conjugates of oligonucleotide

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Bioconjugate

ChemistryMAY/ JUNE 1990Volume I , Number 3

0 Copyright 1990 by th e Amer ican Chemica l Soc ie ty

REVIEW

Conjugates of Oligonucleotides and Modified Oligonucleotides: A

Review of Their Synthesis and PropertiesJoh n Goodchild

Worcester Foundation for Experimental Biology,222 Maple Avenue, Shrewsbury, Massachusetts 01545.Received February21 , 1990

Table of Contents

1. Introduction2. Chemical Synthesis of O ligonucleotides

A. Strategies of SynthesisB. Synthesis of Modified Oligonucleotides

(i) Methylphosphonates(1)

(ii) Phosphotriesters(2 )(iii) Phosphorothioates(3 )(iv) Phosphoramidates(4 )(v) Other Phosphate Modifications

(vi) Non-Phosph ate Internucleoside Linkages3. Synthesis of Conjugates

A. Incorporation of Conjugate and Linker Groupsduring C hemical Synthesis of Oligonucleotides(i ) Incorporation of Modified Nucleotides

(ii) Incorporation of Non-NucleotidesB. Incorporation of Modified Nucleotides during

Enzymatic Synthesis of OligonucleotidesC. Postsy nthe tic Modification of Oligonucleotides

(i ) Reactionsof Primary Alkylamines(ii) Reaction s of Thio ls

(iii) Reactions of Phosphates and Thiophos-phates

(iv) Electrophilic Linkers(v) Reactions at Naturally Occurring Sites in

Nucleic AcidsD. Conjugate Groups

4. Properties of Modified Oligonucleotides and Con-jugatesA. Th e Effect of Modification on Oligonucleotide

HybridizationB. Th e Effect of Modification on Nuclease Resis-

tanceC. Cellular Uptakeof Modified Oligonucleotides

D. Modified Antisense Oligonucleotides5 . Concluding Com ments

1. INTRODUCTION

In th e molecular processes of living things, nothing sur-passes Watson-Crick base pairing in importan ce. It is

fundame ntal to the events th at define l ife: the storage,transmission, an d translation of genetic information. Th esimplicity of these hydrogen-bonded bridges and th e smallnumber of bases involved gives a predictability to theinteractions of nucleic acids that is unattainable as yetwith other biological molecules.

This applies even to the shortest oligonucleotides whichhybridize like their larger relatives and can be viewed a sinformational molecules containing fragmentsof t ruegenetic code. Thes e are valuable models to investigateth e physical and biological prope rties of DNA and RNAth at would be intractable otherwise. Due to the l imitednumber of bases used, oligonucleotides can contain thefull range of functional groups an d show similar chemi-cal reactivity to true nucleic acids. Thi sis useful for elu-cidation of the reactions of mutagens, carcinogens, andantitumor drugs with DN A and RNA.

As a result of their ability to base pair, oligonucle-otides are used extensively in molecular biology a s link-ers, probes, and primers in such activitiesas sequencing,amplification by PCR , determ ination of second ary struc-ture, engineering mutations, tailoring RN A with ribonu-clease H , and assembling DNA constructs.

In o ther a pplication s, “antisense ” oligonucleotides caninhibit expression of viralor mRNA and, more recently,double -stranded DNA. Useful for genetic analysis, thisalso has potential for therapeutic application.

Th e recent de ma nd for oligonucleotides resulted fromimprovements in their chemical synthesis th at also made

1043-1802/90/2901-0165$02.50/0 0 990 American Chemical Society



166 Bioconjugate Chem., Vol. 1 , No. 3, 1990 Goodchild

SchemeIB

RO 0-P-0- +J-",-B

RO 0-P-0- +4 R

B

+O O - P - N i . P r 2OR

B

ROi: -P-0 +

HOJOR

HOJOR

HOJOR

HOJOR

ArS0 ,CI

f 1-RO 0 - P - 0 OR

OX

OR ---*

availa ble a wide range of conjugates. Th e few, simplederivatives of dinucleotides that were available at firsthave grown 10 times in length a nd can be festooned w itha plethora of different pendant groupsif so desired.

