis of siRNA & oligonucleotide therapeutics in biological fluids & tissues
ligation probe, as is the case for many, but not
all, OGN test articles. A schematic of the ligation
assay is shown in Figure 3.
In the ligation assay, a template probe is
designed to be complementary to the OGN test
article. However, in addition to the OGN test
article complementary sequence, the template
probe has a generic extension at its 5´ end. This
extension is complementary to a generic 5´ phos-
phorylated ligation probe. The 3´ end of a nine
nucleotide-long ligation probe is labeled with,
for example, digoxigenin, whereas the 5´ end
of the template probe can be biotinylated for
immobilization to the plate.
The OGN test article is first denatured and
then hybridized to the template probe. The
hybridization mixture is then bound to a strep-
tavidin-coated microtiter plate. The ligation
ensues on a solid support (Figure 3) . T4 DNA
ligase serves to bind the ligation probe onto the
intact 3´ end of the hybridized OGN test article.
After stringent washes to remove the nonligated
ligation probe, the amount of signal is measured
downstream of digoxigenin via reporter enzymes
and substrates.
The main advantage of the ligation-based assay
is that 3´ end, n-truncated metabolites are hardly
detectable since the efficiency of T4 DNA ligase
is very low in the presence of a gap. The 5´-end
metabolites will be detected; however, appar-
ently, the vast majority of metabolites present
in plasma are truncated at the 3´ end via 3´→5´
exonuclease activity [64,65] . The method is also
very sensitive and compares favorably with the
simpler sandwich hybridization method in this
regard. The ligation reaction is versatile enough
to enable a number of nucleotide chemistries,
such as PS or LNA. Also, in addition to DNA,
the ligation reaction is compatible with RNA
OGN test article substrates, such as siRNA.
„„Nuclease-based hybridization assay
The nuclease-based hybridization method, also
known as the S1 nuclease cutting assay, takes
advantage of the properties of a single strand-
specific nuclease, namely S1 nuclease, to degrade
the free capture probe and nonfully matched
hybrids, leaving only the full-length capture
probe–OGN test article duplex intact for down-
stream quantification. It is a variant of the classic
nuclease protection assay, developed for OGNs
and on a microtiter plate format.
The key advantage of the cutting assay is that,
in theory, only the full-length OGN test article
should be detected, making the assay specific
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| Review
Label
Ligation
probe
3´OH Ligation site
Ligation
Oligonucleotide
test article 5´PO4
Template probe
Biotin Biotin
Streptavidin- Streptavidin-
coated plate coated plate
Bioanalysis © Future Science Group (2009)
Figure 3. Schematic representation of the ligation step for the
hybridization–ligation assay. Briefly, the ligation probe is ligated to the 3’ end
of the oligonucleotide test article with the 5’ end of the template probe serving as
a template for ligation.
for the complete active pharmaceutical ingredi-
ent. However, in practice, the sequence context
and the intrinsic characteristics of S1 nuclease
commonly used with the cutting assay may also
allow the detection of n-1-truncated OGN,
since the enzyme only partially degrades nicked
DNA hybrids, for example [66] . Optimization
of conditions and use of different single-strand-
specific nucleases may help to improve discrimi-
nation [67] . Both the nuclease-based and liga-
tion–hybridization assays are patented by Isis
Pharmaceuticals (CA, USA) [201,202] .
„„Competitive hybridization assay
The competitive hybridization assay format
involves the competition between the OGN
test article and a tracer OGN (i.e., label probe),
bearing the same sequence composition as that
of the OGN test article for hybridization to a
solid phase-tethered complementary OGN [59] .
The tracer OGN can be labeled at the 3´ end
with either a direct or an indirect label, depend-
ing on the sensitivity required. Direct labels
include radiolabels or fluorescent and chemilu-
minescent substrates. Indirect labels, such as anti-
gens (e.g., digoxigenin), can selectively interact
with reporter enzymes, enabling reaction with an
enzymatic substrate, resulting in a colorimetric
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Review | Tremblay & Oldfield
or fluorescent signal. As the concentration of the
test article increases, the amount of competitively
bound label probe decreases, thereby resulting in
a signal profile (calibration curve) that is inversely
proportional to the concentration of OGN test
article in the matrix.
The competitive assay can be used as a third
option if sandwich or ligation assays are not fea-
sible. For example, it may be difficult to develop
an assay using a sandwich hybridization for a
short 12-mer OGN because the capture and
detection probes will be too short and will not
enable sufficient hybridization specificity, or the
3´ end of the OGN test article may be blocked
or inaccessible, in addition to it being very short.
„„Hybridization-based fluorescence assay
A method for quantification of single-strand
DNA OGNs in solution based on intercala-
tion of fluorochromes, such as Hoechst 33258
or ethidium bromide, has been developed [68] .
It does not rely on enzymatic amplification, as
the usual hybridization assays do. The method
makes use of the relative affinity of fluorescent
