Nucleosides in RNA, Modied

N. Dinuka Abeydeera and Christine S. Chow,

University, Detroit, Michigan
doi: 10.1002/9780470048672.wecb688

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Background to Modied Nucleosides in RNA Synthesis of Modied Nucleosides Incorporation of Modied Nucleosides into RNA Applications of Modied RNAs Summary and Outlook

Wayne State

Natural and unnatural modied nucleosides play important roles in chemistry and biology. Over 100 different functional group modications occur on natural nucleosides, which range from sugar methylations to base isomerization reactions. Unnatural modications are also broad in their range of possible structures and applications. Both types of modications have been incorporated site specically into RNAs to understand their biological roles or to be used as biophysical, structure, and mechanistic probes. More recently, modied RNAs have been applied as therapeutic agents. This article summarizes the approaches toward generation of modied nucleosides and their incorporation into RNA using chemical, biochemical, or combined (semisynthesis) approaches. Some examples of applications with modied RNAs will also be discussed.

Background to Modied Nucleosides in RNA

The expansive array of RNA functions discovered to date is highly dependent on the ability of RNA to fold into unique structures, undergo large conformational changes, or participate in specic interactions with macromolecules (e.g., RNA and proteins), metal ions, and small organic ligands. Although the core nucleotide structures are sufcient for many biological roles of RNAs, over 100 natural modications in RNA have been discovered over the past six decades, which seem to modulate RNA function (1, 2). In addition, a wide variety of unnatural nucleosides has been synthesized and incorporated into RNA. These modications often provide RNA with unique properties, such as expanded or altered hydrogen-bonding; van der Waals, base-stacking, or electrostatic interactions; as well as unique metal-binding sites, chemical reactivity, or uorescent characteristics. The synthetic routes to generate modied nucleosides vary considerably, as do the routes for incorporation of the nucleosides into small oligonucleotide fragments or large, full-length RNAs. The syntheses of modied RNAs serve many purposes, such as developing a better understanding of the biological roles of natural modications, generating tools for studies of RNA structure and function, and producing novel therapeutic agents. The types of nucleoside or nucleotide modications range from alterations of the base component to changes in the sugar or phosphate moieties. The natural modications include pseudouridylation (generation of a C -glycoside), methylation,

deamination, thiolation, and alkylation (1, 2). Hypermodications also exist in which multiple components of the nucleotide are altered. Some unnatural modications include uorescent bases, such as 2-aminopurine, or novel nucleobases to expand the genetic code. This review summarizes some of the most common approaches taken by chemical biologists toward generation of modied nucleosides and their incorporation into RNA using chemical, biochemical, or combined (semisynthesis) approaches. It is possible to use these techniques to construct RNA molecules with a wide variety of functional groups at highly specic positions. Although the applications of modied RNAs are numerous and far reaching, it is not possible to give a comprehensive view in this short article; therefore, representative or recent examples of applications have been selected for discussion.

Synthesis of Modied Nucleosides

The chemical synthesis of novel nucleosides or those that mimic natural modications will provide biologists with new probes for RNA function. Thousands of modied bases have been described in the literature, which range from synthesis of both natural and unnatural nucleosides to their incorporation into short oligonucleotides and full-length nucleic acids. Numerous different approaches have been taken to generate modied nucleosides (36), which clearly cannot all be described in this brief review. Therefore, several examples have been selected to highlight some general strategies for preparing nucleoside analogs. Traditional methods for modied nucleoside synthesis 1

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Nucleosides in RNA, Modied

involve either a glycosylation reaction between a purine or pyrimidine base and an activated sugar, or derivatization of a standard nucleoside.

Generation of modied nucleosidescoupling strategies

Most nucleosides contain N -glycosidic linkages (i.e., linkages through the base nitrogen atom). The N -glycosides can be generated by reactions with a suitably protected and activated sugar component and a silylated base (purine or pyrimidine) fragment. The most commonly used reaction in N -glycoside synthesis is the Hilbert-Johnson-type sugar-base condensation in the presence of a Friedel-Crafts catalyst, such as SnCl4 , which can be used to produce pyrimidine, purine, as well as sugar- and/or base-modied nucleosides (Fig. 1a) (7, 8). Several improvements to these methods have been made since the original reports, which include the use of microwave-assisted reactions to generate nucleoside libraries (9). Maydanovych et al. (10) have used similar coupling strategies to generate adenosine analogs for the study of RNA editing, and they reported the detailed procedures in a recent review article. The less-common C -glycosides contain a C-C linkage between the carbohydrate moiety and the heterocyclic base. A wide range of aryl C -glycosides can be generated by using the Heck coupling reaction, as recently reviewed by Wellington and Benner (11). Pseudouridine (Fig. 1b), which is the most abundant natural modication found in RNA, is a C -glycoside generated by posttranscriptional isomerization of uridine residues at specic sites along the oligonucleotide chain. Although numerous groups have reported on the synthesis of pseudouridine, Hanessian, and Machaalanis stereocontrolled synthesis is the most convenient and efcient route to date (12). Their synthesis involves a reaction between a suitably protected D-ribonolactone and lithiated pyrimidine, followed by sequential ring-opening and ring-closing steps to produce either or pseudouridine in high yields. The ratio of / anomers is controlled by the presence of L-selectride and zinc chloride.