Not all modifications to oligonucleotides qualify as con-jugates. In this review, a conjugate is considered to resultfrom th e coupling of twoor more molecules with distinctpropertiesso th at som e of th e characteristics of each areretained in the produ ct. An example is the union of anoligonucleotide with a fluorescent dye t o give a fluores-cent oligonucleotide.A distinction is drawn between con-jugates an d other m odifications of oligonucleotides tha tdo not result in such a combination of properties. How-ever, th e oligonucleotide component of th e conjugate mayitself be modified in other ways and may carry more th anone conjugate group.

Conjugates may be designed to improve some alreadyexisting feature of th e oligonucleotide, for examp le, thestrength of hybridizationor uptake by cells. More often,th e oligonucleotide is endowed with some completely new

property , either physical or chemical, while retaining itsability to base pair. In this way, new applications havebeen developed t ha t were not possible previously.

Conjugate groups combined with oligonucleotides fallinto thre e major categories.

1. Chemically Reactive Groups. Groups th at cleaveor cross-link with oth er nucleic acidsor proteins are usedto study interactions between these molecules and olig-onucleotides, to modifyor cleave nucleic acids at partic-ular sites, an d to create possible therapeu tic agents.

2. Fluorescent or Chemiluminescent Groups. Theseare used in nonradioactive probesor primers for auto-mat ed sequencing and in physical-chemistry studie s includ-ing potentially pow erful new application s involving com -

binations of differe nt conjugates.3. Groups Promoting Intermolecular Interac-tions. The best known example is probably biotin thatbinds to streptavidin . Th is has been used particularlyas a reporter system for nonradioactive probes. Otherexamples are intercalating agents used to strengthen thehybridization of the oligonucleotide with its comple-ment and polylysine used to enhance cellular uptake.

This review is divided into three main parts. Th e firsttwo cover the chemical synthe sis of modified oligonucle-otides and conjugates and the third is concerned withtheir properties. This includes a discussion of antisenseinhibition in general terms, but applications in areas suchas diagnosisor genetic analysis are no t covered here andth e reader is referred t o other sources.

2. CHEMICAL SYNTHESISOF OLIGONUCLEOTIDES

Much of the chemistry described in this review hasbeen developed around the metho ds for synthesizing oli-gonucleotides. These are introduced in sectionA onlyin sufficient detail to provide th e necessary background

for what follows.Th e oligonucleotide compone nt of a conjugate may con-tain all natural nucleotidesor may itself be modified.SectionB reviews meth ods for the synth esis of modifiedoligonucleotides.

A. Strategies of Synthesis. Th e impetus for the cur-rent interest in oligonucleotides derives from develop-me nts in two areas. Advance s in molecular biology overthe last decade orso created uses for these compoundswhile advances in chemical synthesis made them avail-able for practical applications.A synthesis that took m an-years of work in 1979( I ) could be done today in a fewman-hours.

This resulted from improvem ents in two aspects of oli-gonucleotide sy nthesis. One was the deve lopment of solidsupports tha t made possible the automation of the pro-cess and led to m icroprocessor-controlled synthesizers.The other was improvement in the synthesis of phos-pha te esters to give the coupling efficiencies of 98% an dabove that are necessary to take full advantage of thebenefits tha t solid supports offer. Th e historical devel-opment of reactions used for nucleotide coupling is indi-cated in Scheme I and fuller accounts of oligonucleotidesynthesis may be found in refs2-5.

Th e first of these reactions is called the d iester appro achan d was developed by Khorana( I ) . Ester forma tion waseffected withdicyclohexylcarbodiimideor an aryl sulfo-nyl chloride. Th is coupling reactio n is no longer used,but despite some shortcomings, the amide protecting

groups developed for the bases and th e dim ethoxytritylfor 5'-OH a re still standa rd.This synthesis was improved by protecting the phos-

phate group to give the triester approach in reaction2.This not only prevented side reactions but greatly facil-itated workup and purification by enabling much largerscale and m ore rapid purification by chromatography onsilica gel in organic solvents. Wi th improved cou plingreagents such as sulfonyl tetrazolides, this method wassufficiently efficient for solid support synthesis and isstill used to some exten t today(6).

