A Brief History, Status, and Perspective of

Modified Oligonucleotides for

Chemotherapeutic Applications
Modification of oligonucleotides for drug
applications has a very short history (for highlights see Table 4.1.1 and Table 4.1.2). Intensive efforts to modify short strands of nucleic
acids to enhance their properties as drug candidates began in earnest in the late 1980s. The
notion that oligonucleotides could be made into
drugs along with the discovery and development of two essential enabling technologies
automated DNA sequencing (Connell et al.,
1987) and commercial automated oligonucleotide synthesis and purification in the early
1980sset the stage for oligonucleotide drug
discovery (reviewed by Zon, 1993). Subsequently, oligonucleotide medicinal chemistry and pharmacology, which form the basis of
structure-activity relationship studies, have
been highly developed in the process of converting oligonucleotides into drugs. Before
this, little value was given to synthetically
changing the structure of short strands of DNA
or RNA except for some early modifications
primarily directed to enhance structural studies
and diagnostic applications. Now great interest
in making drugs out of oligonucleotides has
spurred intense structure-activity/property relationship (SAPR) studies to optimize drug
properties (Cook, 1998a,b).
Given that oligonucleotides are gene-based
materials (or informational materials; Cohen,
1991) and considering the emergence of
genomic target selection, which is also gene-

Table 4.1.1

UNIT 4.1

based, it is surprising that it has taken so long

for the drug discovery community to recognize
the potential value of modified oligonucleotides as drugs. However, when traditional
medicinal chemists first consider modifying
oligonucleotides, they are confronted with a
number of daunting chemical, biophysical, and
biochemical properties of oligonucleotides
(Table 4.1.3), which has surely slowed progress
in this area. In addition to the need to apply
novel ligands to drug discovery, the nucleic acid
targets that serve as receptors, particularly RNA
molecules, are novel as well. In spite of the
early difficulties in exploring the use of oligonucleotides as drugs, however, much progress has been made in recent years. The first
antisense oligonucleotide drug, Vitravene, was
recently approved by the Food and Drug Administration (FDA) for treating cytomegalovirus retinitis in AIDS patients, and several
additional oligonucleotides are in late phase II
and III clinical studies for anti-cancer and antiinflammatory indications (Table 4.1.4).

OVERVIEW OF BASE PAIRING

AND GENE EXPRESSION
A single strand of nucleic acid has a polymeric sugar-phosphate backbone with ribofuranosyl (in RNA) or deoxyribofuranosyl (in
DNA) residues connected at the 3 and 5 hydroxyls by phosphoric acid to form phosphodiester linkages. Each sugar residue has one of

Historical Listing of Oligo- and Polynucleotides as Therapeutic Agents

Milestone

References

DNA alkylating agents

Poly(rI) poly(rC)
Macromolecular antimetabolites
Antitemplates
Antisense oligonucleotides (targeting RNA)
Heterocycle modified oligonucleotides:
prodrugs for nucleosides
Antigene oligonucleotides: triple strand
formers
Aptamers

Belikova et al. (1967); Summerton (1979)

De Clercq et al. (1969)
Chandra and Bardos (1972); reviewed by
Bardos and Ho (1978)
See Table 4.1.2
Alderfer et al. (1985)

Sense approach

Moser and Dervan (1987); LeDoan et al.

(1987)
Ellington and Szostak (1990); Tuerk and Gold
(1990)
Morishita et al. (1995)

Contributed by P. Dan Cook

Current Protocols in Nucleic Acid Chemistry (2000) 4.1.1-4.1.17
Copyright 2000 by John Wiley & Sons, Inc.

Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.1

Table 4.1.2

Highlights of the Development of Chemotherapeutic Oligonucleotides

Milestone

References

Antisense experiments in cell culture

Stephenson and Zamecnik (1978); Zamecnik

and Stephenson (1978)
Merrifield, Letsinger, Caruthers (1962-1987)
Tso et al. (1987)
Eckstein, Stec, Zon (1967-1981)
Cech (1986); Altman (1989)
Isis Pharmaceuticals (1992-1994)

Automated synthesis, protocols, reagents

Methylphosphonate oligonucleotides
Phosphorothioates
Ribozymes
Initiation of first clinical trial; first report of
clinical efficacy
Twenty five oligonucleotides entered into
clinical development
First New Drug Application (NDA) for an
antisense oligonucleotide
First approved antisense oligonucleotide drug

five types of heterocycles (nucleobases) attached at the C1 position of either the deoxyribofuranosyl or ribofuranosyl sugar. These heterocycles are the purines (guanine and adenine)
and the pyrimidines (cytosine and thymine,
with uracil replacing thymine in RNA).
Complementary DNA describes a chemical complex of two strands of deoxyribonucleic acid that are bound together by WatsonCrick base-pair hydrogen bonding. This essences of life bonding specifies that guanine
in one DNA strand specifically binds to cytosine in the other strand, and that adenine in
one DNA strand specifically binds to thymine
in the other strand (Fig. 4.1.1). In RNA, uracil
specifically binds to adenine. A sequence (or
a specific ordering of nucleobases) of an
RNA or a DNA strand will bind specifically

(1992-present; see Table 4.1.4)

Isis Pharmaceuticals (April 1998)
Isis Pharmaceuticals (August 1998)

(hydrogen bond) to another sequence only if

the nucleobases match up (base pair). The
strands are then said to be complementary to
one another.
The complementary DNA strands twist into
a helical motif with one strand having its bases
ordered in the 3 to 5 direction and the other
strand having its bases ordered in the opposite
direction (5 to 3). This is referred to as antiparallel orientation (Fig. 4.1.2). The doublestranded DNA (dsDNA) serves as the template
for synthesis of either complementary DNA
molecules, to provide a process of self-replication, or complementary RNA molecules (from
the sense strand) for the process of transcription. The complementary DNA strand, referred
to as the antisense strand, is itself rarely transcribed to give an RNA molecule.

