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1 Nature Uses Nucleic Acid Polymers for Storage, Transfer, Synthesis, and Regulation of Genetic Information

1 Nature Uses Nucleic Acid Polymers for Storage, Transfer, Synthesis, and Regulation of Genetic Information

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12 Unnatural Nucleoside Analogs for Antisense Therapy



O



O



N

R1



N



R1



O



N



H N



NH

O



N

N



O P

O

O



O

O



N



NH2

NH2



N

R1



H

H N



N H



N



NH



R1



O



O



N



O



N

O



N



O

A T base pair



O

N



R2



N H



N



R2



O



O

O P

O

O



N



N



N

O

O P

O

O



H



NH2



N



N H

H



N



N



O



G C base pair



N

O



R1 = H, R2 = Me, DNA, 1

R1 = OH, R2 = H, RNA, 2

Figure 12.1 Structures of DNA and RNA and of the ASO—RNA drug—receptor interaction.



tissues such as the central nervous system (CNS), as much as 98.8% of the genome

is transcribed into RNA. The vast majority of this RNA does not code for protein

but is believed to be involved in intricate regulatory functions, which may very well

be responsible for the biological complexity observed in higher species. Thus, the

role of nucleic acid polymers within biology has grown from information storage

and heredity for DNA to information transfer, protein synthesis, and regulatory

mechanisms for RNA.



12.2

The Antisense Approach to Drug Discovery



The field of antisense was born several decades before the regulatory role of RNA in

cellular biology became apparent. Zamecnik and Stephenson showed that

externally delivered short pieces of chemically modified DNA targeted to their

complementary RNA inside the cell could suppress protein synthesis [9]. These

oligonucleotides came to be known as antisense oligonucleotides (ASOs) and are

defined as short (12–25 nucleotides in length) chemically modified oligonucleotides

that bind to their complementary RNA using Watson–Crick base pairing and modulate

RNA function to produce a pharmacological effect [10]. Just like a small-molecule drug

designed to bind a protein receptor, an antisense oligonucleotide has to first bind to



12.2 The Antisense Approach to Drug Discovery



its biological receptor (i.e., RNA). Once hybridization is accomplished, the

oligonucleotide has to elicit a functional response. These functional responses

can be broadly classified into two general categories of antisense mechanisms

that (i) promote RNA degradation or (ii) interfere with RNA function without

degradation (Figure 12.2).

Numerous pathways for degrading RNA exist inside a cell [11]. The precise

pathway by which an ASO promotes RNA degradation is fundamentally

determined by the specific chemical design features of the ASO. Single-stranded

(ss) ASOs, which contain stretches of >5 DNA nucleotides, are able to promote

RNA degradation via ribonuclease H (RNase H)-mediated cleavage [12,13]. RNase

H refers to a family of enzymes that preferentially cleaves the RNA strand in an

RNA/DNA heteroduplex [12]. In contrast, dsRNA duplexes (20–22 nucleotides in

length) downregulate gene expression through the RNA interference pathway [14].

In this mechanism, one of the RNA strands of the duplex (guide strand) is loaded

into an endonuclease Argonaute 2 (Ago2), which is a component of the RNAinduced silencing complex (RISC). Ago2 cleaves the RNA strand of a fully

complementary RNA/RNA duplex (siRNA (small interfering RNA) pathway),

thereby inhibiting gene expression [15]. In the event of partial complementarity

between certain regions in the 30 -untranslated region (30 -UTR) of the RNA and the

RISC-loaded ASO, the RNA is transported into vesicles known as P-bodies where it

is eventually degraded by RNA metabolizing enzymes. This is known as the



Figure 12.2 Several antisense applications can be harnessed for therapeutic applications [10].



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12 Unnatural Nucleoside Analogs for Antisense Therapy



microRNA (miRNA) pathway and a single miRNA can regulate the expression of

hundreds of genes [16].