The next improvement wasto replace th e reacting phos-phat e with a trivalent phosphite. Originally, a phospho-rochloridite was used(7) but now the phosphoramiditein reaction 3 is preferred (8). Activation is by a mild



Review

proton donor, usually triazole, and after each couplingreaction th e phosphite is oxidized to th e pentavalent level.This is the route employed most often today and per-mits synthesis of chain lengths considerably in excess of100.

The four th and most recent method( 9 , 1 0 ) eturns topentavalent phosphorus, this time a hydrogen phospho-nate after which this approach is named and which wasfirst recognized a s a potential sy nthon som e tim e ago(11).A carboxylic acid chloride is used as condensing agent.

This m ethod is comparable in efficiency to th e previousone althoughit is generally considered inferior for verylong sequences.It has the advantage tha t the oxidationstep does not have to be performed after each couplingreaction but can be left until the end of the synthesis.This simplifies and speeds up th e process but, more impor-tant ly, it facilitate s the sy nthesis of modified oligonucle-otides where sulfur, nitrogen, or other elem ents replaceoxygen in the reaction a t phosphorus.

These synthetic approaches have been adap ted for usein the rib0 series(12-14) and for nucleosides with unn at-ural L-sugars( 1 5 )or a-glycosidic linkages( 1 6 ) .

B. Synthesis of Modified Oligonucleotides. Thissection reviews some of the options t ha t are available formodification of th e oligonucleotide component within t heconjugate. Th e consequences of these changes for bio-chemical properties are discussed in section3.

Modifications to th e oligonucleotide have been employedmost often for use in antisense inhibition w hereit is nec-essary for oligonucleotides to survive in cell cultures orother biological environments and also to cross the cellmem brane. Nucleases are widespread and the lipophiliccell mem brane is an effective barrier against passive dif-fusion of polyelectrolytes.

Often, modifications can be chosen to im prove perfor-mance in both areas. For examp le, increasing lipophilic-ity to improve uptake is likely to decrease the rate ofdegradation by nucleases tha t are designed to degradepolyanions. Th e conjugate group itself may prove ben-

eficial in these regards ifit is lipophilic or otherwise inhib-its the action of nucleases. As yet, the numbe r of exam-ples of conjugate groups combined with othe r modifica-tions to the oligonucleotides is sma ll but increasing rapidly.

Changes might be made a t the bases, the sugars, theends of the chain, or at the p hospha te groups of the back-bone. Those at the bases and sugars are generally themost difficult chemically an d require the greatest amo untof synthesis. In addition, they must not disrupt the abil-i ty of bases to form hydrogen bonds. One example isthe use of a-nu cleotides discussed later.

Modifications to the ends and backbone of the mole-cule are easier synthetically. Because the 5’-terminus isthe most common site for conjugation, the phosphategroups are often available for further derivatization. Asthey are th e site of action of nucleases and also carry thecharges t ha t inhibit cellular uptake, this presents the mostdirect approach to improvement in these areas.

Th e type of phosph ate mod ification most studied is toreplace or block t he negatively charged oxygen atom t ogive structures1-4. The syntheses of these and othermodifications are discussed below.

Bioconjugate Chem., Vol. 1, No. 3, 1990 167

are one of the most extensively studied classes of oligo-nucleotides because of thei r useful chemical an d biolog-ical properties. Older metho ds for their synthe sis werebased on modifications of the triester approa ch(1 -23) .Following the introduction of phosphine reagents(24-2 6 ) , solid-support synthesis based on phosphoramiditechemistry has become standard(27) . Phosphonamiditereagents react as efficiently as the usual phosphora-midites and can be used interchangeably during auto-mated synthesis to insert the uncharged linkage at any

or all positions within the sequence. Produc ts from thisapproach were found to give better melting curves thanthose from a synthesis based on triester type chemistry(2 8 ) .

Because of the d ifferent natur e of the backbon e, spe-cial metho ds for their characterization h ave been devel-oped ( 2 9 ) . Like purification, this is still not as easy aswith fully charged phosphodiesters.

Only the m ethyl substituent on phosphorus has beeninvestigated t o any ex tent.A phenyl phosphonate wasprepared early on(18) and a 4,4’-dimethoxytriphenyl-methanephosphona te was obtained unexpectedly from anattempted Arbusov reaction( 3 0 ) . More recently, a (dif-1uoromethyl)phosphonate was synthesized to mimic th epolarity of the natu ral oxygen ato m m ore closely( 3 1 ) .