intercalating agents towards double-stranded
DNA hybrids as opposed to single-stranded
DNA. It is claimed that the method reaches the
low nanomolar range.
Quantitative whole-body
autoradiography
For distribution of OGN test articles in ani-
mals, quantitative whole-body autoradiography
(QWBA) has been applied. QWBA enables sub-
organ and whole-body distribution with mini-
mal sample disruption. Labeling of the OGN
can be performed, for example, with 3H or 14C.
The elimination phase of the OGN can be
studied with QWBA [69] . The OGN test article
penetration of cells in tissues can also be quali-
tatively examined by micro-autoradiography [70] .
The results of QWBA have a limited selectiv-
ity due to the fact that metabolites/breakdown
products will be measured along with the full-
length OGN active pharmaceutical ingredient.
Therefore, this technique provides good pre-
liminary data but fails to differentiate between
the parent compound and its metabolites. In
addition, because only the associated radio­
activity is measured, should there be any (e.g.,
tritium exchange with the biological medium), it
would invalidate the results obtained. However,
should the OGN test article bind to any previ-
ously unknown target, QWBA would be likely
to detect this.
602 Bioanalysis (2009) 1(3)
Nuclear imaging emission tomography
PET and single-photon emission tomography
are high-resolution, sensitive imaging tech-
niques that can be used for the repeated, non-
invasive quantification of OGNs in animals
and humans. These techniques have the same
limitations as QWBA regarding lack of selectiv-
ity for the parent compound versus metabolites.
One advantage is their ability to study molecular
interactions and functional studies in vivo [71,72] .
HPLC
The challenges encountered with quantification
of OGN using HPLC have been to improve
the resolution between parent and metabolites
and to increase the sensitivity of the technique.
Improvement in separation has been performed
mainly using ion-pair reversed-phase HPLC [73]
but also anion-exchange HPLC [74] . Fluorescent
detection has improved the sensitivity of
HPLC. Sensitivities reported were 250 ng/ml
by UV detection and 40 ng/ml with fluorescent
detection [75] .
Liquid chromatography coupled to
mass spectrometry
The introduction of novel soft-ionization
techniques, such as electrospray ionization
(ESI), enabled the ana­lysis of both low- and
high-molecular-mass, nonvolatile and fragile
compounds by LC–MS. LC–MS combines
the chromatographic separation of the compo-
nents of an OGN mixture with the selectivity
and sensitivity of detection found in MS, along
with molecular mass and molecular structure
information [76–78] .
The use of ESI-LC–MS has been applied to
the quantitative ana­lysis of OGN. LC mobile-
phase modifiers and ion-pairing reagents, such
as tetraethylammonium acetate [79] , hexafluoro-
2-isopropanol and triethylamine [80] , have been
used to improve chromatography and signal
intensity with MS. This approach has also been
used for the ana­lysis of PN-modified OGNs
using LC–MS [81] .
Matrix effects, including ion suppression
from salts, small organic and inorganic compo-
nents, proteins and nonprotein macromolecules,
have detrimental effects on the ESI signal [82,83] .
Therefore, purification steps are usually carried
out using phenol/chloroform-based LLE and
solid-phase extractions. Purification steps limit
the applicability of LC–MS for the quantifica-
tion of OGNs in biological matrices, especially
in tissues.
future science group
 Bioana­lysis of siRNA & oligonucleotide therapeutics in biological fluids & tissues
Recently, quantification of OGNs by LC–
MS/MS, where the OGN test article was pre-
extracted from the plasma samples using LLE
followed by solid-phase extraction, has been
reported with promising results [84] . The quan-
tification of a phorphorothioate OGN in tissues
by LC–MS/MS with a LLE extraction has also
been established [85] .
Capillary gel electrophoresis
Capillary gel electrophoresis (CGE) is an ana-
lytical technique with high resolution suitable
for the ana­lysis of therapeutic OGNs and their
metabolites in biological matrices in support of
TK/PK studies [86] .
The capillary in CGE is filled with a gel that
separates molecules by size and charge with the
application of a voltage. Small, negatively charged
OGN analytes move away from the anode faster
than larger OGN analytes. The results are
expressed as an electropherogram (similar to a
chromatogram) with separation peaks and can
usually resolve the parent compound from the
| Review
n-1 metabolite right down to the n-9 metabolite.
An example of results obtained with CGE–UV
performed on human samples of a single-stranded
PS ODN is shown in Figure 4 [87] .
Similar to chromatographic methods, study
samples require elaborate pre-extraction proce-
dures prior to injection into the system, hence
the need for an internal standard. In addition,
the exact nature of size-separated peaks cannot
always be determined, especially when it is not
possible to distinguish comigrating OGN.
Typically, the UV detection lower limits of
quantitation are in the order of 30–100 ng/ml.
However, the use of laser-induced fluores-
cence detection [88] has enabled a lower limit
of quantification of approximately 250 pg/ml
in human blood plasma with an acceptable
signal-to-noise ratio.
CGE coupled to MS
The development of ESI-MS enabled interfacing
to CGE systems in addition to LC systems for
the bioana­lysis of OGNs [89] .