Table 4.1.3 Chemical, Biochemical, and Biophysical Properties of First-Generation

Oligonucleotides

Parameter

Property

Molecular weight
Charge
Chirality in backbone
Instabilities
Solubility

High (20- to 25-mers, MW 7000 to 9000)

Highly negative (19 to 24 charges) or neutral
P=S, Me-P, N-Pa
Biological and chemical (nucleases, pH, depurination, dethiation)
Insoluble in common organic solvents depending on backbone
modification (P=S versus Me-P)a
No
Complex synthesis of reactive monomers
Unfamiliarity with instrument synthesis of oligomers
Purification by ion exchange chromatography
Analysis and characterization atypical

Crystalline?
Ease of synthesis
Modified
Oligonucleotides
for
Chemotherapeutic
Applications

aMe-P, methylphosphonate; N-P, phosphoramidate; P=S, phosphorothioate.

4.1.2
Current Protocols in Nucleic Acid Chemistry

Table 4.1.4

Oligonucleotides Entered into Clinical Developmenta

Disease
indications

Oligonucleotide

Molecular target

ISIS 2105

HPV 6 and 11 E2 Genital warts

genes product
HCMV IE gene-2 CMV retinitis

ISIS 2922

Statusb

Sponsor

Terminated

Isis

NDA approved
(August 1998)
III, Crohns;
II for others

Isis/Ciba Vision

Renal allograft,
rheumatoid
arthritis, Crohns
disease,
ulcerative colitis,
psoriasis
Cancer
II

Isis/Boehringer
Ingleheim

Cancer

II

Isis/Novartis

HCMV IE gene
Gp120
Ha-ras
gag gene
gag gene
UL36 and UL37
Protein kinase A
p53
c-myc
c-myb
gag gene
HIV integrase
Bcl-2
gag gene

CMV retinitis
AIDS
Cancer
AIDS
AIDS
CMV retinitis
Cancer
Cancer
Restenosis
Cancer
AIDS
AIDS
Cancer
AIDS

I/II
Terminated
I
Terminated
I
I
II
Terminated
Terminated
Terminated
Terminated
I/II
II
Terminated

Cancer

MG-98
Resten-NG

Ribonucleotide
reductase R2
subunit
DNA methylase
c-myc

Isis
Isis
Isis
Hybridon
Hybridon
Hybridon
Hybridon
Lynx/Inex
Lynx/Inex
Lynx/Inex
Chugai
Aronex
Genta
Novopharm
Biotech
GeneSense

Heptazyme

IRES region

Angiozyme

Vascular
endothelial
growth factor
receptor
Immune system

ISIS 2302

Intercellular
adhesion
molecule
(ICAM-1)

ISIS 3521/CPG
64128A
ISIS 5132/CPG
69846A
ISIS 13312
ISIS 5320
ISIS 2503
GEM-91
GEM-92
GEM-132
GEM-231
OL(1)p53
LR-3280

Protein kinase
C-
c-raf kinase

GPs 0193
Zintevir
G3139
GPI-2A
GTI 2040

CPG-x

Solid tumors
I
Restenosis and
I
proliferative
diseases
Hepatitis C virus I

Isis/Novartis

MethylGene
AVI BioPharma

Antiangiogensis
(cancer)

Ribozyme
Pharmaceuticals
Ribozyme

Hepatitis B

CpG

aAbbreviations: E, early gene; HCMV, human cytomegalovirus; HPV, human papillomavirus; IE, immediate early gene;

IRES, internal ribosomal entry site; NDA, New Drug Application; UL, unique long.
bI, II, and III indicate phase of clinical trials at time of printing.

Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.3
Current Protocols in Nucleic Acid Chemistry

H
O
O

O
O

Figure 4.1.1

O
P
HO

O O O
P

N
O O

O
O

HO

N
H

Watson-Crick hydrogen-bonding (base-pairing) rules (G to C, A to U or T).

The transcribed RNA molecules, in turn,

serve as the templates that allow the ordering
of the amino acids within the polypeptide chain
of proteins, during the process of translation.
Translation is an appropriate term because the
nucleotide sequence information (language) of
nucleic acids is translated into the amino acid
sequence (language) of protein. The complete
processDNA makes RNA makes protein
has been referred to as the central dogma of
molecular biology. A more common term for
the process is gene expression. Thus, the two
stages of gene expression are transcription (the
conversion of DNA to RNA) and translation
(the conversion of RNA to proteins). DNA also
self-replicates to maintain an organisms
genome. Furthermore, regulation of gene expression in living organisms involves extensive
recognition and binding to specific nucleic acid
sequences by nucleic acidbinding proteins.

INFORMATIONAL DRUG
DISCOVERY APPROACHES AND
THEIR SUCCESS

Modified
Oligonucleotides
for
Chemotherapeutic
Applications

H(CH3)

N U/T
N
O

N G

A N
H
N
H
N H O

N H

Disease states (genetic, oncogenic, or infective) are typically a result of the production of
abnormal proteins, the end-stage product of
gene expression. Thus, it is not surprising that
drug discovery has historically focused on interfering with the functions of the abnormal
proteins rather than on preventing abnormal
protein formationtreating the disease symptoms rather than the cause. In contrast, treatment approaches involving informational chemicals, such as oligonucleotides, attempt to prevent
the formation (gene expression) of abnormal protein by targeting DNA, RNA, or regulatory proteins that are required for transcription and translation of the abnormal protein. Having a set of
binding rules (Watson-Crick base-pairing rules)
that allows one to rapidly and precisely select
a molecule to synthesize that will inhibit gene