Methylation at the N 2 position on guanosine to yield m2 G and m2 2 G can be achieved following sugar and O 6 protection and generation of a 2-uoro intermediate. The 2-uoro intermediate is treated with CH3 NH2 or (CH3 )2 NH to yield m2 G or m2 2 G, respectively (Fig. 2a, steps iii-vii) (13). Alternatively, m2 G can be generated through reduction of a suitably protected p -thiocresol intermediate (Fig. 2a, steps iii, viiix) (14). Methylated adenosine analogs can be obtained using inosine as a starting material. Generation of the 6-chloro intermediate allows for conversion into m6 A or m6 2 A with CH3 NH2 or (CH3 )2 NH, respectively (Fig. 2b, steps xi, xii, vi, vii) (13). Methylated cytidine analogs can be obtained from 5-methyluridine (m5 U) as the starting material (15). Suitably protected m5 U is reacted with P2 S5 to yield a 4-thio intermediate that is then converted to 5-methylcytidine (m5 C) during treatment with NH3 (Fig. 2c, steps xiiixv). Uridine can be used as a starting material for the synthesis of N 4 -methylcytidine (m4 C), N 4 -2 -O -dimethylcytidine (m4 Cm) and 2 -O -methylcytidine (Cm) (Fig. 2d, xvixx). A tetrazole intermediate is generated (16, 17) that can be converted to m4 C or m4 Cm with the appropriate methylating agent. The 2 -O -methyl group is installed through an anhydronucleoside intermediate and opening with Mg(OCH3 )2 in methanol (18). Methylated uridine analogs can be generated using related methods, such as treatment of uridine with NaH and MeI (Fig. 2d, steps i, ii) (13) to give m3 U or conversion to 2 -O -methyluridine (Um) through an anhydronucleoside intermediate and opening by the appropriate nucleophile (18). Several unnatural nucleoside phosphate modications (Fig. 2e) have been generated, which include phosphorothioate, phosphoroamidate, phosphorothiolate, and methylphosphonate derivatives. The syntheses of these modications have been reviewed by Verma and Eckstein (19). A modication of those procedures can be used for the generation of phosphoroselenoate RNAs (20).

Convertible nucleotides
Convertible nucleotides are modied nucleotides that contain reactive functionalities. They are incorporated into RNA at specic locations and allow for convenient postsynthetic introduction of chemical probes, isotope labels, or cross-linking agents, such as disulde linkages. In this approach, the nucleotide derivative contains a leaving group on its base moiety and is incorporated into the oligonucleotide of interest at a dened location (21). Once the preparation of the desired precursor RNA is completed, the oligonucleotide is treated with an appropriate nucleophile (e.g., amine), which can displace the leaving group and become attached to the specic base moiety (Fig. 3). As will be discussed in more detail later, the convertible nucleosides have many applications, and they can be used to control the RNA structure and function, as well as to regulate RNA conformational switching and dynamics.

Nucleoside modications
An alternative route to modied nucleosides involves derivatization of the standard nucleosides, guanosine, adenosine, cytidine, and uridine. Among the large repertoire of modications identied to date, the methylated nucleotides are highly abundant and occur frequently in the functionally important regions of RNA (1, 2). Methylation typically occurs on the sugar or base portion of the nucleoside. Although many approaches have been taken to methylate nucleosides, site-selective introduction of a methyl group can be challenging. A recent report by H obartner et al. (13) showed the synthesis of a wide variety of methylated ribonucleosides, which included 1-methylguanosine (m1 G), N 2 -methylguanosine (m2 G), N 2 ,N 2 -dimethylguanosine (m2 2 G), 1-methylinosine (m1 I), 3-methyluridine (m3 U), N 4 methylcytidine (m4 C), N 6 -methyladenosine (m6 A), and N 6 ,N 6 dimethyladenosine (m6 2 A). Guanosine methylation at the amido nitrogen to yield m1 G can be achieved by treatment of guanosine with NaH in DMSO followed by addition of methyl iodide (Fig. 2a, steps i, ii) (13). 2

Enzymatic approaches to nucleoside triphosphate synthesis

Nucleotides can also be prepared by using enzymatic reactions. Modied nucleoside triphosphates are useful as precursors for

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Nucleosides in RNA, Modied

(a)

O NH HO OSiMe 3 OSiMe 3 N N OSiMe 3 BzO BzO O

iv i ii, iii

N O HO OH

N N O uridine NH2 N HO N O NH2 O

OBz

BzO OAc O BzO OBz

i, iii

N HO Bz N N N SiMe 3 N SiMe 3 O OH OH adenosine N N

HO HO cytidine

(b)
OtBu PGO O O O O N + OtBu I HO
i iv

O HN N HO pseudouridine NH O O OH

PG = protecting group OtBu N PGO O OH O O

ii

OtBu N OtBu PGO OtBu O O

iii

N HO

OtBu PGO N

N OtBu

HO

O O O

Figure 1 A general scheme for the synthesis of N- and C -glycosides is shown. a) The preparation of N-glycosidic linkages for pyrimidines and purines is outlined: i) SnCl4 , ClCH2 CH2 Cl, 2270 C, 45 hours; ii) saturated NaHCO3 ; iii) NH3 in CH3 OH, rt, 16 hours; iv) excess NH3 (7, 8). b) The preparation of a C -glycoside, pseudouridine, is shown: i) t-BuLi, THF, 78 C; ii) ZnCl2 , L-Selectride, DCM/THF, 78 C to rt; iii) PPh3 , DIAD, THF, 0 C to rt, 24 hours; iv) 70% AcOH, 50 C, 5 hours (12).

the synthesis of RNA through a modied DNA template and T7 RNA polymerase. The chemoenzymatic synthesis of nucleoside triphosphates was recently reviewed by Wu et al. (22). A recent example is the synthesis of 8-azaguanosine (8azaGuo), which is a guanosine analog with N8 in place of C8 that displays uorescent properties, and the conversion to the corresponding

triphosphate (8azaGTP) (23). The strategy for 8azaGTP synthesis involves the use of enzymes from the pentose phosphate and nucleotide salvaging pathways, many of which are commercially available. The presence of the triphosphate allows for the incorporation of 8azaGuo into RNA using a DNA template and T7 RNA polymerase. 3