Other than the RNA degrading mechanisms of gene expression outlined

previously, single-stranded ASOs that do not contain stretches of DNA nucleotides

or are chemically precluded from being loaded into RISC can also modulate gene

expression by binding to and interfering with the function of endogenously

produced microRNAs. Such oligonucleotides (miRNA antagonist) generally result

in derepression of gene expression by antagonizing miRNAs, which are natural

repressors of gene expression [17]. ASO chemical designs that do not produce RNA

degradation can also be used to modulate RNA splicing in cells. Almost all

eukaryotic RNA transcripts undergo alternative splicing, which can be redirected by

targeting ASOs to splice junctions [18]. Once again, depending on the chemical

design of the ASO, splicing can be orchestrated to produce exon exclusion or

inclusion resulting in the formation of alternatively spliced gene products [19].

While the majority of antisense mechanisms discussed above have historically

targeted mRNA, which code for proteins, other classes of RNA, which can function

as targets for therapeutic intervention, continue to be discovered. These include the

family of long noncoding RNAs, which regulate gene expression at the transcriptional level in a cell- and tissue-specific manner [20]. Recently, toxic RNAs that are

directly causative of disease pathology have also been identified [21]. Thus,

targeting RNA directly to modulate its function or to control the formation of

downstream gene products presents an attractive avenue to expand the universe of

druggable targets and to discover newer classes of pharmacological agents that are

orthogonal to small-molecule and protein-based therapeutics.

There is now unequivocal evidence that shows that Nature uses antisense as a

mechanism to control gene expression. In fact, microRNAs could be considered as

Nature’s own ASOs. Therefore, synthetic oligonucleotides, which bind to RNA by

Watson–Crick base pairing and modulate its function agnostic of the antisense

mechanism, could be considered as derivatives of naturally occurring ASOs.

However, while natural nucleic acids such as DNA and RNA are stable and can

function while inside a cell, they make for very poor drugs [22]. This has

necessitated the use of medicinal chemistry strategies to improve the drug-like

properties of oligonucleotides for use in therapeutic applications.



12.3

The Medicinal Chemistry Approach to Oligonucleotide Drugs



The simplicity of the antisense concept, which promises exquisite control of gene

expression using chemical agents that can be readily prepared using automated

equipment in high-throughput fashion, has enticed researchers in the field for

several decades. Moreover, since the nature of the drug–receptor interaction

(Watson–Crick base pairing) of antisense oligonucleotides with their biological

receptor is well understood, one can, in theory, design an oligonucleotide to

modulate the function of any gene, as the nucleotide sequences are available from



12.4 Structural Features of DNA and RNA Duplexes



gene banks. In practice, however, this has proved to be significantly more difficult

to achieve [23].

Natural nucleic acids are extremely labile in extracellular biological fluids and

undergo rapid nuclease-mediated degradation upon administration to animals.

They also have poor intrinsic pharmacokinetic properties causing them to be

filtered by the glomerulus and excreted in the urine rapidly after administration to

an animal [24,25]. Moreover, given that much of the RNA in a cell is bound up in

complex secondary and tertiary structures and that RNA/RNA duplexes are more

stable compared to RNA/DNA duplexes, shorter DNA-based ASOs do not possess

sufficient affinity to invade these structures and form productive drug–receptor

interactions. This is less of a problem for RNA-based ASOs, which function via the

RISC pathway, since the argonaute proteins assist with hybridization to complementary RNA. However, as mentioned earlier, unmodified single- or doublestranded RNA oligonucleotides are extremely labile in biological media and

distribute poorly to tissues. As a result, all oligonucleotides have to be stabilized by

using chemical modifications or encapsulated within cationic lipid formulations

for use as therapeutic agents.

For an externally delivered ASO to function as a drug, it needs to travel intact

from its site of administration into the cytoplasm or nucleus of a cell. During this

process, the ASO has to survive being digested by ubiquitous endo- and

exonucleases, distribute into tissues, cross cellular and/or nuclear membranes,

bind to its target RNA, and elicit a functional response. Given that most ASOs are

polyanionic molecules with molecular weights ranging from 45 to 200 kDa, this is

not a trivial process. From the perspective of a medicinal chemist, the use of

chemical modifications to improve the drug-like properties of ASOs has to

accomplish at least four distinct objectives: (i) improve affinity for complementary

RNA, (ii) improve stability versus nuclease-mediated digestion, (iii) impart

favorable pharmacokinetic properties, and (iv) maintain or confer the ability to

produce a functional response.