expression at the DNA or RNA level, and that

offers exquisite specificity for its receptor, is
unique in the history of drug research. Thus,
the employment of informational drugs to complement genomic target validation in modern
drug discovery is revolutionary.
Several discovery approaches are being pursued that employ the encoded information of
modified oligonucleotides as the drug agent
(Tables 4.1.1 and 4.1.2). In the process of transcription, complementary RNA (message RNA
or mRNA) is derived from the sense DNA
strand by Watson-Crick base-pair recognition
and binding. The antisense approach targets the
initial sense complementary RNA strand (primary transcript) as well as many downstream
sites that are available as RNA is metabolized
in the process of protein production (e.g., splicing, transport, translation; Fig. 4.1.2).
A strict definition of the antisense approach
describes the inhibition of gene expression
through targeting of a predetermined sequence
in an RNA. There are several types of antisense
approaches, which may be differentiated on the
basis of their modes of action (Fig. 4.1.2).
Antisense oligonucleotides primarily operate
by sequence-specific binding to the targeted
RNA. The resultant heteroduplex recruits a
ubiquitous endonuclease, ribonuclease H
(RNase H), to cleave the targeted RNA strand.
Other potential antisense modes of action are
by direct sequence-specific binding of the oligonucleotides to the targeted RNA (or merely
occupying a site on the RNA) such that the
function of essential RNA-binding proteins is
disrupted or that secondary structure required
for gene expression of the RNA is disrupted. A
variety of distinct sites in the intermediary
metabolism of RNA can potentially be targeted
by antisense oligonucleotides with RNase H
dependent or independent modes of action
depicted in Figure 4.1.2.

4.1.4
Current Protocols in Nucleic Acid Chemistry

Figure 4.1.2

Informational drug-targeting sites. CAP, m7Gppp; W/C, Watson-Crick.

RNase L, like RNase H, is another cellular

nuclease that cleaves RNA. Its endonucleolytic
cleavage is triggered by binding to (2-5)linked polyadenylic sequences (Fig. 4.1.3).
Torrence and co-workers have conjugated the
RNase L activating moiety to oligonucleotides
to provide sequence-specific cleavage of targeted RNA (Torrence et al., 1994).
Ribozymes are RNA molecules with catalytic RNA-cleaving activity (Cech, 1986) and
thus represent another type of antisense oli-

gonucleotide. These are typically much longer

RNA molecules that contain both a catalytic
region (which can cleave another RNA segment) and an adjacent sequence or two sequence arms that allow Watson-Crick base-pair
binding to the target. A variety of ribozymes have
been described that differ primarily in their threedimensional structures and lengths (reviewed by
Rossi, 1998). The sequence-specific cleavage of
a predetermined RNA target is accomplished
without assistance from any protein.

NH2
N
O
O
O
HO P O P O P O
O
O
O

N
NH2

HO

O
O P O
O

N
O

N
N
NH2

HO

O
O P O
O

HO

Figure 4.1.3

(2-,5)-Adenylic oligonucleotides.

N
N

O
O

N
N

oligonucleotide

Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.5
Current Protocols in Nucleic Acid Chemistry

A
X = O,S,C
5'
Heterocycle

A,C,G,T

Sugar

3'
O
Linkage

R
2'-Position

O P O
O

4'

Connection sites

A,C,G,T
Pendants

Replace sugar-phosphate
(e.g., amide linkage, PNA)
Subunits of oligonucleotide to modify

B
Phosphorous modifications
a.
b.
O
O
O P O
O P S
O
O

O
O P BH3
O

d.

e.
O
O
O P NHR O P OR
O
O

O
O P CH3
O
h.

g.

f.

c.

O
O P Se
O

O
S P S
O

Linkage modifications
i.
j.
Me N
O

NH
O P O
O

HN

n.

m.

l.

k.

CH2
O P O

Sugar modifications
p.

o.

r.

q.
base

base
O

s.

O
OMe

base

base

OMe
NH2

t.
u.

base

v.

base

base

base
O

O
O

w.

NMe2

x.
base

N
NMe2 O P NHR

O
NMe2
base

O
O

Heterocycle modifications
y.
Me

z.
O

NH2
Me

NH
N
cc.
Me

aa.

N
N

NH

NH2

NH2

Connection modification
ff.

NH2

Sugar-phosphate replacement
gg.

base
N

Modified
Oligonucleotides
for
Chemotherapeutic
Applications

NH

N
N

N
NH2

ee.

dd.

NH2

bb.

NH2

O
base
HN

base
N

4.1.6
Current Protocols in Nucleic Acid Chemistry

Oligonucleotides may also inhibit gene expression at the DNA level by several antigene
approaches. The most often examined approach
is triple strand formation by oligonucleotides
binding in the major groove of dsDNA via several
Hoogsteen base-pairing rules (Helene, 1993). A
less often examined approach is the use of
oligonucleotides to bind via Watson-Crick
base-pairing rules to a single strand of DNA
available from the formation of a transcription
bubble or locally open-chain site of dsDNA.
Utilizing nucleic acids as decoys to compete
with natural cis-acting sites on dsDNA for essential regulatory proteins is referred to as the
sense approach. In this case, synthetic oligonucleotides (typically dsDNA) are designed to bind
to proteins in a sequence-specific manner.
In another protein-binding approach, aptamers, derived from nucleic acid selection
processes (see Chapter 9), can specifically target regulatory proteins. Common steps to all
selection methods for nucleic acidbinding
species are: (1) generation of a large pool of
sequence diversity from chemically synthesized DNA pools, (2) transformation of the
pools by enzymatic manipulations such as polymerase chain reaction (PCR) or in vitro transcription, (3) selection of functional shapes,
and (4) amplification. As each RNA sequence
folds into a distinct three-dimensional shape,
and because of the large number of structures
(millions) generated, complementary binding
to a protein target may occur, providing useful
biological properties (reviewed by Bacher and
Ellington, 1998).
Of those drug discovery approaches based
on oligonucleotides, the antisense efforts are
clearly the most advanced, in that twenty three
first-generation phosphorothioate oligonucleotides have entered human clinical trials and
one antisense oligonucleotide has achieved
FDA approval (Table 4.1.4). Much less success
has been accomplished by targeting DNA with

triple-strand-forming oligonucleotides, antisense ribozymes, and RNase Lmodified oligonucleotides. From a chemical point of view,
these and other less successful approaches
listed in Tables 4.1.1 and 4.1.2 suffer from not
being amenable to the readily available, firstgeneration backbone-modified oligonucleotides such as methylphosphonates, amidates, phosphorothioates, and oligomers
(Fig. 4.1.4B). Success in these drug discovery
approaches, assuming that the biological rationale is valid, will require much more intensive chemical efforts.