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Nucleosides in RNA, Modied

(a)
N HO O OH OH m1G
i, ii

O N N CH3 ONPE NH2 HO O OH OH m2 G N NH2

v, vii

N N N

N NH CH 3

ONPE
v, vi

ONPE N N N CH 3

O N HO O AcO OH OH guanosine (G)

iii, viii

N NH AcO N O OAc N

N HO O

NH2

iii, iv

CH 3

O N N NH HO N NH CH2 S AcO OAc OH OH m2G

ix, x

O NH N

OH OH m 22 G

AcO O

N O

NH CH3

CH3

(b) N HO O Cl O N N HO O AcO OH OH inosine (l) OAc N N NH AcO

xi, xii vi

NHCH3 N N

OH N N
vii

OH m6A H3C N CH3

N O

N HO O OH OH m62A N N

Figure 2 Nucleoside base, sugar, and phosphate modications are shown. The syntheses of methylated guanosine (a), adenosine (b), cytidine and uridine (c and d) nucleosides are highlighted. Reaction conditions: i) NaH, DMSO, rt, 2 h; ii) MeI, rt, 5 h (13); iii) Ac2 O, DMAP, Et3 N, CH3 CN, 0.5 hours; iv) NPE, PPh3 , DEAD, dioxane, rt, 2 hours; v) HBF4 , NaNO2 , acetone/water, 20 C to rt, 3 hours; vi) 8 M CH3 NH2 , ethanol, 712 hours; vii) (CH3 )2 NH in ethanol/water, rt, 39 hours (13); viii) p-thiocresol, CH3 CO2 H, HCHO, ethanol, reux, 4 hours; ix) NaBH4 , DMSO, 100 C, 1 hour; x) NH3 in CH3 OH, rt, 16 hours (14); xi) Ac2 O, DMAP, pyridine, rt, 16 hours; xii) chloromethylenedimethyliminiumchloride, CHCl3 , reux, 4 hours (15); xiii) benzoyl chloride, pyridine, 55 C, 3 days; xiv) P2 S5 , pyridine/water, reux, 4 hours; xv) NH3 in CH3 OH (sealed), 100 C, 24 hours; xvi) tetrazole, TsCl, diphenyl phosphate, pyridine, rt, 36 hours (16, 17); xvii) CH3 NH3 + Cl , KOH, (C2 H5 )3 N, CH3 CN/water, rt, 24 hours; xviii) (PhO)2 CO, NaHCO3 , DMF, 80 C, 3 hours; xix) Mg(OCH3 )2 , CH3 OH, reux, 5 hours (18); xx) NH4 Cl, KOH, (C2 H5 )3 N, CH3 CN/water, rt, 24 hours. e) Variations of nucleoside phosphate modications are shown.

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Nucleosides in RNA, Modied

(c) H3C

O NH N O OH OH BzO O
xiii, xiv

S H3C NH N O OBz O
xv

NH2 H3C N N O OH OH m5 C O

HO

BzO

HO

5-methyluridine (m5 U)

(d)

N N

N N NHCH3 N N O
xvii, x

AcO O
xi, xvi

HO O

AcO

O NH HO O OH OH uridine (U)
xviii

OAc O N CH3

OH OH m4 C

i, ii

HO O

OH OH m3U O N HO O OH N O
xix

N O NH HO O OH OCH3 Um NHCH3 N HO O OH OCH3 m4 Cm N O HO N O AcO O AcO

xvii, x

N N N N N O

xi, xvi

OCH3
xx, x

NH2 N N O OH OCH3 Cm O

(e)

S O

O P O

O Base O
O

Se O

O P O

N P O

phosphorothioate

O P O

OR

phosphoroselenoate

H3C P O

O O

Base phosphoramidate
O O S P O

O OR

methylphosphonate

phosphorothiolate

Figure 2 (continued )

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Nucleosides in RNA, Modied

single site incorporation X 5 3 :Nu (amine, probe, etc.) 5 X

multiple site incorporation X 3 :Nu-S-S-Nu:

Nu 5 3

S-S

3
Figure 3 The convertible nucleoside approach is shown (21). On the left, a single convertible nucleoside that has been incorporated site-specically into the RNA is treated with a nucleophile to displace the leaving group, X. On the right, an RNA with multiple convertible nucleosides can be used to generate a cross-linked analog.

Incorporation of Modied Nucleosides into RNA

Through a combination of chemical and biochemical approaches, modied nucleosides can be incorporated into RNA either globally (i.e., at many sites) or in a site-specic manner. Depending on the particular application of interest, either a global or site-specic incorporation strategy may be desirable.

Enzymatic incorporation
Modied nucleotides in RNA can be generated by using site-specic enzymes. Several naturally occurring modifying enzymes have been isolated and used to modify either small oligonucleotides or full-length RNA templates at specic sites (Fig. 4a) (24). The enzyme reactions are useful for understanding the biological roles of the modied nucleosides; however, they are not as useful for generating modied RNAs for biophysical studies or as therapeutic agents because the reactions do not go to completion, and the modied RNAs are often difcult to separate from the unmodied RNAs. RNAs in bacteria and eukaryotes are modied by different pathways. Individual modications in bacterial RNAs are mediated by one of many unique protein enzymes that are typically site specic and target particular structures or sequences of RNA (24). The most common enzymes are methyltransferases and pseudouridine synthases. Because of the greater number of modications in RNAs from higher organisms, an alternative pathway of insertion is used that employs fewer site-specic enzymes. Noncoding RNAs known as small nucleolar RNAs (snoRNAs) combine with a set of proteins to generate a small nucleolar ribonucleoprotein (snoRNP) complex that guides the site of enzyme modication on the target RNA (25). The discovery of snoRNAs has been extremely important for understanding the biological roles of RNA modications, but the use of snoRNPs is not as convenient for generation of site-specically modied RNAs for biophysical studies. 6

RNA segments can be modied at their 3 ends by using a bisphosphate analog of the modied nucleoside and T4 RNA ligase (Fig. 4b) (26). This reaction is general and essentially can be applied to any RNA sequence and modication type that can be converted into a bisphosphate. The 3 -modied RNA segment can then be dephosphorylated with calf intestinal phosphatase and ligated with another 5 -phosphorylated RNA fragment to generate larger RNAs with site-specic modications (Fig. 4b) (26). Methods for ligation include the use of a DNA splint with either T4 RNA ligase or T4 DNA ligase (27, 28). The presence of the complementary DNA fragment promotes formation of the desired RNA product and minimizes dimer or circular RNA formation. A second enzymatic approach employs T7 RNA polymerase and a DNA template that directs the addition of specic ribonucleotides according to standard Watson-Crick base-pairing rules. This transcription method can be used to generate RNAs of any length and sequence. The T7 RNA polymerase will often accept modied nucleoside triphosphates. The method is useful for global modication, but typically it does not allow for site-specic incorporation of modied nucleotides. This limitation has been overcome in several cases, in which nonstandard nucleotides are used to direct the insertion of modied nucleotides through alternative base-pairing schemes (Fig. 4c) (29).