A B 0.016

0.016 Full-length PS ODN

0.014
0.014

0.012
0.012

0.010
0.010
UV absorbance (260 nm)

UV absorbance (260 nm)

IS IS
(n-) 0.008 (n-)
0.008
metabolites metabolites

0.006 0.006

0.004 0.004

0.002 0.002

0.000
0.000

7.5 10.0 10.0 12.5

Retention time (min) Retention time (min)

Figure 4. Example of a capillary gel electrophoresis–UV electropherogram of a PS ODN in human plasma sample following
(A) 4 h of infusion, or (B) in human urine at day 15 of the study, pre-end of infusion.
CE system: Beckman P/ACE System MDQ; Injection: electrokinetic; 10 kV; 5–20 s;
IS: Internal standard; ODN: Oligodeoxynucleotide; PS: Phosphorothioate.

future science group www.future-science.com 603

Review | Tremblay & Oldfield
Regulatory filing
Application filed with the
US FDA and/or other regulatory
authorities prior to the
evaluation of a medicinal
therapeutic in humans
Capillary gel electrophoresis is considered to
be less expensive and faster than HPLC. Smaller
sample volumes and higher separation efficiency
can be achieved. CGE–MS therefore combines
the advantages of both techniques [90] . Freudeman
et al. successfully separated and detected 12–20-
base long homo-oligodeoxyribonucleotides and
their PN counterparts using CGE interfaced with
ESI-MS [89] . The coated capillaries used for the
separation contained an entangled polymer solu-
tion consisting of PEG. One problem with the use
of CGE–MS is the need for high-concentrated
buffer solutions, which dramatically decrease
the sensitivity of MS detection. Also, and again,
elaborate extraction methods are required [90] .
Quantitative PCR
Quantitative PCR (qPCR), also referred to as
real-time PCR, is a variation of the PCR per-
formed in capillaries, where the product forma-
tion of a PCR reaction is monitored in real time
using various detection methods. qPCR enables
amplification of nucleic acids up to the zepto-
mol range  [91] , compared with the femtomol
range found with immunoassays, for example.
Different methods have been implemented for
the quantitative determination of miRNA and
siRNA, including primer extension [92] , invader
assay [93] , ligation assay [94] , stem–loop reverse
transcriptase PCR assay [95] and competitive
qPCR [96] .
Quantitative PCR is a very sensitive tech-
nique that may be useful for the determina-
tion of therapeutic OGNs in small amounts
of samples, such as human biopsies obtained
in the course of clinical trials. One advantage
of qPCR is that there may be little or no need
for sample processing and purification [96] . On
the other hand, owing to the inherent nature
of the PCR and due to the background that
can be obtained with the formation of primer
dimers, extensive optimization of the method
may be required to improve precision and
accuracy. In addition, the method should be
selective enough to mitigate the interference of
truncated OGN metabolites.
Bioanalytical method
development & validation
Bioanalytical methods for the quantitative deter-
mination of OGN test articles are compared in
Table 1. These methods are used in the pharma-
ceutical industry to generate results and evaluate
TK/PK profiles and the bioequivalence of the
test article. The quality of these studies, which
are often used to support regulatory filings, is
directly related to the quality of the underlying
bioanalytical data and therefore to the quality of
the method-validation process.
Unlike proteins, OGNs may be much less prone
to lot-to-lot variability in purity and potency,
which compares in this regard to small molecules.
Specificity is the ability to measure the therapeutic
OGN unequivocally in the presence of other com-
ponents in the assay matrix. Both ligand-binding
assays and/or chromatographic methods may
be used dependent upon the therapeutic OGN
of interest. Prior to any validation, it is always

Table 1. Comparison of bioanalytical methods for the determination of oligonucleotides.