MODIFICATIONS AND DRUG

PROPERTY ALTERATIONS
A diverse range of modifications at all possible modification sites of an oligonucleotide
have been reported (reviewed in Cook, 1998a).
Figure 4.1.4A illustrates a dimer of an oligonucleotide depicting possible modifications for
enhancing oligonucleotide drug properties.
These include alterations of heterocycles, carbohydrates, linkages (backbones), and connection and conjugation sites, as well as the complete removal of the sugar-phosphate backbone. Most of the positions available in a GC
or AT dimer (26 positions for each dimer) that
do not directly interfere with Watson-Crick
base-pair hydrogen bonding have been modified at one time or another.
The nucleobases or heterocycles of nucleic
acids provide the recognition points for Watson-Crick base pairing and must maintain this
specific interaction in any modification. Thus,
the scope of heterocyclic modifications is quite
limited; this is verified by the fact that only five
or six modified heterocycles have demonstrated useful oligonucleotide drug properties.
These can be characterized as lipophilic modifications at C5 of pyrimidines and C7 of 7deazapurines. Enhanced stacking has been suggested to be the reason for increased binding

Figure 4.1.4 Oligonucleotide modification. (A) Dimer structure showing potential modification
sites. (B) Examples of oligonucleotide modifications: (a) natural phosphate diester; (b) chiral
phosphorothioate; (c) chiral methyl phosphonate; (d) chiral phosphoramidate; (e) chiral phosphate
triester; (f) chiral boranophosphate; (g) chiral phosphoroselenoate; (h) phosphorodithioate; (i)
methylenemethylimino (MMI); (j) 3-amide; (k) 3 achiral phosphoramidate; (l) 3 archiral methylene
phosphonate; (m) thioformacetal; (n) thioethyl ether; (o) 2-fluoro; (p) 2-O-methyl; (q) 2-O-(3amino)propyl; (r) 2-O-(2-methoxy)ethyl; (s) 2-O-2-(N,N-dimethylaminooxy)ethyl (DMAOE); (t) 2O-2-[2-(N,N-dimethylamino)ethyloxy]ethyl (DMAEOE); (u) 2-O-N,N-dimethylacetamidyl; (v)
N-morpholinophosphordiamidate; (w) hexose nucleic acid; (x) locked nucleic acid (LNA); (y)
5-propynyluracil-1-yl; (z) 5-methylcytosin-1-yl; (aa) 2-aminoadenin-9-yl; (bb) 7-deaza-7-iodoadenin-9-yl; (cc) 7-deaza-7-propynyl-2-aminoadenin-9-yl; (dd) phenoxazinyl; (ee) phenoxazinylG-clamp; (ff) deoxyribofuranosyl; (gg) peptide nucleic acid (PNA). See Cook (1999).
Current Protocols in Nucleic Acid Chemistry

Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.7

Modified
Oligonucleotides
for
Chemotherapeutic
Applications

affinity with this type of heterocycle modification. 2-Aminoadenine represents another

type of modification that provides enhanced
binding affinity. In this case, an additional
hydrogen bond is formed to uracil, providing
significant increases in melting temperature
(Tm). All of these modificationspositioned to
lie in the major or minor groove of the heteroduplexdo not affect sugar conformation of
the heteroduplex, and do not provide useful
nuclease resistances, but will support an
RNase H cleavage mechanism.
Modifications in the ribofuranosyl moiety
have provided the most value in the quest to
enhance oligonucleotide drug properties. In
particular, certain 2-O modifications have
greatly increased binding affinity, increased
nuclease resistance, and altered pharmacokinetics, and are potentially less toxic than phosphorothioate oligonucleotides (Cook, 1998a).
Preorganization of the sugar into a 3-endo
pucker conformation is responsible for the increased binding affinity. Unfortunately, no
sugar modification has been reported that is
useful in supporting RNase H cleavage.
Linkage or backbone modifications involve
changes to the nonbridging oxygen atoms, such
as phosphorothioate (P=S), methylphosphonate (Me-P), phosphoramidate (N-P), and others
shown in Figure 4.1.4B, as well as extensive
changes in which the entire four-atom linkage
between sugar moieties is replaced. The most
useful linkage-replacement modification,
MMI (Fig. 4.1.4B), provides greatly enhanced
binding affinities, removes the nuclease-cleavable phosphodiester linkage and the chirality
of the phosphorothioate linkage, and allows
adjustments of oligonucleotide lipophilicity
by controlling the negative charge. Of the
linkage changes, only phosphorothioates induce
RNase H cleavage.
Several examples of connection modifications in oligonucleotides are -nucleoside linkage (reverse connection of the heterocycle at
C1) and (2-5)-phosphodiester bonds (in place
of the normal (3-5)-phosphodiester bond).
Peptide nucleic acid (PNA) is an example of
the complete removal of the sugar-phosphate
backbone and replacement with a peptide linkage. In addition to the above modifications,
pendants or conjugation groups can be attached
at many positions in the various subunits to
enhance drug properties.
The antisense concept implies well-defined
structural requirements for the oligonucleotide
ligand that binds to a reasonably characterized
RNA receptor. Although this knowledge of