(ii) Phosphotriesters (2) . These com pounds were usedas nonionic analogues of oligonucleotides by Miller et al.before the methylphosphonates( 3 2 ) .They a re more dif-ficult to synthesize and, as a result, most studies havebeen limited to oligonucleotides containing only a singletriester or only thymine bases.

Th e problem is the lability of the triester function dur-ing the basic conditions used in deblocking or cleavingfrom the solid support. Use of milder conditions(3 2 ,3 3) or more labile amine protecting groups(34-37) ormore stable triesters( 3 8 , 3 9 ) ave not yet given a methodas versatile as synthesis of the p hosphonates.

Originally, met han ol or ethano l in the presence of tosylchloride was used to esterify internucleoside phosp hatesaf ter each coupl ing in shor t sequences( 3 2 , 40 , 4 1 ) .Recently, i t has been reported th at th is reagent can givecomplete triesterification of heterosequences as long as10-20 bases following tem pora ry protectio n of the amin ogroups with 9-fluorenylmethoxycarbonylchloride (9-fluorenylmethyl carbonochloridate) in solution(3 6 ) . Thisis the only me thod t ha t gives extensively esterified prod-ucts of this size. Met hyl methane sulfona te has been usedalso as a methylating agent( 4 2 ) .

Different triesters have been introduced as the pro-tec t ing groups dur ing synthesis by the t r ies ter( 4 3 ) ,phosphite( 3 8 , 3 9 ) , r amidite( 3 3 , 3 4 , 3 7 , 4 4 ) pproachesor by oxidation of hydrogen pho sphonate s with alcohols( 4 5 ) . In addition, transesterification in the presenceof

fluoride ions can replace aryl protecting groups on phos-pha te by alkyl to give more stable esters(41 , 46-48).Groups tha t have been used other than the usual methyl

and ethyl includel,l-dimethyl-2,2,2-trichloroethyl 3 8 ,39, 431, isopropyl ( 3 3 ) ,neopentyl (48 , 491, n-butyl (451,and 2,2,2-trifluoroethyl( 3 7 ) .

( i i i ) Phosphorothioutes ( 3 ) . Used extensively by Eck-stein and his co-workersfor the st udy of enzyme mech-anisms, these derivatives are the closest to the naturalnucleic acids in terms of structure and charge density.Fo r reviews of thei r pr epa ratio n a nd uses see refs 50-52.Both DNA and RNA polymerases accept the appropri-a te 5’-0-( -thiotriphosp hates) as substrates to give prod-ucts containing just th eR , stereoisomers of phosphorus.

Chemical synthesis i s a lso s t ra ightfo rward. Eck-

e 9 e e-0-P-0- -0 -P -0 - -0-P-0- -0 -P-O-

cH, O R S- NR ,

1 2 3 4

( i ) Methylphosphonutes ( 1 ) . These nonionic deriva-tives were introduced by Miller et al. in 1979(1 7 ) and



168

stein’s use of elemental sulfur to oxidize dinucleosidephosphites is applicable to phosphoramidite synthesis onsolid suppo rts giving oligonucleotides with p hosphorothio-ate l inkages throughoutor jus t a t selected positions( 5 0 ,5 3 , 5 4 ) . A concern is tha t the phosphorothioate func-tions are subjected to an iodine oxidation step after e achsubseq uent roun d of coupling which could lead t o replace-ment of some sulfur by oxygen( 5 5 ) . In practice, phos-phorothioate could still be isolated after48 rounds ofcoupling( 5 3 )and NMR studies indicated that about one

sulfur atom per molecule was lost to oxidation duringthe synthesis of a 20-mer( 5 6 ) .Eleme ntal sulfur can also be used for oxidation follow-

ing the hydrogen phosphonate approac h( 4 5 , 5 7 - 6 1 ) . Thisoffers some practical advantages over the phosphora-midite method w here oxidation has to be performed a ftereach coupling. Th e usual solvent for sulfur is carbon dis-ulfide, which is volatile, malodorous, an d troubleso me inautomated synthesizers, where the sulfur tends to formsolid deposits. It is preferable to perform the oxidationjust once manually a t the end of the synthesis.