Chemical approaches
The chemical synthesis of RNA has the distinct advantage of allowing for site-selective incorporation of modied nucleosides into the oligonucleotide chain. The early phosphodiester method developed by Khorana (30) was followed by the H-phosphonate and phosphoramidite approaches (31). Although the H-phosphonate approach has distinct advantages over phosphoramidite synthesis, it is not as widely used. Therefore, the focus of this section will be on the more commonly employed

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Nucleosides in RNA, Modied

(a) N N

E (snoRNP)

(b)

+ pXp

T4 RNA ligase

Xp

CIP
T4 RNA ligase

X p (c) DNA promoter Y DNA template

T7 RNA polymerase NTPs + XTP
X O O Z
1) coupling

X RNA transcript

(d)

BasePG 2

O P O

O Y Base P 1 O X O Y Z

2) capping 3) oxidation

O O

Base PG2 AcO

HO O O

Base PG 1 + O Y

O O

O Base PG 2

O P O

O O

O Y + Base PG 1 O O O O Y

Base PG1 O O Y

O P N

O Y

PG = base protecting group

3 nucleoside on solid support

HO O
steps 1 to 4 repeated

Base PG 2

O P O

O O

O Y Base PG 1 O

4) 5 deprotection

X = 5 protecting group Y = 2 protecting group Z = phosphate protecting group O O Y

Figure 4 The site-specic incorporation of modied nucleotides into RNA is depicted. a) Modifying enzymes (E) convert standard nucleotides (N) into modied versions (N-X) (e.g., methylation, pseudouridylation) (24). Eukaryotes require a snoRNP complex along with the modifying enzyme to direct the site of modication, whereas bacteria use unique enzymes to modify each site (25). b) T4 RNA ligase is used to add a single modied nucleotide (X) in the form of a bisphosphate (pXp), or to ligate a modied fragment to another piece of RNA (26, 27). c) Enzymatic incorporation employs T7 RNA polymerase and a modied DNA template, in which deoxynucleotide Y makes a unique base pair with ribonucleotide X (in the form of XTP) to incorporate the modied nucleotide at a single, specic site on the RNA chain (29). d) The steps for incorporation of modied nucleotides by solid-phase oligonucleotide synthesis are shown (3234). e) The most common protective groups for the phosphoramidite building blocks are highlighted.

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Nucleosides in RNA, Modied

(e) TBS chemistry

X
DMT MeO TBS

Y
CNEt

NC Si

P N

MeO

DMT

TOM MeO

CNEt

TOM chemistry

O Si

NC

P
N

MeO

BzH

ACE

Me

ACE chemistry

O O Si O Si O Si O O O O O H3C

P N

Figure 4 (continued )

phosphoramidite method. The chemical synthesis of RNA occurs on a solid support, such as a controlled-pore glass or polystyrene bead (Fig. 4d). The synthesis typically goes in the 3 to 5 direction, building from the 5 -hydroxyl group at each cycle. The 5 OH is protected with a suitable organic moiety that can be removed quickly and efciently. The 2 hydroxyl is also protected until the end of the synthesis. The monomer unit that reacts with the 5 OH is a nucleoside with a phosphoramidite moiety at the 3 hydroxyl. The phosphoramidite method involves four major steps after deprotection of the 5 -hydroxyl group from the 3 nucleoside attached to the solid support: 1) coupling at the 5 OH with a suitably protected phosphoramidite, 2) capping the unreacted 5 -OH groups, 3) oxidation of the phosphite linkage to generate a phosphate, and 4) removal of the 5 -protecting group of the newly added nucleotide unit (Fig. 4d). The nal steps involve removal of the oligonucleotide product from the solid support and deprotection of the 2 -OH groups and phosphate moieties. The nal RNA product is then puried using standard methods, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The choice of 5 and 2 protecting groups is critical for the successful generation of an oligonucleotide product. The chosen phosphoramidites must have high coupling efciencies and be stable during the entire oligonucleotide synthesis, yet easily 8

removed at the end of the synthesis. The synthetic route to generate the phosphoramidites should also be efcient in order to reduce the cost of the oligonucleotide synthesis. A wide range of protecting groups has been introduced for RNA synthesis over the past three decades; however, only the most commonly used methods will be discussed. One of the rst 2 -OH protecting groups to be employed was tert -butyldimethylsilyl (TBDMS or TBS) (32). More recently, [(triisopropylsilyl)oxy]methyl (TOM) (33) and benzhydryloxy-bis(trimethylsilyloxy)silyl (BzH) (34) have been developed for 2 -OH and 5 -OH protection (Fig. 4e). Whereas each method has distinct advantages and disadvantages, they complement one another because unique chemical modications on the nucleoside may be more compatible with one particular method. Modications that are chemically incompatible with these protecting groups would require alternative methods. Several key factors in choosing the type of phosphoramidite to use include: 1) fast coupling times (<2 minutes), 2) high coupling efciencies (>99.8%), 3) high stability in solution, and 4) the ability to generate long RNA fragments (>20 nucleotides in length). Many recent reviews on RNA synthesis protocols are available that provide more details regarding phosphoramidite preparation (6, 34). The rationale for choosing chemical synthesis to generate modied RNAs is that a wide range of both natural and unnatural modications can be incorporated at single or multiple

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Nucleosides in RNA, Modied

sites, and highly pure RNAs can be produced on a large scale (>mg). As mentioned earlier, convertible nucleotides can be generated in which a group of modied nucleosides that contain reactive functionalities are incorporated into RNA and allow for subsequent attachment of chemical probes, such as isotopic labels, tools for disulde cross-linking, or uorescent moieties (21). This postsynthetic modication allows for generation of site-specically modied RNAs in fewer steps compared with complete enzymatic or chemical synthesis (Fig. 3).