rather precise binding is of great value and

certainly sets this drug discovery approach
apart from current approaches, additional
modifications are limited to modifications
that do not interfere with Watson-Crick basepairing rules.
Modifications of oligonucleotides may be
expected to address essentially every facet of
antisense drug properties. Biophysical and biochemical properties that may be affected by
modifications include binding affinity, basepair specificity, nuclease resistance, chemical
stability, lipophilicity, solubility, endonucleolytic cleavage of the RNA of a heteroduplex, biological pharmacokinetic properties,
toxicological properties, and pharmacological
properties. Drug properties that will be affected
by altering biophysical and biochemical parameters include the general areas of pharmacokinetics, pharmacodynamics, and toxicology. In addition, certain modifications may
represent cost and proprietary patent advantages.
Modifications of short strands of DNA and
RNA for drug purposes should be rationally
directed by drug property deficiencies of the
parent or first-generation oligonucleotides (Table 4.1.5). Biological deficiencies are exposed
by pharmacologic, pharmacokinetic, and toxicologic studies of the parent oligonucleotides
at the biophysical, biochemical, in vitro, in
vivo, and clinical levels. In addition, as modified oligonucleotides move from discovery into
the development pipeline, chemical development deficiencies in, for example, larger-scale
synthesis, purification, and analytical processes will emerge that must be resolved. Finally,
in contemplating research in oligonucleotides,
one should search the patent literature in addition to examining the traditional scientific literature, as considerable proprietary positions
of various aspects of the technology have been
achieved. Hopefully, the days of incorporating
nucleosides, novel or known, into oligonucleotides based merely on availability are over.
This nonrational approach was of value in the
beginning of oligonucleotide structure-activity
modifications simply to develop an information
base. Now that more knowledge is available
about what is required to achieve enhanced
properties, current and future oligonucleotide
modifications and studies of their structure-activity relationships should be soundly and rationally based on the need to resolve one or
more of the deficiencies listed in Table 4.1.5,
or to overcome emerging drug process or production problems.

4.1.8
Current Protocols in Nucleic Acid Chemistry

Table 4.1.5

Limitations of First-Generation P=Sa Oligonucleotides

Area

Limitation

Pharmacodynamics

Low affinity per nucleotide unit:

low potency
limited sites to target
Inhibition of RNase H at high concentrations
RNase H mechanism implicated
Limited oral bioavailability
Limited blood-brain barrier penetration
Dose-dependent pharmacokinetics
Release of cytokines
Complement-associated effects on blood pressure?
Clotting effects
Accumulation in kidneys?

Pharmacokinetics

Toxicologics

aP=S, phosphorothioate.

OPTIMIZATION OF BINDING
AFFINITY AND NUCLEASE
RESISTANCE
Oligonucleotide Binding Affinities
Based on the ligand-receptor theory of pharmacological activity, increasing the affinity of
an oligonucleotide for its RNA target should
increase potency. A relatively simple physicochemical experiment is employed to determine
the level of binding and specificity of a modified oligonucleotide. A complementary oligonucleotide is the simplest target to which an
antisense oligonucleotide can hybridize. Thus,
using length-matched complementary oligonucleotides at stoichiometric concentrations in a
medium that mimics the intracellular environment as closely as possible (salt, pH, temperature, concentration), melting curves are employed to determine how tightly a modified
oligonucleotide binds to its complement. This
involves a spectrophotometric analysis to
measure absorbance versus temperature. The
melting temperature (Tmthe temperature at
which the mixture is half duplex and half single-stranded) is determined (Freier et al.,
1992). Oligonucleotide affinity, as measured by
melting curves, increases with the length of the
oligonucleotide-RNA heteroduplex. Thus, 15to 25-mers are typically used in antisense experiments rather than shorter oligonucleotides,
which may have Tm values close to or below
physiological temperature and, therefore, may
only form low levels of the required heteroduplex.
A few short oligomers (12-mers or less)
have exhibited interesting biological activity,
and these require modifications leading to high
affinity per nucleotide unit. One interesting

example was the report of potent and selective

inhibition of gene expression by phosphorothioates (7- and 8-mers) containing only
5-propynyl-substituted uracil and cytosine
bases (Fig. 4.1.4B; Wagner et al., 1996). Furthermore, several recent studies correlate binding affinities of a series of 2-O-modified oligonucleotides with increased in vitro and in
vivo activity (Kawasaki et al., 1993; Morvan et
al., 1993; Monia et al., 1993, 1996; Altmann et
al, 1996a,b,c, 1997). An extensive comparative
listing of binding affinities of 2-O modifications
was provided by Freier and Altmann (1997).

Oligonucleotide Nuclease Resistance

A number of in vivo pharmacokinetic studies in several animal species indicate that P=S
oligonucleotides are not as stable as initially
thought (Zhang et al., 1996; Nicklin, 1998).
Although the stability of P=S oligonucleotides
may be sufficient for many drug applications,
greater stability will be helpful in expanding
dosage regimens (longer duration of action
relates to less frequent dosing) and in developing oral bioavailability. Additionally, less degradation of modified oligomers will minimize
metabolite toxicity. In summary, the preponderance of published antisense biological data
suggests that oligonucleotides with higher
binding affinities and greater stabilities towards
nucleases are important medicinal chemistry
objectives.

STANDARDS ESTABLISHED BY
RECENT SAPR STUDIES
The intense oligonucleotide research performed in the 1990s has provided a remarkable
enhancement of several of the desired antisense

Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.9
Current Protocols in Nucleic Acid Chemistry