In the following sections, we will discuss nucleic acid modifications commonly

used to improve the drug-like properties of ASOs, which are natural products

themselves or whose design was inspired by some inherent structural feature of

natural nucleic acids such as DNA and RNA. However, prior to embarking on that

discussion, it is important to familiarize the reader with some fundamental

structural features of oligonucleotide duplexes and other terminology routinely

used in nucleoside and nucleic acid parlance [26].



12.4

Structural Features of DNA and RNA Duplexes



Precise conformational preferences around several rotatable bonds in the sugarphosphate backbone create the distinctive three-dimensional helical architectures

of oligonucleotide duplexes. Natural nucleic acids such as DNA and RNA form

duplexes with a right-handed double helix. The heterocyclic bases stack along the



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12 Unnatural Nucleoside Analogs for Antisense Therapy



center of the helix while the sugar-phosphate backbone runs along the periphery in

an antiparallel orientation. This structural arrangement results in the formation of

distinct grooves along the double helix, which are used to classify nucleic acid

duplexes into A- and B-type helices (Figure 12.3) [27]. Double-stranded DNA

typically forms B-type helices, which are characterized by a wide and shallow major

groove and a narrow minor groove. In contrast, double-stranded RNA forms A-type

helices, which have a narrow and deep major groove and a wide and shallow minor

groove. In both cases, the heterocyclic nucleobases are positioned in the major

groove, while the sugar-phosphate backbone is oriented toward the minor groove.

DNA/DNA duplexes are relatively flexible and can adopt several different

conformations depending on the external factors such as humidity, salt concentration, counterion, and sequence [27]. In contrast, RNA/RNA duplexes are more

stable and almost always adopt the A-type helical geometry. The distinct shapes

created by DNA and RNA double helices act as determinants of several biological

functions, which are characteristic for each class of nucleic acid polymers.

The conformation of the sugar-phosphate backbone in nucleic acid polymers

starting with P, 50 O, C50 , C40 , C30 , 30 O, P is described using torsion angles a, b, c,

d, e, and j, respectively (Figure 12.4a) . The rotational equilibrium around the

torsion angles is expressed in ranges (syn $0 , synclinal (sc) $60 , anticlinal (ac)

$120 , and antiperiplanar (ap) $180 ) and can be depicted with the help of

Newman projections (Figure 12.4b). Interestingly, the canonical ranges for all the

torsion angles are essentially identical in DNA and RNA (Àsc for a, ap for b, ỵsc for

c, ỵsc for d, ap for e, and Àsc for j). Yet, the overall three-dimensional structures of

these helices are distinctly different. Greater insights into these differences can be

gained by examining the fundamental structural difference at the monomer level

between DNA and RNA, which is the conformation of the furanose ring.

The furanose ring in DNA and RNA is not planar and exists in an envelope (E)

form with four atoms in one plane and one out of plane, or in a twist (T) form with



Figure12.3 Structuresof (a)B-typeDNA/DNAand(b) A-typeRNA/RNAduplexes showing position

and relative widths of the major and minor grooves [27].



12.4 Structural Features of DNA and RNA Duplexes



(b)



(a)



O

O Pα

O

O β γ



ε

O

O Pξ

O

O

(c)



X



O χ Bx

δ



O

2' Bx

Bx

5'

5'

O 1'

O 1'

4' 3'

4'

2'

2

3

O



3'



O

O P

O O



(d)



~5.9 Å



O

5'



O



4'



O



Bx



O

P



O



γ

4'



O 3'



O 1'



Bx



2'



R



R = H, DNA

R = OH, RNA



H H O

O



H

O



R



I (γ in +sc)



Figure 12.4 Conformational descriptors for

nucleotides and nucleic acids. (a) Backbone

torsion angle descriptors. (b) Torsion angle

ranges depicted using Newman projections.