Semisynthesis approaches
The enzymatic and chemical approaches can be easily combined to generate >100 nucleotide-length RNAs that contain either natural or unnatural modications at specic sites. Either chemically or enzymatically produced fragments can be joined or ligated with T4 DNA ligase or T4 RNA ligase along with a DNA splint, as mentioned above (Fig. 4b) (27, 28). Together, these methods can be applied to generate large, full-length RNA molecules, such as 23S ribosomal RNA (approximately 3000 nucleotides in length), with site-specic modications to study RNA function (35).

Applications of Modied RNAs

Numerous motivations can be cited for generating site-specically modied RNAs. The rst reason is to use modied RNAs for structurefunction studies in either natural or model systems. For this purpose, many different biophysical approaches are employed, such as uorescence spectroscopy, NMR spectroscopy, and X-ray crystallography. For mechanistic studies, site-specic changes to the oligonucleotide allow for single-atom or functional-group changes and subsequent biochemical studies to be conducted and compared with wild-type RNA. A second major reason for constructing modied RNAs is to use them as therapeutic agents with enhanced stability or improved biological activity. Antisense oligonucleotides were recognized long ago as having potential therapeutic properties for downregulating gene expression. More recently, RNA interference (RNAi) has been discovered as another tool for regulation of mRNA activity. Other therapeutic agents include RNA aptamers and ribozymes. Also of recent interest is the use of modied nucleosides to expand the genetic code, as well as to understand the basic principles of Watson-Crick base pairing and recognition of certain RNA structures by enzymes, proteins, and other biological ligands.

to affect the stability or structure of the RNA. An enzymatic synthesis of large RNAs (up to 100 nucleotides) that contain 2 -methylselenium labels (Table 1) (3650) was reported by H obartner et al. (37), which was combined with chemical synthesis using the 2 -O -TOM methodology and a 2 -Se -methyladenosine phosphoramidite. More recently, they have used the ACE methodology and modications of all four standard nucleosides to generate RNAs suitable for X-ray analysis (38). In addition to the sugar modications, Carrasco et al. (20) have also generated selenophosphate modications. Nuclear magnetic resonance (NMR) methods have also been developed for three-dimensional structure analysis of RNA. The major challenge in RNA structure determination by NMR is the size limitation because of spectral overlap with large RNAs. Consequently, heteronuclear NMR has become increasingly important for solving the structures of large RNAs and/or RNA complexes with proteins and other ligands. Although RNAs modied at all or multiple sites with 13 C or 15 N labels can be generated using enzymatic methods (51, 52), site-specic placement of these labels is often desirable. Numerous approaches have been used in which modied nucleosides with specic 13 C and/or 15 N labels (see for example, Reference 17) are converted into the corresponding phosphoramidites and used for site-selective incorporation into the desired RNA fragment, which includes convertible nucleotides (21). In one recent example, an RNA hairpin that represents a region of the self-splicing Tetrahymena group I intron was generated with [8-13 C-7-15 N]-guanosine (Table 1) at one position and [7-15 N]-guanosine at another position, such that the two 15 N7 NMR signals from the RNA could be unambiguously assigned (39). The specically labeled sample was used to identify specic metalion binding sites that are important for RNA folding and catalysis.

Biophysical probes
A variety of nucleoside modications can be used to monitor RNA folding, stability, and structure, as well as ligand interactions. Fluorescent analogs, such as 2-aminopurine (2-AP) (Table 1), have proven to be highly useful to monitor RNA conformational changes and dynamics in solution (4042). The 2-AP modication causes very little perturbation to the RNA structure, and it has been used to monitor drug (e.g., aminoglycoside antibiotic) interactions with target RNA (53). In contrast, attachment of a bulky substituent, such as 8-bromoguanosine (8-BrG) (Table 1), to the RNA base may limit rotation about the glycosidic bond, favoring one conformation (e.g., syn ) over another (e.g., anti ). Such an approach has been used to stabilize YNMG RNA hairpin motifs and enhance the RNA folding rate, because of preorganization of the denatured state of the RNA (43). Generation of site-specically modied RNAs that can perform disulde cross-linking allows for RNA folding pathways to be examined (21, 54). Photoactivated cross-linking is another strategy that uses naturally occurring or synthetic modications, such as 4-thiouridine, 6-thioguanosine, 5-bromouridine, and 5-iodouridine (Table 1), to study RNA-RNA interactions in folded RNAs (19). 9

Structure studies
X-ray crystallography is a widely used technique for the three-dimensional structure analysis of RNA molecules. The extraction of information from X-ray diffraction patterns from RNA crystals requires proper phase determinations using methods such as multiwavelength anomalous dispersion in which placement of specic heavy atoms is required. Selenium-modied ribonucleotides have been used for these purposes by Du et al. (36), because the selenium atom does not seem

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Nucleosides in RNA, Modied

Table 1 Some representative modied nucleosides and nucleotides are shown Modication Application phasing tool in X-ray crystallography Reference(s) 3638

unambiguous NMR peak assignments during 3D structure analysis

39

uorescent probe of RNA conformational changes

4042

stabilize nucleoside conformations about the glycosidic bond

43

photoactivated cross-linking probe of RNA folding to study RNA-RNA interactions

19

photoactivated cross-linking probe of RNA folding to study RNA-RNA interactions

19

natural modied nucleoside

4447

mechanistic probe of RNA structure and function

48

10

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Nucleosides in RNA, Modied

Table 1 (Continued ) Modication Application probe for RNA-mediated catalysis in functional RNAs Reference(s) 49

photolabile moiety for the temporal and spacial control of siRNA targeting

50

Modied nucleosides incorporated into small RNA model systems can also be used to investigate the global versus individual effects of modied nucleotides on natural RNAs, such as rRNA or tRNA. For example, in some early studies, Yarian et al. (44) demonstrated that pseudouridine (Table 1) leads to increased thermal stability of the tRNA anticodon stem-loop region. Later, Meroueh et al. (45) demonstrated that pseudouridines have opposing effects on rRNA helix 69 stability, which depends on their specic locations and sequence contexts. These effects on stability may be important for conformational switching mechanisms in functional RNAs (46, 47).