Method Sample Sensitivity Selective for Metabolite Robustness Class of OGN
processing parent OGN quantification
QWBA Extensive tissue Moderate No No Good All
preparation
HPLC Moderate Low No No Good Not good for PEG aptamers
CGE–UV Extensive Moderate Yes Yes Good Not good for PEG aptamers or
double-stranded OGN
CGE–LIF Extensive High Yes Yes Poor Not good for PEG aptamers
or double-stranded OGN
LC–MS Moderate High Yes Yes Good All
Hybridization None for plasma Very high Yes No (unless a Good All
assay samples specific probe is
(moderate for designed to
tissues) that effect)
qPCR Moderate Highest No No Poor All; ligation method required if
the 3’ end is blocked
CGE: Capillary gel electrophoresis; LIF: Laser-induced fluorescence; OGN: Oligonucleotide; q: Quantitative; QWBA: Quantitative whole-body autoradiography;
UV: Ultraviolet.

604 Bioanalysis (2009) 1(3) future science group

 Bioana­lysis of siRNA & oligonucleotide therapeutics in biological fluids & tissues
important to ensure that the bioanalytical method
is fully developed and that any major pitfalls are
resolved before the actual validation starts. First,
one determines what is expected from the method
in order to choose the right assay platform, that
is, hybridization, HPLC, MS, CGE and so forth.
Recommended development parameters will
include specificity and selectivity, accuracy and
precision, and in-process stability. The method
can be validated once these parameters are tackled
within a fine-tuned experimental procedure.
For a ligand-binding method development, as
would be the case for a hybridization assay, the
following items should be considered:
n Assay format selection (sensitivity vs selectivity)
n OGN class and formulation considerations
n Probe design
n Reagent selection
n Standard reference material
n Matrix selection
n Method optimization
n Reagent concentrations
n Incubation conditions
n Prevalidation assessments
For the validation itself, there are many
extensive Guidance Documents and White
Papers already published [97–99] . Generally, the
parameters listed in Box 1 are assessed.
Future perspective
siRNAs have probably become the leading class
of investigational therapeutic OGNs owing to
their relatively high potency [1] . New types of
OGN and RNAi-related therapeutics, such
as miRNA [100] or piwi-interacting (pi)RNA
[101] , are expected to emerge from the wave of
small RNA therapeutics. Current bioanalyti-
cal methods will need to evolve according to
these new compounds and the new regulatory
paradigm that it entails. Nevertheless, siRNA
will benefit from the experience of its predeces-
sors, as current quantification methods of AS
ODNs are, for the most part, readily adaptable
to siRNA indications.
In parallel to siRNA therapeutics, several
nanotechnology delivery vehicles are currently
under development [44] . The qualitative and
quantitative determination of delivery vehicles,
in addition to the active therapeutic OGNs con-
tained in the formulation, may become a topic of
future science group
| Review
interest from a regulatory standpoint; for exam-
ple, to verify the selective delivery of a siRNA to
tissues and organs and to document the extent of
nonspecific siRNA release in plasma.
For a number of applications, including
the determination of multiple OGN indica-
tions, multiplexed determination of therapeutic
OGNs is currently implemented. For instance,
not only can multiple siRNA indications be
determined in one study sample, but so can
cytokine expression, biomarkers, therapeutic
target(s) and delivery vehicles. The determina-
tion of the two strands of a siRNA molecule can
also benefit from multiplexing technologies.
Luminex™ xMAP microsphere-based tech-
nology enables the simultaneous quantification
of up to 100 analytes, such as cytokines. Nucleic
acid quantification methods have been developed
for the Luminex platform, including single nucle-
otide polymorphism genotyping, genetic disease
screening, gene-expression profiling, HLA DNA
typing and microbial detection [102] . Analysis of
siRNA in multiplex with the Luminex platform
has also been reported [103] .
Box 1. Validation parameters of quantitative methods
for oligonucleotides.
Method validation
„„ Prove the reliability, robustness and reproducibility of the assay
„„ Acceptance criteria for validation parameters are predefined
„„ Accuracy should be within ±20% compared with the nominal concentration, and
precision should be ≤20%
„„ QC samples (QC1, QC2 and QC3) and standard curve prepared in the same
biological matrix at anticipated study samples
Range of calibration and QC sample ranges
Interbatch precision and accuracy
„„ Five QC levels (LLOQ, QC1, QC2, QC3 and ULOQ) in replicates of three performed
on six occasions
Selectivity/specificity
„„ Ten independent lots of matrix (e.g., plasma, urine and tissue samples) assessed
blank and spiked with the analyte
„„ Metabolite cross-reactivity
Dilution linearity
„„ Use a concentrated standard in the matrix dilute with a blank sample/matrix and
check for linearity of dilution
„„ Check for parallelism using an incurred sample with a high concentration and
dilute with a blank sample/matrix and check for linearity
Prozone (hook effect)
„„ Use a concentrated standard at least 100-times the concentration of the highest
calibration standard to be assayed without dilution to ensure the absence of a
prozone effect
Stability
„„ In-process: room temperature, 4°C, freeze-thaw
„„ Long-term storage (-20/-80°C)
LLOQ: Lower limit of quantification; QC: Quality control; ULOQ: Upper limit of quantification.
www.future-science.com 605
Review | Tremblay & Oldfield
Meso Scale Discovery (MSD™) multiplex-
ing technology is a promising electrolumines-
cence-based detection system making use of the
microtiter plate format, with favorable sensitivity
and range of detection. Also, the volume of study
samples can be reduced using MSD due to a lower
volume requirement for each well of the plate.
The throughput with hybridization assays
can be scaled-up using new miniaturization and
automated liquid handling technologies. For
example, the Gyrolab™ bioanalytical system
miniaturizes and integrates assay steps for quan-
titative immunoassays on CD-microlaboratories
processed with automated workstations.
The assessments of immunogenicity and/or
mechanism of action of siRNA therapeutics can
be developed in support of regulatory filing. For
a siRNA aimed at PCSK9, a 5´-rapid amplifi-
cation of cDNA ends (RACE) assay has been
used for demonstrating RNAi-mediated silenc-
ing activity by investigating the cleavage site of
the target mRNA extracted from dosed animal
tissues [15] . 5´-RACE has also been applied to
monitor the duration of siRNA activity targeted
at PLK1 in mice with hepatic tumors [104] .
Off-target effect assessment of siRNA thera-
peutics, that is, the impact of an unforeseen asso-
ciation of siRNA to untargeted RNA [105] , may
also become a subject of concern, since siRNA
drugs are relatively new and considering the
importance of mechanisms of action for pharma-
cological entities and the potential unforeseen side
effects associated with this class of compounds.
In conclusion, bioanalytical methods will need
to adapt to multiple indications for a variety of
therapeutic OGNs formulated with increasingly
complex delivery vehicles. Delivery targeted at
specific organs and tissues will require sensitive
methods for lower therapeutic doses. Sample
volume can also be reduced with technologi-
cal innovation, which will reduce the burden of
sample collection on patients in the course of
clinical trials.
Acknowledgements
The authors would like to thank Helen Legakis for critical
review of the manuscript.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial
involvement with any organization or entity with a finan-
cial interest in or financial conflict with the subject matter
or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or
pending, or royalties.
No writing assistance was utilized in the production of
this manuscript.