(c) Envelope and twist conformations of the



3'

4'



3

2T



O

O



Bx



O



1'

2'



Bx



O3',O4'-gauche

O

O

P

O

O

C2'-endo or South

preferred in DNA



H

Bx



1'



X



-sc



O

2' Bx

5'



~7.1 Å



C3'-endo or North

preferred in RNA



(e)



Y



-ac



3'

2

3T



O



O O

O P

OH

O2',O4'-gauche

O O

O3',O4'-trans



5'



Y

ap



E



E



X

Y



Y

+ac



+sc



O



X



X



Y



O



Bx



H



H

O



H H O



O



R



II (γ in ap)



O

O



Bx



H

R



III (γ in -sc)



furanose ring. (d) Conformational equilibrium

of the furanose ring showing the North

(C30 -endo) and South (C20 -endo) sugar puckers.

(e) Conformational equilibrium around the

exocyclic C40 —C50 bond.



three atoms in one plane and two out of plane (Figure 12.4c). This conformational

equilibrium is described with the help of two descriptors: (i) the pseudorotation

phase angle (P), which defines the type of deviation from planarity, and (ii) the

amplitude of puckering (tm), which defines the magnitude of deviation from

planarity [28]. These conformations are represented as a pseudorotation cycle,

where the E and T forms alternate every 18 . The top of the pseudorotation cycle is

designated as North and after rotation of 180 the mirror image conformation or

South is encountered. The furanose rings in DNA and RNA generally prefer the



409



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12 Unnatural Nucleoside Analogs for Antisense Therapy



two ranges in the pseudorotation cycle: C30 -endo or North (P $ 0–36 ) and C20 -endo

or South (P $ 144–190 ) with the preference being largely determined by the

presence or absence of the hydroxyl group at the C20 -position.

The furanose rings in DNA and RNA are also replete with stereoelectronic effects

due to the presence of an electron-withdrawing oxygen atom at every carbon atom

of the five-membered ring with the exception of DNA, which lacks the hydroxyl

group at the C20 -position [29–31]. This atom arrangement ensures that every

oxygen atom is spaced within two carbon atoms of each other. As a result, the

oxygen atoms tune the conformation of the furanose ring to position themselves in

a gauche orientation relative to each other (gauche effect) [32]. This orientation of

two electronegative atoms on adjacent carbons is stabilized by hyperconjugation of

the electron-deficient sà orbital of the CÀÀO bond with the s orbital of the anti

CÀÀH or CÀÀC bond.

In DNA, the absence of the C20 -oxygen atom ensures that the C30 - and C40 oxygen atoms position themselves in a gauche orientation, which drives the

conformation of the furanose ring toward the S-type or C20 -endo sugar pucker

(Figure 12.4d). This arrangement increases the distance between the 30 - and 50 phosphodiester (PO) linkages and results in the formation of a B-form duplex with

$10 bp per helical turn. In contrast, the presence of the 20 -oxygen atom in RNA

steers the conformation of the furanose ring toward the N-type or C30 -endo sugar

pucker, which positions the C20 - and C40 -oxygen atoms in a gauche orientation. This

shortens the distance between the 30 - and 50 -phosphodiester linkages resulting in a

more compressed duplex with $11 bp per helical turn [27]. In addition to the

electronic effects outlined above, the presence of the 20 -hydroxyl group in RNA also

results in the formation of a water lattice network in the minor groove, which

further stabilizes RNA/RNA duplexes by dissipating some of the backbone charge

density at the edge of the minor groove [33].

In addition to the conformational equilibrium of the furanose ring, there also

exists a rotational equilibrium around the exocyclic C40 –C50 single bond in DNA

and RNA nucleotides (Figure 12.4e). This equilibrium results in three main

conformations where the C50 -OH and the C40 -O ring oxygen are in a staggered

orientation (I, II, and III). Of these, conformations I and II are favored as they

position the C50 -OH and the C40 -O in a gauche orientation while conformation III is

least favored as it positions the electronegative oxygen atoms in an anti orientation.