Functional RNAs
RNAs have an enormous range of functions. Those functions have been the subject of numerous publications over the past several decades. One example is RNA aptamers, which are oligonucleotides that have been selected for and tailored to bind with high afnity and selectivity to a variety of ligands, which include nucleotides, peptides, proteins, vitamins, and drugs (57). Aptamers are engineered from random nucleic acid libraries through repeated rounds of in vitro selection. Modied nucleotides play important roles in aptamer function, because they provide enhanced stability to the RNA-based aptamers and can also endow them with desirable chemical reactivities or control of their folded structures, which would be benecial for the development of novel targeting agents. Several modied aptamers have already been developed as potential therapeutics that are currently in clinical trials (57). Nucleic acid selection methods have also been exploited for the development of novel RNA enzymes or ribozymes (58). An in-vitro selected RNA that contains the modied nucleotide 5-(4-pyridylmethyl)-uridine (Table 1) can catalyze carboncarbon bond formation in a Diels-Alder cycloaddition, with an 800-fold rate acceleration compared with a random RNA (49). Modied RNAs that contain the same uridine modication have also been selected to mediate metalmetal bond formation in the synthesis of palladium nanoparticles (59). Modied RNAs are likely to have many other applications as novel ribozymes that catalyze important biological reactions or can be used to create novel materials.

Mechanistic probes
Site-specically modied RNAs have been used in many applications to examine RNA structurefunction relationships, RNAprotein interactions, RNAligand interactions, and RNAcatalysis mechanisms. Some earlier studies demonstrated the use of synthetic oligonucleotides to probe the roles of specic functional groups and detailed mechanisms in ribozyme catalysis (55). The synthesis of nucleoside analogs allows for a full-range of chemical diversity (e.g., inductive effects, space-lling capacity, etc.) to be explored, such that quantitative structure activity relationships can be determined for RNA enzymes and other biologically important RNAs (56). Nucleotide analog interference mapping (NAIM) is an alternative method to conventional single-atom substitution experiments, which provides biochemical information related to the RNA structure and function (48). NAIM has the ability to screen rapidly the effects of chemical group substitution rapidly on RNA function. This method uses an array of modied 5 -O -(1-thio)-nucleoside triphosphates (Table 1) to assess concurrently the contributions of functional groups at every nucleotide position along the RNA backbone. The applications of this method range from mapping of RNA tertiary contacts and substrate contacts in ribozymes to quantication of the energetic contributions of RNAligand interactions.

RNAi/siRNA
Short interfering RNA (siRNA) is a class of molecules composed of double-stranded RNA molecules (2025 nucleotides) that are involved with the interference of specic genes (i.e., RNA interference or RNAi). During the RNAi process, the siRNA is incorporated into a protein complex, which unwinds the siRNA by an ATP-dependent mechanism to generate an 11

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active RNA-induced silencing complex (RISC) (60). The unwound antisense strand of RNA will then guide the RISC to the target mRNA and bind according to standard Watson-Crick base-pairing schemes. Strand scission of the target mRNA is induced by RISC, which leads to a decreased level of protein expression by the corresponding mRNA. The overall success of gene downregulation depends on the stability and structure of the duplex formed between the siRNA and target mRNA. This idea has led to the design and synthesis of numerous modied siRNAs with the anticipation that appropriate changes in the nucleotide composition will lead to enhanced stability, both in terms of chemical stability (i.e., to avoid nuclease degradation) and thermal stability (i.e., to generate a duplex with more favorable free energy, enthalpy, and/or entropy contributions). The modied oligonucleotides are also designed to have increased cellular uptake and properties that will enhance their biodistribution or entry into specic cell types (61, 62). Another type of nucleotide modication involves the addition of a photolabile moiety (Table 1), such that temporal and spatial control of siRNA targeting is possible (50).

Acknowledgments
We are grateful for support from the National Institutes of Health (Grants GM054632 and AI061192).

References
1. 2. 3. Limbach PA, Crain PF, McCloskey JA. Summary: the modied nucleosides of RNA. Nucleic Acids Res. 1994;22:21832196. Rozenski J, Crain PF, McCloskey JA. The RNA modication database: 1999 update. Nucleic Acids Res. 1999;27:196197. Beaucage SL, Iyer RP. The synthesis of modied oligonucleotides by the phosphoramidite approach and their applications. Tetrahedron 1993;49:61236194. Eaton BE, Pieken WA. Ribonucleosides and RNA. Annu. Rev. Biochem. 1995;64:837863. Gallo M, Montserrat JM, Iribarren AM. Design and applications of modied oligonucleotides. Braz. J. Med. Biol. Res. 2003;36:143151. Cobb AJA. Recent highlights in modied oligonucleotide chemistry. Org. Biomol. Chem. 2007;5:32603275. Vorbr uggen H, Bennua B. A new simplied nucleoside synthesis. Chem. Ber. 1981;114:12791286. Lichtenthaler FW, Voss P, Heerd A. Stannic chloride catalysed glycosidations of silylated purines with fully acylated sugars. Tetrahedron Lett. 1974;15:21412144. Bookser BC, Raffaele NB. High-throughput ve minute microwave accelerated glycosylation approach to the synthesis of nucleoside libraries. J. Org. Chem. 2007;72:173179. Maydanovych O, Easterwood LM, Cui T, V eliz EA, Pokharel S, Beal PA. Probing adenosine-to-inosine editing reactions using RNA-containing nucleoside analogs. Methods Enzymol. 2007;424: 369386. Wellington KW, Benner SA. A review: synthesis of aryl Cglycosides via the Heck coupling reaction. Nucleosides Nucleotides Nucleic Acids 2006;25:13091333. Hanessian S, Machaalani R. A highly stereocontrolled and efcient synthesis of - and -pseudouridines. Tetrahedron Lett. 2003;44:83218323. H obartner C, Kreutz C, Flecker E, Ottenschl ager E, Pils W, Grubmayr K, Micura R. The synthesis of 2 -O -[(triisopropylsilyl)oxy] methyl (TOM ) phosphoramidites of methylated ribonucleosides (m 1 G , m 2 G , m 2 2 G , m 1 I , m 3 U , m 4 C , m 6 A, m 6 2 A) for use in automated RNA solid-phase synthesis. Monatshefte f ur Chemie 2003;134:851873. Bridson PK, Reese CB. A novel method for the methylation of heterocyclic amino groups. Conversion of guanosine into its 2-N -methyl and 2-N ,2-N -dimethyl derivatives. Bioorg. Chem. 1979;8:339349. Fox JJ, Van Praag D, Wempen I, Doerr IL, Cheong L, Knoll JE, Eidinoff ML, Bendich A, Brown GB. Thiation of nucleosides. II. Synthesis of 5-methyl-2 -deoxycytidine and related pyrimidine nucleosides. J. Am. Chem. Soc. 1959;81:178187. Reese CB, Ubasawa A. Nature of side-reactions in oligonucleotide synthesis involving arenesulphonyl derivatives of 3-nitro-1,2,4triazole and related condensing agents. Nucleic Acids Symp. Ser. 1980;(7): 521. Ariza X, Vilarrasa J. High-yielding preparation of [3-15 N]cytidine, [4-15 NH2 ]cytidine, and [3-15 N,4-15 NH2 ]cytidine. J. Org. Chem. 2000;65:28272829. Roy SK, Tang J-y. Efcient large scale synthesis of 2 -O -alkyl pyrimidine ribonucleosides. Org. Process Res. Dev. 2000;4: 170171.