For synthesis of chimeric structures containing bothphosphorothioates and phosphodies ters in the back-bone, treatm ent with sulfur partsway hrough the synthe-sis is followed by further rounds of coupling and oxida-tion. Th is proved more successful with the phosphora-m i d i t e t h a n t h e h y d r o g e n p h o s p h o n a t e m e t h o d .Unprotected phosphorothioates generated on sulfur treat-me nt of hydrogen phospho nates survived subseq uent cou-pling a nd ox idation cycles much less well tha n th e blockedintermediates formed from phosphites( 6 0 ) .

( i u ) P h o s p h o r a m id a t e s ( 4 ) . An appealing feature ofphosphoramida tes is the diverse range of amines tha t migh tbe introduc ed. Given a good, general synthesis, this classof compounds should offer the greatest opportunity forstructural variation. Substituents on the amine couldinclude conjugate as well as nonconjugate groups. It migh tbe desired to introduce just one phosphoramidate at aspecific positionor to modify all the phosphates with eitherthe same or different substituents.Such precision andcontrol is attainable with current synthetic methods.

A number of reactions for preparing phosphorami-date s have been a pplied to oligonucleotides with greateror less success as discussed in ref62. Th e prefered methoduses an amine in the p resence of carbon tetrachlorid e oriodine as the oxidant in the hydrogen phosphonateapproach (45 ) . By leaving this st ep until the end of thesynthesis , phosphoramidate l inkages are in t roducedthroughout th e sequence( 1 5 ,5 8 , 6 3 , 6 4 ) . Alternatively,further rounds of coupling can be performed after ami-dation t o generate a heterogeneous backbone( 4 5 , 6 1 , 6 4 ) .By alternating hydrogen phosphonate coupling steps withphosphoramidite, various unmodified a nd modified link-ages can be inserted at specific sites throughout thesequence ( 6 1 ,6 3 ) .

Another attractive fea ture of phosphoramidates is th atthey may be readily converted to phosphodiesters for char-acterization by the usual means( 4 5 , 6 4 ) .

Th e P- N bond is hydrolyzed in acid and, unless thenitrogen is substituted,it is also too base labile to sur-vive th e deblocking reaction( 6 4 , 6 5 ) . This bond is morelabile in the ribo series( 6 5 ) . The oligonucleotide ami-dates made by this method are l isted in Table I .

( u ) O t h e r Phosphate M o d i f i c a t i o n s . The four typesof phosphate modification discussed above have all beenused fairly extensively to modify the properties of oligo-nucleotidesor their conjugates. Other modifications tha thave been made but, for themost part, not yet studied

6ioconjugate Chem., Vol. 1, No. 3, 1990 Goodchild

Table I. Amidates Made by the Hydrogen PhosphonateApproach

refs45,6445, 58, 6262626462,6463

structure of theamine component refs

NHCHzCHz-morpholinyl 63NH(CH2)zNHCO- 61

cholesterylpiperidinyl 45morp holiny l 45, 58, 64piperazinyl 58N-methylpiperazinyl 45

Table 11. Modified Internucleoside Phosphates0II

-A-P-C-ID

A B C D refsNH000000000000S0CHzSS0

00S00NPrSSeSSSSS0000S0

0NH00S00000000

00S

00CH3Se0NEtzNEtzNEt,‘ 3 3SNHROPrOEt00000CH3

66,6767,686953, 707172727273747433, 7544, 7677.67787979267

Table 111. Groups Used To Replace InternucleosidePhosphates

€!oup refs

-0coo- 85-88-0CHzCONH- 89-0CHzCOO- 90-0CONH- 89,91-93-0SiRzO- 94-96

in this way are listed in Table11. Furthe r examples includederivatives with pentava lent phosph orus(80 ,811 ,but manymore possibilities remain unsynthesized.