Modified
Oligonucleotides
for
Chemotherapeutic
Applications

drug properties. The results achieved with

modified oligonucleotides concerning binding
affinity, base-pair specificity, nuclease resistance, and support of RNase H cleavage of the
targeted RNA is impressive. One should consider the level of binding affinity (as represented by Tm), nuclease resistance (as represented by t1/2), and support of an RNase H
mechanism that a new modification should possess in order to be of interest as a potential drug.
A number of winners have been identified
(Fig. 4.1.4B) and discussed in several reviews
(Cook, 1998a,b).
Certain modifications of oligonucleotides
provide an increase in Tm of >1.5C/modification (C/mod) relative to a P=S oligonucleotide
(1.0C/mod relative to an unmodified P=O
oligonucleotide), and nuclease resistance (t1/2)
of >24 hr with snake venom phosphorodiesterase (SVPD; about the same as for P=S). In
view of these values, the author suggests that a
novel modification should exhibit a Tm
>1.5C/mod compared to its P=S oligonucleotide parent. In evaluation of binding properties, the modified oligonucleotides should be
hybridized with an RNA complement, as this
is the receptor required for the antisense approach. A clear correlation between Tm values
derived from hybridization to a DNA complement or to an RNA has not been established.
Also, correlations of Tm values have not been
established for oligonucleotides having just
one modification (point or pendent modification) or several modifications distributed
throughout the sequence, or for those having a
contiguous placement of the modification in the
sequence (Cook, 1991; Lesnik et al., 1993;
Buhr et al., 1996; Matteucci and Krosigk,
1996). Since the application of a modification
will likely require its uniform placement in the
sequence, or at least several contiguous bases
in a row for a gapmer strategy (described below,
Cook, 1993), binding affinity measurements
(Tm) should be taken with these types of modified oligonucleotides. Furthermore, the modification must not compromise base-pair specificity. In this regard, information from a number
of papers suggests that base-pair specificity
actually increases as Tm values are increased.
A specificity that is comparable to base mismatches of a P=S phosphorothioate would appear to be a useful standard.
Nuclease resistance of a novel modification
in a P=O oligonucleotide backbone should at
least be at the level of uniformly modified P=S
oligonucleotides. Given that measurements of
t1/2 values, unlike Tm measurements, are deter-

mined using several procedures (e.g., by incubation with heat-inactivated fetal bovine serum,
purified enzymes such as SVPD, cell or tissue
extracts; or by in vivo dosing followed by
extraction and analysis) under different conditions, the use of P=S oligonucleotide controls
(standards) is necessary. In addition, several
concentrations of nuclease should be employed
to identify and minimize complications leading
to enzyme inhibition (Cummins et al., 1995).
Half-lives of 24 hr are often reported for P=S
oligonucleotides in SVPD assays. When the 3
end of an oligonucleotide is modified to have
sufficient resistance to 3-exonucleases, endonucleolytic cleavage becomes evident. Thus,
modifications should also protect against endonucleases. In a gapmer strategy, this is accomplished by a phosphorothioate gap, which
will also support an RNase H cleavage mechanism. Nuclease resistance of a modified oligomer, if not provided by the modification, may
in many cases be enabled by employing a P=S
oligonucleotide backbone.
In considering the relative importance of the
nuclease resistance of an antisense oligomer
and its affinity level for its RNA target, recent
biological results suggest that it may be more
important to enhance the stability of an oligonucleotide than its binding affinity (Crooke
et al., 1996a). Modifications that provide highbinding oligonucleotides with low nuclease resistance have not provided significant biological activity, whereas oligonucleotides such as
phosphorothioates have. Although some modifications provide high binding affinities and
high nuclease resistance, they may not exhibit
useful antisense activities because they do not
support an RNase H mechanism. A modification that supports an RNase H mode of action
and provides high Tm and nuclease resistance
t1/2 values has not been reported. Thus, an ideal
oligonucleotide modification would provide an
oligomer that hybridizes to target RNA with
high binding affinity and specificity, would be
stable to nucleolytic degradation, and would
allow RNase H cleavage of the RNA target.
This has led to the theory that to optimize the
antisense activity of an oligomer, a combination of oligonucleotide modifications will be
required (Cook, 1991, 1993).
As noted above, after high-binding, nuclease-resistant 2-O-modified oligonucleotides
were developed, it was rather disappointing that
oligomers uniformly modified were inactive or
less active than their first-generation parent
phosphorothioates. It is now well known that
uniformly 2-O-modified oligonucleotides do

4.1.10
Current Protocols in Nucleic Acid Chemistry

not support RNase H cleavage: the 2-O-modified oligonucleotide-RNA heteroduplex presents a structural conformation that is recognized by the enzyme but is not cleaved (Crooke
et al., 1995; Lima and Crooke, 1997). This lack
of activity has led to the development of a
chimeric strategy (gapmer technology; Cook,
1993; Monia et al., 1993; Yu et al., 1996).
This approach focuses on the design of highbinding, nuclease-resistant antisense oligonucleotides that are gapped with a contiguous
sequence of 2-deoxyribonucleoside phosphorothioates (2-deoxy/PS)(Fig. 4.1.5). On
hybridization to target RNA, a heteroduplex is
presented that supports RNase H cleavage of
the RNA strand. The stretch of the modified
oligonucleotide-RNA heteroduplex that is recognized by RNase H may be placed anywhere
within the modified oligonucleotide. The modifications in the flanking regions of the gap
should not only provide nuclease resistance to
exo- and endonucleases, but should also not
compromise binding affinity and base-pair
specificity (Hoke et al., 1991).
Modifications of the phosphorus atom of the
natural phosphodiester linkage (producing
methylphosphonates, phosphorothioates, and
phosphoramidates) destabilize heteroduplexes
0.7 to 1.5C per modification (Fig. 4.1.4B;
Agrawal et al., 1990; Dagle et al., 1991; Guinosso et al., 1991). The decreased binding affinity of these modified oligonucleotides could
be expected to reduce antisense effectiveness.
In the case of chimeric 2-O-methyl- or 2fluoro-modified oligonucleotides, an enhancement in the binding affinity of 2.0 to 2.3C
(compared to P=S oligonucleotides) for each
modification is obtained (Sproat et al., 1989;
Guinosso et al., 1991; Miller et al., 1991;
Kawasaki et al., 1993). However, it is now clear
that 2-O-methyl- and 2-F-modified DNA are
not sufficiently nuclease resistant to have antisense value as P=O backbones (Sproat et al.,
1989; Morvan et al., 1993; Sproat and Lamond,
1993; Sands et al., 1995; Prasmickaite et al.,
1998). The potential problem in this area can
be circumvented by the use of 2-O-methyl- or
2-F-modified phosphorothioates in the flanking regions (doubly modified; Miller et al.,
1991; Kawasaki et al., 1993).
Flank