Conformation I is further stabilized by a CH Á Á Á O type interaction between the 50 O

and H6 in pyrimidines or H8 in purines [34]. As a result, I is the most commonly

observed conformation in DNA and RNA duplexes.



12.5

Improving Binding Affinity of Oligonucleotides by Structural Mimicry of RNA



As mentioned previously, RNA duplexes are inherently more stable than DNA

duplexes. Oligonucleotide medicinal chemists have used this intrinsic property of

RNA duplexes to design novel analogs that mimic the C30 -endo conformation of the



12.5 Improving Binding Affinity of Oligonucleotides by Structural Mimicry of RNA



furanose ring in RNA, as a general strategy to improve ASO affinity and nuclease

stability. These strategies are exemplified by (i) analogs that exist predominantly in

the C30 -endo sugar pucker such as 20 -modified RNA, (ii) analogs that are locked in

the C30 -endo sugar pucker such as the class of 20 ,40 -bridged nucleic acids (BNAs),

and (iii) analogs that mimic the C30 -endo sugar pucker using a non-furanose ring

system such as hexitol nucleic acids (HNAs).

12.5.1

20 -Modified RNA



Over 100 post-transcriptionally modified nucleosides have been identified in various

types of RNA [35], several of which are modified at the 20 -position of the nucleoside

furanose ring. Nature uses 20 -modified RNA to improve nuclease stability and to

prevent metabolic modification of key nucleotides in tRNA and ribosomal RNA.

Introducing alkyl substituents on the 20 -hydroxyl group of RNA also prevents

autocatalytic hydrolysis, which can occur by nucleophilic attack of the 20 -hydroxyl

group onto the 30 -phosphodiester linkage resulting in strand cleavage. Over the

years, modifying the 20 -position of RNA has been a prolific area of research and has

yielded a plethora of modifications for the medicinal chemist toolbox [36].

Introducing substitution on the 20 -hydroxyl group projects the appended moiety into

the minor groove while maintaining the C30 -endo sugar pucker. Moreover, since the

minor groove in RNA duplexes is wide and shallow, it can accommodate a host of

substitutions without affecting duplex stability [37]. The list of 20 -modified RNA

analogs described in the literature is perhaps too long for a complete summary here.

Instead, we will focus on some important 20 -modifications that have been extensively

investigated in the context of nucleic acid therapeutics.

12.5.1.1 20 -O-Me RNA

20 -O-Me RNA (3, Figure 12.5a) is a naturally occurring RNA analog that is found

ubiquitously in tRNA and ribosomal RNA. 20 -O-Me RNA was one of the earliest

sugar modifications employed in antisense medicinal chemistry and still remains a

widely used modification in nucleic acid therapeutics. ASOs containing 20 -O-Me

RNA have been used for RNase H-based antisense [38], for improving the nuclease

stability and reducing the off-target effects of siRNA [39,40], and for modulating

RNA splicing [18]. The antisense mechanism employed is largely determined by

the design of the oligonucleotide. For example, chimeric ASOs comprising of a

central gap region of phosphorothioate (PS) DNA flanked on either side with 20 -OMe RNA were originally used for RNase H applications (Figure 12.5c). This

chimeric configuration came to be known as a “gapmer” and remains the most

extensively studied ASO design to date [13]. The 20 -modified nucleotides in the 30 and 50 -flanks of a gapmer improve affinity of the ASO for RNA and also protect the

ASO from nuclease-mediated degradation, while the central DNA gap region

supports RNase H-mediated digestion of the targeted RNA. For RNAi applications,

introducing 20 -O-Me RNA at positions 2–3 in the guide strand has been shown to

reduce off-target effects [40]. Alternately, a more audacious use of this modification



411



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12 Unnatural Nucleoside Analogs for Antisense Therapy



(a)

O



O



O

O



OH

RNA



O



O



Bx

O



O



OMe



O O



R

C3'-endo or North

R = OH, OMe, F,

O-methoxyethyl



O

O



O



O



(c)