(ui) N o n - Ph o s p h a t e I n t e r n u cl e o s id e L i n k a g e s . In earlywork, the entire sugar phosp hate backbone was replacedas in poly( 1-vinyluracil) or in poly(acry1icacid) hydrazidederivatives(82-84). These suffered from solubility prob-lems and spacing of the bases along the chain was dif-ferent from th at of natural nucleic acids. Most recentapproaches have involved the less extensive modifica-tion in 5 (97) or replacing jus t the bridging phosph ate by

B a s e

I&C -

5

the groupsin Table 111. As yet, synthesis of these typesof compound is still a t an early stage. Solubility in wateris a common problem an d hybridization is different fromtha t of natural oligonucleotides in some cases.



Review

3. SYNTHESISOF CONJUGATES

Conjugate groups may be coupled t o oligonucleotideseither thro ugh sites present naturally in nucleic acids orthrough some other reactive linker group introduced spe-cifically for the purpose. Th e naturally occurring groupsth at can be used are amino groups on th e bases, hydroxylgroups on the sugars, and ph osphate groups, both termi-nal and internal. Linker groups attached to the oligonu-cleotide for derivatization are most commonly primaryamines, thiols, or aldehydes, but th e possibilities are m any.Often, the linker is attached to th e oligonucleotide by aspacer arm e ither to facilitate coupling or to distance th econjugate group from th e oligonucleotide.

Eith er the conjugate group or the linker may be intro-duced at one of three stages during oligonucleotide syn-thesis. Thes e each have advantages and disadvantages.

1. The group can be attached to a nucleotide beforeincorporation into the growing chain. Th is approach canbe used for either chemicalor enzy matic synth esis of oli-gonucleotides.

2. Molecules other than nucleotides can be intro-duced du ring th e synthesis of the oligonucleotide.

3. A linker or conjugate group can be attached to anatural nucleic acidor to a synthetic oligonucleotide after

deblocking.SectionsA and B below describe incorporation durin gchemical or enzymatic synthesis, respectively. SectionC covers coupling following synthesis a nd section D listssome conjugate groups that have been used.

A. Incorporation of Conjugate and Linker Groupsduring Chemical Synthes is of Oligonucleotides. Froma synth etic point of view, incorp oration of conjugates andlinkers during th e assembly of a n oligonucleotide rathertha n afterward is the most rigorous approach. It givesgreatest control over the num ber a nd location of th e mod-ifications; side reactions are minimized by the protect-ing groups on th e nucleotides, and advantage is taken ofthe benefits of solid-support synthesis for workup andpurifica tion. Two strategies will be considered in whichthe conjugate groupor linker mayor may not be part ofa nucleotide synthon. In both cases, the reactions areperformed under anhydrous conditions, unlike the postsyn-thes is modif ica t ions descr ibed la ter, which are per-formed largely in aqueous solution.

( i) I n c o r p o r a t i o n o f M o d i f i e d N u c l e o t i d e s . Th i sapproach is appealing for chemical synthesis as th e nucle-otide building block carrying the desired modifier canbe introduced precisely at any interna l or terminal posi-tion in the oligonucleotide with assurance that modifi-cation a t tha t position is complete. As a result, concernsover th e uniformity of the produc t a nd its identity shouldbe less tha n those with some other methods. However,it is necessary to first pre pare an d purify th e nucleotide,

and the modifications m ust be able to withstand the cou-pling reaction and th e rigors of acid an d basic deblock-ing. In some cases, nons tand ard protecting groups maybe necessary. Probably as a result of the greater syn-thetic effort required, this approach has not been usedas widely a s others.

Substituents may be attached to nucleotides a t the base,sugar,or phos pha te residues, bu t ideally, changes shouldnot interfere with hybridization. While there is poten-tial for using phosphate-modified precursors such as sub-stituted phosphonatesor phosphotriesters, little work hasbeen done in this regard other tha n with the simple block-ing groups described in th e previous section.

Two sites on bases which are easy to manipulate chem-ically witho ut necessarily preventing base pairing a re C(5)

Bioconjugate Chem., Voi. 1, No . 3, 1990 169

Table IV . Linkers Incorporated into Oligonucleotides asSubstituents on Pyrimidine Nucleoside PhosphoramidateSynthons

substituent refsAt N(4)of dC

A t C(5 ) of dU(CH2hNHz 100-102C=C(CHz)ZNHz 103

C=CCH~NHCO(CH~)ENHZ 103CH=CHCONH(CHz)aNHz 10 4

of uracil a nd N(4) of cytosine, an d several nucleotide phos-phoramidi te synthons have been prepared with pro-tected linkers at these positions (Table IV).