4. 5.

6. 7.

Other applications
RNAs that employ the four standard bases have more limited opportunities for hydrogen-bonding interactions and functionality, even though the range of tertiary interactions is broad (63). The use of additional base pairs would not only increase the structural complexity of RNA but would also allow for enzymatic replication. This concept has been exploited by several research groups to expand the genetic code, as well as to generate novel, site-specically modied RNAs (e.g., biotinylated, uorescent, cross-linked RNAs) (6, 29, 64).

8.

9.

10.

11.

12.

Summary and Outlook

This article highlights methods for synthesis of modied nucleosides and their site-specic incorporation into RNA. RNAs of practically any size can now be engineered with a broad range of natural or unnatural modications to the base, sugar, or phosphate portions of the nucleotides. Using chemical, biochemical, or combined approaches, a change of a single atom at a desired location can be accomplished, as well as global modication. Because of the diversity of modications accessible through chemical or enzymatic synthesis, the modied RNAs also have a tremendous range of applications, some of which have been summarized in the last portion of this review. Future efforts will continue to give researchers a better understanding of the importance of natural modied RNAs, provide new tools for biophysical and structure analysis for structure-function studies of RNA, and lead to novel RNA therapeutics or catalysts. On a more practical note, novel methodologies for cost-effective, large-scale preparation of highly pure modied RNAs will also be of importance (65), and improved catalysts for RNA ligation (66) will allow for larger, biologically relevant RNAs to be generated with greater ease. 12

13.

14.

15.

16.

17.

18.

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Nucleosides in RNA, Modied

19. 20.

21.

22.

23.

24. 25. 26. 27. 28.

29.

30. 31.

32. 33.

34. 35.

36.

37.

38.

39. 40.

Verma S, Eckstein F. Modied oligonucleotides: synthesis and strategy for users. Ann. Rev. Biochem. 1998;67:99134. Carrasco N, Caton-Williams J, Brandt G, Wang S, Huang Z. Efcient enzymatic synthesis of phosphoroselenoate RNA by using adenosine 5 -(-P -seleno)triphosphate. Angew. Chem. Int. Ed. Engl. 2006;45:9497. Allerson CR, Verdine GL. Synthesis and biochemical evaluation of RNA containing an intrahelical disulde crosslink. Chem. Biol. 1995;2:667675. Wu W, Bergstrom DE, Jo Davisson V. Chemoenzymatic preparation of nucleoside triphosphates. Curr. Protoc. Nucleic Acid Chem. 2004; Chapter 13: Unit 13.2. Da Costa CP, Fedor MJ, Scott LG. 8-Azaguanine reporter of purine ionization states in structured RNAs. J. Am. Chem. Soc. 2007;129:34263432. Ferr e-DAmar e AR. RNA-modifying enzymes. Curr. Opin. Struct. Biol. 2003;13:4955. Ye K. H/ACA guide RNAs, proteins and complexes. Curr. Op. Struct. Biol. 2007;17:287292. England TE, Uhlenbeck OC. Enzymatic oligoribonucleotide synthesis with T4 RNA ligase. Biochemistry 1978;17:20692076. Moore MJ, Sharp PA. Site-specic modication of pre-mRNA: the 2 -hydroxyl groups at the splice sites. Science 1992;256:992997. Stark MR, Pleiss JA, Deras M, Scaringe SA, Rader SD. An RNA ligase-mediated method for the efcient creation of large, synthetic RNAs. RNA 2006;12:20142019. Hirao I, Kimoto M, Mitsui T, Fujiwara T, Kawai R, Sato A, Harada Y, Yokoyama S. An unnatural hydrophobic base pair system: site-specic incorporation of nucleotide analogs into DNA and RNA. Nat. Methods 2006;3:729735. Khorana HG. Nucleic acid synthesis. Pure Appl. Chem. 1968;17: 349381. Gait MJ. An introduction to modern methods of DNA synthesis. In: Oligonucleotide Synthesis: A Practical Approach. Gait MJ, ed. 1984. Washington, D.C.: IRL Press Ltd. Ogilvie KK, Theriault N, Sadana KL. Synthesis of oligoribonucleotides. J. Am. Chem. Soc. 1977;99:77417743. Pitsch S, Weiss PA, Jenny L, Stutz A, Wu X. Reliable chemical synthesis of oligoribonucleotides (RNA) with 2 -O -[(triisopropylsilyl)oxy]methyl(2 -O -tom)-protected phosphoramidites. Helv. Chim. Acta 2001;84:37733795. Scaringe SA. Advanced 5 -silyl-2 -orthoester approach to RNA oligonucleotide synthesis. Methods Enzymol. 2000;317:318. Erlacher MD, Lang K, Shankaran N, Wotzel B, H uttenhofer A, Micura R, Mankin AS, Polacek N. Chemical engineering of the peptidyl transferase center reveals an important role of the 2 -hydroxyl group of A2451. Nucleic Acids Res. 2005;33:16181627. Du Q, Carrasco N, Teplova M, Wilds CJ, Egli M, Huang Z. Internal derivatization of oligonucleotides with selenium for X-ray crystallography using MAD. J. Am. Chem. Soc. 2002;124:2425. H obartner C, Rieder R, Kreutz C, Puffer B, Lang K, Polonskaia A, Serganov A, Micura R. Syntheses of RNAs with up to 100 nucleotides containing site-specic 2 -methylseleno labels for use in x-ray crystallography. J. Am. Chem. Soc. 2005;127:1203512045. Puffer B, Moroder H, Aigner M, Micura R. 2 -Methylselenomodied oligoribonucleotides for x-ray crystallography synthesized by the ACE RNA solid-phase approach. Nucleic Acids Res. 2008;36:970983. Fan Y, Gaffney BL, Jones RA. RNA GGUU motif binds K+ but not Mg2+ . J. Am. Chem. Soc. 2005;127:1758817589. Ward DC, Reich E, Stryer L. Fluorescence studies of nucleotides and polynucleotides. J. Biol. Chem. 1969;244:12281237.