Executive summary
Hybridization-based assays have several key advantages
„„ High target specificity
„„ In general, no sample cleanup is required (e.g., plasma samples)
„„ Minimal cleanup for tissue samples (extraction)

„„ Good accuracy and precision/reproducibility (15–25%)

„„ Assay designs selective for parent oligonucleotide (OGN)

„„ Low reagent cost

„„ Low instrumentation cost

„„ Equipment is not the limiting factor

„„ Methods are easily automated for high throughput

„„ High sensitivity (pg/ml to ng/ml levels) 100–10000-times more sensitive than capillary gel electrophoresis–UV

Key limitations
„„ Quantification of parent/total detectable OGN metabolites (shortmers) not quantifiable in parent assay
„„ Narrower calibration range than chromatographic methods (20–50-fold)
„„ High reagent quality imperative (assay robustness)

„„ Cannot detect intact double-stranded OGNs

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„„Websites
301 US FDA: drug approval package: vitravene
(fomivirsen sodium intraveal injectable)
injection
www.fda.gov/cder/foi/nda/98/
20961_vitravene.htm
302 US FDA: FDA approves new drug treatment
for age-related macular degeneration
www.fda.gov/bbs/topics/news/2004/
new01146.html
303 Fire and Mello win Nobel Prize
www.the-scientist.com/news/print/24964
609