Bx



O



Bx



MeO

2'-O-Methoxyethyl RNA

4 (MOE)



2'-O-Me RNA, 3



(b)

O



O



O



Bx



OH



Bx

F



2'-F-RNA, 5

(FRNA)



O



Bx



Bx

R



O

C2'-endo or South



O

O P

O

S



R

O



O

O P

O

S

HO



n = 2-5

Bx

m = 6-14

O



Bx

R

n = 2-5



0



Figure 12.5 (a) Structures of some 2 -modified analogs of RNA. (b) Conformational equilibrium of

the furanose rings in 20 -modified RNA. (c) Structure of a “gapmer” ASO.



in conjunction with the 20 -deoxy-20 -fluoro RNA (20 -F RNA, described later) involved

replacing every alternate RNA nucleotide in the guide and the passenger strand

with 20 -O-Me and 20 -F RNA [41]. This ASO design greatly improved the thermal

stability, nuclease stability, and the potency of siRNA in cells and remains one of

the only designs where each RNA nucleotide in the siRNA duplex was replaced

with a modified nucleotide. More recently, it was shown that an appropriately 50 phosphate-modified alternating 20 -OMe/20 -F fully modified single-stranded RNA is

capable of activating RNAi in cell culture and in animals by loading into Ago2 and

cleaving targeted RNA. Importantly, the activity in animals was achieved without

the need of cationic lipid formulations to deliver the ASO into hepatocytes [42].

Prior to this report, it was generally perceived that only dsRNA could be loaded into

the RISC complex and induce gene silencing. This report represents an outstanding example of using chemical modifications to engineer nuclease stability

and binding affinity into an ASO while imparting mechanistic competency.

12.5.1.2 20 -O-Methoxyethyl RNA

Despite the many uses of 20 -O-Me RNA, this modification has largely been

supplanted by the 20 -O-methoxyethyl (MOE, 4, Figure 12.5a) RNA modification,



12.5 Improving Binding Affinity of Oligonucleotides by Structural Mimicry of RNA



especially for RNase H-based antisense applications [43]. The methoxyethyl

substituent essentially represents an ethylene glycol moiety appended at the 20 position of RNA [44]. The addition of a second oxygen atom within two carbon

atoms of the 20 -oxygen of RNA imparts restricted rotation around the ethylene

linker and positions the oxygen atoms in a gauche orientation relative to each other.

The crystal structure of a MOE RNA duplex showed that the MOE side chain traps a

molecule of water and increases the hydration and steric hindrance around the 30 phosphodiester linkage resulting in a significantly improved nuclease resistance

profile relative to 20 -O-Me RNA [45]. The restricted rotation of the MOE side chain

further rigidifies the ribose C30 -endo sugar conformation (Figure 12.5b) and

improves affinity for complementary nucleic acids relative to 20 -O-Me RNA. Firstgeneration PS DNA [46] ASOs are known to bind to many proteins resulting in

hybridization-independent nonspecific toxicities such as stimulation of immune

receptors. The ability of the MOE side chain to create a sphere of hydration around

the phosphorothioate backbone has been useful for mitigating the nonspecific

interaction of PS DNA-modified ASOs. Next only to the PS backbone modification,

MOE is the most widely evaluated nucleic acid modification in human clinical

trials. Several gapmer ASOs containing MOE nucleotides are in human clinical

development. One particular ASO, mipomersen (KynamroTM), targeting apolipoprotein B 100 (Apo-B 100), has recently completed several phase III clinical trials

and is awaiting registration by regulatory authorities in Europe and the United

States [47–49]. In addition, a fully modified MOE ASO that modulates the splicing

of the survival of motor neuron (SMN) protein has entered human clinical trials for

the treatment of spinal muscular atrophy (SMA) [50–52].