More recent research has focused on 2-O

modifications such as methoxyethyl (Altmann
et al., 1996a,b,c) and aminopropyl (Griffey et
al., 1996), which not only provide relatively
high binding affinities but also a level of nuclease resistance that allows replacement of thiophosphates with natural phosphodiester linkages. With a favorable combination of Tm and
t1/2, 2-O modifications can be employed in the
chimeric strategy (gapmer technology; Fig.
4.1.5), which allows a significant portion of
P=S linkages to be replaced with P=O linkages.
Just how many sulfurs can be replaced depends
on the length of the oligomer and the gap size
or RNase H cleavage site. Typically, a 21-mer
with a 7-nucleotide gap has 65% of the P=S
linkages replaced with P=O linkages. As noted
in the discussion of limitations of P=S oligonucleotides, reduction of the sulfur content in a
P=S oligonucleotide could have important implications in the pharmacokinetic and pharmacodynamic properties as well as the toxicity
profiles of oligonucleotides (Altmann et al.,
1996a).
A very important aspect of gapmer technology is that the gap or RNase H cleavage site
must be protected from endonucleolytic cleavage. Phosphodiester linkages and even an alternating P=S/P=O motif do not provide a useful
level of nuclease resistance for biological activity (Dagle et al., 1991; Hoke et al., 1991).
The recently reported lack of activity of a
gapped 3-amidate phosphodiester is likely
due to endonuclease degradation (Heidenreich
et al., 1997). Phosphorothioates are the only
useful modification to allow a reasonable combination of binding affinity and nuclease resistance and also support an RNase H mechanism.
Hence, as noted above, most antisense activities
require an RNase H mechanism, which in turn
requires sulfur in the form of thiophosphate
somewhere in the chimeric molecule for nuclease resistance.
An additional issue that should be kept in
mind is the impact that oligonucleotide modifications may have on the cost of future antisense drugs. Again, as a standard, the cost of P=S
oligonucleotides should be considered. The
cost of phosphorothioates has been dramatically reduced due to improvements in the process, cost reduction of key reagents, and eco-

Gap

5'- 2'-Modifications/PS or PO

Flank
2'-Modifications/PS or PO

2'-deoxy/PS

Figure 4.1.5

Gapmer technology. PO, phosphodiester; PS, phosphorothioate.

-3'

Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.11
Current Protocols in Nucleic Acid Chemistry

nomics of larger-scale syntheses. As of 1999,

the cost of materials for a 20-mer P=S oligonucleotide was <$300/gram; it is projected to
decline to <$50/gram as larger quantities are
required. The 2-O-methoxyethyl-modified
oligonucleotides are derived from ribonucleosides and should eventually be substantially less expensive than modified oligonucleotides derived from deoxyribofuranosyl nucleosides. P=S oligonucleotide antisense drugs
are expected to be cost competitive when considering parenteral (intravenous) treatment three
times/week with a 1 mg/kg dose. Modified
oligonucleotides must be cost effective and will
need to be less expensive to synthesize (e.g.,
shorter), or provide a greater therapeutic index
(increased potency) or other important advantages to offset an increase in the cost of synthesis.
When considering research to modify oligonucleotides, the author believes it is important to know the extent to which proprietary
protection of a modification can be obtained.
Important patent positions for many types of
modifications have been established during the
1990s as designing drugs from oligonucleotides has become of interest (Crooke et al,
1996b; Sheffery and Gordon, 1996; Craig et al.,
1997).

GENERAL GUIDELINES FOR

ENHANCING
OLIGONUCLEOTIDE DRUG
PROPERTIES

Modified
Oligonucleotides
for
Chemotherapeutic
Applications

The excitement roused by the prospect of

using oligonucleotides as drugs stems from the
fact that, unlike other drug discovery approaches, they are informational materials
i.e., chemicals having a specific set of rules (the
Watson-Crick base-pairing rules) that clearly
govern their binding to a specific nucleic acid
receptor. In addition, the fact that DNA and
RNA molecules are, compared to proteins,
relatively new molecular targets is of great
interest.
The first test in a SAPR study to determine
whether a modified oligonucleotide is worth
pursuing is whether it maintains its sequence
specificity according to Watson-Crick rules
(Fig. 4.1.6). A newly modified oligonucleotide
that does not possess an acceptable level of
specificity (comparable to that of the P=S oligonucleotide) is an immediate failure. The next
step is to determine how tightly the modified
oligonucleotide binds to its target nucleic acid.
As noted, the binding affinity of a modified
oligomer is a physicochemical property, determined by measuring the oligomers sequence-

specific interaction with its length-matched

RNA complement. The hybridization or melting process is performed under a rather standard
set of conditions designed to mimic an intracellular environment (Freier et al., 1992). Because
this calculation is done under artificial conditions, it may not accurately represent the binding of an oligomer to a native RNA inside a cell.
The next essential property of an oligonucleotide is sufficient resistance to degradation
by exo- and endonucleolytic plasma and tissue
nucleases (Fig. 4.1.6). Nuclease resistance is
determined in various assays, such as incubating the oligonucleotide in heat-inactivated fetal
bovine serum, cellular extracts, or purified exonucleases or endonucleases (also see Standards
Established by Recent SAPR Studies). These
results are also likely to differ from the stability
of an oligomer in vivo. However, these Tm and
t1/2 assays do allow SAPR studies to proceed,
and thus provide reasonable methods to compare various oligonucleotide modifications at
an early developmental stage. This is quite
different from traditional drug discovery approaches.