41. 42.

43.

44.

45.

46.

47.

48. 49. 50. 51.

52.

53.

54.

55.

56.

57. 58. 59.

60.

Millar DP. Fluorescence studies of DNA and RNA structure and dynamics. Curr. Op. Struct. Biol. 1996;6:322326. Ballin JD, Bharill S, Fialcowitz-White EJ, Gryczynski I, Gryczynski Z, Wilson GM. Site-specic variation in RNA folding thermodynamics visualized by 2-aminopurine uorescence. Biochemistry 2007;46:1394813960. Proctor DJ, Ma H, Kierzek E, Kierzek R, Gruebele M, Bevilacqua PC. Folding thermodynamics and kinetics of YNMG RNA hairpins: specic incorporation of 8-bromoguanosine leads to stabilization by enhancement of the folding rate. Biochemistry 2004;43:1400414014. Yarian CS, Basti MM, Cain RJ, Ansari G, Guenther RH, Sochacka E, Czerwinska G, Malkiewicz A, Agris PF. Structural and functional roles of the N1- and N3-protons of at tRNAs position 39. Nucleic Acids Res. 1999;27:35433549. Meroueh M, Grohar PJ, Qiu J, SantaLucia J Jr, Scaringe SA, Chow CS. Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA. Nucleic Acids Res. 2000;28:20752083. Newby MI, Greenbaum NL. A conserved pseudouridine modication in eukaryotic U2 snRNA induces a change in branch-site architecture. RNA 2001;7:833845. Abeysirigunawardena SC, Chow CS. pH-dependent structural changes of helix 69 from Escherichia coli 23S ribosomal RNA. RNA; 2008 14:782792. Ryder SP, Strobel SA. Nucleotide analog interference mapping. Methods 1999;18:3850. Tarasow TM, Tarasow SL, Eaton BE. RNA-catalysed carboncarbon bond formation. Nature 1997;389:5457. Shah S, Rangarajan S, Friedman SH. Light-activated RNA interference. Angew. Chem. Int. Ed. Engl. 2005;44:13281332. Batey RT, Inada M, Kujawinski E, Puglisi JD, Williamson JR. Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA. Nucleic Acids Res. 1992;20:45154523. Nikonowicz EP, Sirr A, Legault P, Jucker FM, Baer LM, Pardi A. Preparation of 13 C and 15 N labelled RNAs for heteronuclear multi-dimensional NMR studies. Nucleic Acids Res. 1992;20:45074513. Shandrick S, Zhao Q, Han Q, Ayida BK, Takahashi M, Winters GC, Simonsen KB, Vourloumis D, Hermann T. Monitoring molecular recognition of the ribosomal decoding site. Angew. Chem. Int. Ed. Engl. 2004;43:31773182. Sigurdsson ST, Tuschl T, Eckstein F. Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme. RNA 1995;1:575583. Earnshaw DJ, Gait MJ. Modied oligoribonucleotides as sitespecic probes of RNA structure and function. Biopolymers 1998; 48:3955. Gordon PM, Fong R, Deb SK, Li N-S, Schwans JP, Ye J-D, Piccirilli JA. New strategies for exploring RNAs 2 -OH expose the importance of solvent during group II intron catalysis. Chem. Biol. 2004;11:237246. Lee JF, Stovall GM, Ellington AD. Aptamer therapeutics advance. Curr. Opin. Chem. Biol. 2006;10:282289. Joyce GF. Forty years of in vitro evolution. Angew. Chem. Int. Ed. Engl. 2007;46:64206436. Gugliotti LA, Feldheim DL, Eaton BE. RNA-mediated metal-metal bond formation in the synthesis of hexagonal palladium nanoparticles. Science 2004;304:850852. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specic genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 1998;391:806811.

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

13

Nucleosides in RNA, Modied

61. 62. 63.

64.

65.

66.

Manoharan M. RNA interference and chemically modied small interfering RNAs. Curr. Opin. Chem. Biol. 2004;8:570579. Beaucage SL. Solid-phase synthesis of siRNA oligonucleotides. Curr. Opin. Drug Discov. Devel. 2008;11:203216. Hendrix DK, Brenner SE, Holbrook SR. RNA structural motifs: building blocks of a modular biomolecule. Q. Rev. Biophys. 2005;38:221243. Piccirilli JA, Krauch T, Moroney SE, Benner SA. Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 1990;343:3337. Donga RA, Hassler M, Chan T-K, Damha MJ. Oligonucleotide synthesis using ionic liquids as soluble supports. Nucleosides Nucleotides Nucleic Acids 2007;26:12871293. Purtha WE, Coppins RL, Smalley MK, Silverman SK. General deoxyribozyme-catalyzed synthesis of native 3 -5 RNA linkages. J. Am. Chem. Soc. 2005;127:1312413125.

See Also
Fluorescence to Study Nucleic Acids NMR Tools for Nucleic Acids RNA Interference, Mechanisms and Proteins Involved in

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