12.5.1.3 20 -Fluoro RNA

The 20 -F modification of RNA (FRNA, 5, Figure 12.5a) where the 20 -OH group is

replaced with a fluorine atom has been extensively investigated in the context of

oligonucleotide therapeutics. FRNA has been used to modify hammerhead

ribozymes [53], in the flanks of gapmer ASOs [13], in the sense and antisense

strands of siRNA duplexes [41,54], in ssRNAi [42], in microRNA antagonist [55–57],

and to orchestrate the behavior of splice modulating ASOs [19]. In addition,

Macugen, an oligonucleotide aptamer targeting vascular endothelial growth factor 1

(VEGF1), is partially modified with FRNA and is approved by the FDA for the

treatment of macular degeneration [58]. Fluorine has roughly the same atomic radii

as hydrogen but it is highly electronegative [59,60]. As a result, fluorine behaves as

a polar hydrophobic surrogate for the 20 -hydroxyl atom in RNA and steers the

furanose ring into the C30 -endo sugar conformation. FRNA-modified nucleic acids

show improved duplex thermal stability when paired with RNA complements, and

the duplex resembles the A-form geometry similar to RNA/RNA duplexes [61,62].

However, FRNA-modified duplexes have a considerably drier minor groove since

the fluorine atom does not participate in hydrogen bonding with water molecules

in the minor groove, and the improved thermal stability has been attributed to

increased enthalpy as a result of stronger Watson–Crick base pairing [63]. Modeling

experiments showed that FRNA-modified RNA does not interfere and may even



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12 Unnatural Nucleoside Analogs for Antisense Therapy



enhance interactions with Ago2. This in turn could explain the unique gene

silencing properties of FRNA-modified siRNA [54].

12.5.2

20 ,40 -Bridged Nucleic Acids



In the early 1990s, several groups started to explore the concept of covalent

conformational restriction of the furanose ring to improve the affinity of modified

oligonucleotides for complementary RNA [64,65]. The Wengel and the Imanishi

groups independently explored the concept of restricting the conformation of the

nucleoside furanose rings along different stages of the pseudorotation cycle by

creating covalent tethers between different atoms of the furanose ring system. As

part of these efforts, they found that tethering the methyl group in 20 -O-Me RNA

back to the 40 -position locked the furanose ring in the C30 -endo sugar conformation

(Figure 12.6). This nucleoside analog was termed as “locked nucleic acid” or LNA

(6, also known as 20 ,40 -methyleneoxy BNA) and modified oligonucleotides showed

unprecedented increases in the thermal stability of modified duplexes [66–69].

Depending on the sequence context, duplex thermal stability could be enhanced

between ỵ5 and ỵ9  C/modication when the modified oligonucleotides were

paired with RNA or DNA complements.

Structural studies of LNA/LNA duplexes showed that the helix is compressed and

comprises of $14 bp per helical turn (as opposed to 10 for B-form DNA and 11 for

A-form RNA) [70]. Thus, the improved affinity obtained using LNA-modified

oligonucleotides can be traced to improved base stacking, which results from

locking the furanose ring in the C30 -endo conformation. A number of structural

analogs of LNA that replace the 20 -oxygen atom with an isosteric element have

been reported (Figure 12.6). Replacing the 20 -oxygen atom in LNA with sulfur

(20 -thio-LNA 7) [71,72] is well tolerated while replacing it with carbon (carba-LNA 8)

reduces duplex thermal stability relative to LNA [73]. Replacing the 20 -oxygen atom

O



Bx



O O



2',4'

constraint



O

O



Me

2'-O-Me RNA, 3

O

O

O O



Bx



O S



O

Bx



O



O



O

O



O



O



O

LNA, 6



O



O



Bx



O



O



Bx



Bx



O



Carba-LNA, 8

2'-Thio-LNA, 7

LNA, 6

(ΔT m +5 oC/mod.) (ΔT m +5 oC/mod.) (ΔT m +3 oC/mod.)



O



Bx



O N

R

2'-Amino-LNA 9

(ΔT m +4 oC/mod.)



Figure 12.6 20 ,40 -Bridged nucleic acids are conformationally restricted 20 -modified analogs of RNA.



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