CONJUGATED
OLIGONUCLEOTIDES
The sugar, heterocycle, and backbone (linkage) subunit modifications as depicted in Figure 4.1.4A are core modifications that greatly
enhance binding affinities and nuclease resistance of antisense oligonucleotides. To enhance
other antisense drug properties of an optimized,
core-modified oligonucleotide, a variety of
molecules (pendants) have been attached (conjugated) in a point-modification motif (i.e.,
only one pendant in an antisense oligonucleotide). Pendant modifications have primarily been directed to enhancing oligonucleotide
uptake. Other potential applications of pendants
include increased solubility, lipophilicity, and
means to attach synthetic cleaving agents, intercalators (for improvements in binding affinity),
and cross-linking and alkylating groups (see UNITS
4.2 & 4.3). Several reviews have discussed oligonucleotide pendants (Cook, 1991, 1993; Manoharan, 1993; De Mesmaeker et al., 1995).

PERSPECTIVE
One P=S oligonucleotide drug (Vitravene)
is available as of 1999, and others will follow
in the next several years. However, to continually improve this novel and exciting drug class,
and to overcome certain limitations, structural
changes are required. In the 1990s a diverse
range of modifications, at all possible modifi-

4.1.12
Current Protocols in Nucleic Acid Chemistry

 Synthesis of novel or known nucleosides

Synthesis of novel amidites with appropriate protection
Development of oligomerization protocol process/analysis
Synthesis of modified oligonucleotide in standard sequences
Synthesis of length-matched complementry RNA sequences and single base
mismatches and appropriate control sequences
Obtain binding affinities (change in Tm in degrees/modification) of matched and
mismatched modified oligonucleotide

Sequence-specificity or binding
affinity of modification below standards

Abandon modification

Sequence-specificity and binding affinity of

modification improved compared to standards

Prepare appropriate modified oligonucleotides for

nuclease studies
Determine relative nuclease resistance

Nuclease-resistance of modified
oligonucleotides less than standards

Consider additional modifications, e.g.,

combination of modifications, for enhancing
nuclease resistance while maintaining
binding affinity

Modified oligonucleotide possess greater

nuclease-resistance than standards

Examine modified oligonucleotide

with target sequence (tissue culture
reduction of message and/or protein)

Advanced pharmacology,
pharmacokinetics and toxicology
studies

Figure 4.1.6

General pathway for structure-activity/property relationship studies.

cation sites of an oligonucleotide (Fig. 4.1.4A),

has been reported. This application of traditional medicinal chemistry (SAPR studies) to
drug discovery in antisense oligonucleotides,
and in oligonucleotides in general, has answered many important questions. For example, as a result of this rather intense effort,
modifications are now known that stabilize
oligonucleotides towards nucleolytic degradation (e.g., 2-O-methoxyethyl and 2-O-aminopropyl), that greatly enhance binding affinities
while maintaining base-pair specificity (e.g.,
2-O-methoxyethyl), and that support endonucleolytic cleavage by RNase H (e.g., 5propynyl pyrimidines). Although these are biochemical and biophysical properties, a large
volume of cellular and animal studies support
the notion that enhancing these properties correlates with enhanced antisense biological ac-

tivity in vivo. Unfortunately, a single modification that provides high binding affinity nuclease-resistance and support of an RNase H
mechanism is not available. A modification of
this nature is of current interest in the antisense
approach.
It is also known that changing the structure
of phosphorothioate oligonucleotides provides
an opportunity to alter their pharmacokinetic
profile. Structural changes that remove sulfur
(as thiophosphate) and/or change lipophilicity
(e.g., by 2-O modifications) have resulted in
more favorable toxicity profiles (Altmann et al.,
1996a). Although research has revealed these
important antisense properties (and there may
be many more to learn about), and has shown
how to control them, it has not yet determined
the optimum values at which these modifications should be aimed. In addition, the lack of

Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.13
Current Protocols in Nucleic Acid Chemistry

Modified
Oligonucleotides
for
Chemotherapeutic
Applications

antisense oligonucleotides that are orally available and/or penetrate the blood-brain barrier
represents the most important deficiency of
antisense oligonucleotides. Recent reports of
antisense P=S oligonucleotides doubly modified at the 3 and 5 ends with 2-O-methyl or
2-O-methoxyethylto provide a high level of
nuclease resistancehave provided encouraging
results suggesting that these pharmacokinetic deficiencies will soon be solved by appropriate
chemical modifications (Agrawal et al., 1995).
One should be aware of the level of accomplishments achieved in oligonucleotide medicinal chemistry research in the course of the
1990s. Binding affinities, nuclease resistance,
support of RNase H, and cost of synthesis have
been discussed in this unit, and they should be
considered (as standards) before initiating or
continuing oligonucleotide modification research. In addition, understanding the proprietary patent positions that have been established
is an important research consideration. The
author believes that, at this stage of oligonucleotide medicinal chemistry, it is highly unlikely that a single modification will be discovered that will significantly affect all of the
important drug properties described above. The
types of modified oligonucleotides currently
being pursued (going beyond P=S oligonucleotides) possess a combination of modifications, and this trend will certainly continue as
pendants will be conjugated to oligonucleotides with optimized core subunits to obtain
a completely optimized oligonucleotide drug.
The current winners, or the first modifications most likely to be incorporated into antisense oligonucleotides that will undergo clinical trials, are the RNA mimics 2-Omethoxyethyl and 2-O-aminopropyl, and the
backbone modification MMI (Fig. 4.1.4B).
These will likely be utilized in a gapmer strategy. However, efforts to prepare uniform modifications, such as RNA mimics (2-O-modifications), MMI, and PNA are of considerable
interest, in that reliance on RNase H for a mode
of action would not be required. In addition to
these modifications that act either by direct
binding (RNase H independent) or via RNase
H, the author believes that subunit pendant
modifications (such as cholesterol conjugates
and folic acid conjugates) will become increasingly important for optimizing multiply modified oligonucleotides.

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Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.15
Current Protocols in Nucleic Acid Chemistry

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4.1.16
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Contributed by P. Dan Cook

Isis Pharmaceuticals
Carlsbad, California

Synthesis of
Modified
Oligonucleotides
and Conjugates

4.1.17
Current Protocols in Nucleic Acid Chemistry