The triazole leaving group introduced with reagent6can be displaced by various nucleophiles before the final

0N% R

06-OCNEtk i . P s

6

deblocking of th e oligonucleotide(105-107). This mightbe used a s a general-purpose reagent to minimize th e syn-thetic effort necessary to introd uce a variety of other groupsor for the introduction of a group sensitive to the cou-pling or acidic detritylation steps.

In addition, a number of nucleotides already bearingconjugate groups a t these positions have been used.Exam-ples are EDTA, used in the generation of free radicals(IO€!), and biotinyl, dinitrophenyl, pyrenyl, and dansylreporter groups(109). -Bromo-2‘-deoxyuridine has beenincorporated by triester synthesis for its ability to cross-link with DNA-binding proteins on UV irradiation(111).

In th e case of purines, C(8) of a den ine was used a s anattach men t site for th e photoactivatable cross-linkingreagent psoralen(112).

Other nucleotide phosphoramidites have been pre-pared with O(5’) of the sugar replaced by nitrogenor sul-fu r (113-115). Used in t he final cou pling, these give oli-gonucleotides with a thiol or primary amine a t the 5’-endfor subs eque nt reaction with electrophiles. For situa-tions where a spacer was required between th e 5’-aminoand the oligonucleotide, nucleotide7 was used with tri-ester coupling(116).

0

0

2-CI Ph b

0.6-0-

7

Also in the triester series, protected thymidine 3‘,5’-diphospha te was used for the introductionof a terminal5’-phosphate residue a s a site for postsynthetic modifi-cation (117).

Unlike with solid-support synthesis, with th e older solu-tion-phase triester method nucleotides can be added toth e 3‘-end of th e oligonucleotide. Nucleotides carrying



170

various 3’-bound conjugate groupsor linkers have beenintroduced this way(117-1 20 ).

To accomplish this by solid-support synthesis, a spe-cial supp ort bearing the m odified nucleoside may be pre-pared as in the case of cytidine with an am ine linker atN(4) (121).

(ii) Zncorporationof Non-Nucleotides. A number ofreagents other th an nucleotides have been developed forincorporation during oligonucleotide s ynthesis. The se aregenerally much simpler synthetic targets for the intro-duction of conjugatesor linkers. They are used most com-monly to couple a group at the 5’-end of an oligonucle-otide while it is still fixed to th e solid support. Thi s reducesthe potential for side reactions at the bases, which arestill protected, and simplifies purification. Th e reagentmust be soluble in an organic solvent suitable for thecoupling reaction an d, again, must w ithstand the deblock-ing procedure.

Most groups introduced in this way have been used togive, after deblocking, a nucleophilic linker for prepara-tion of a conjugate. One reason for using nucleophiles isthat many suitable electrophilic derivatives of the con-jugate groups are available commercially.

Often, the s ame coupling chemistry is used for nucle-

otides and non-nucleotidesso that the latter may be addedfrom a spare reservoir of the synthesizer using the stan -dard program.

Reagents 8-11 have a protected amine, thiol, or car-boxyl a t one end of a spacer and a phosphoramidite a tthe other (122-128). Reagent 12 is a cyclic analogu e of

Bioconjugate Chem., Vol. 1 , No. 3, 1990 Goodchild

8

Ro P -0 ’k i - p r ,

14

10

I 7O Q J C O C F ,

OCH,

1 2

Tr - S (CH2 k O-P0 C H,

N&.Pr,

11

0 - P - O C N EtNI.Pr,

F M o c - N H

D M TO

13

7%C I, P O C C H , C N

I

c H,

15

9 tha t uses the sam e nitrogen atom both as the leaving

group and as th e linker(129). These compounds can beused interchangeably with the stan dard nucleoside phos-phoramidites. Analogous reagents are available for H-phos-phonate (130) or triester synthesis(120, 131) or linkingthrough a methylphosphonate(132). Hence, these groupscan be incorporated readily during autom ated synthesis,whichever approach is employed, without the necessityfor chang ing reagents. After deblocking, all give oligo-nucleotides with a nucleophilic linker a t the 5‘-end.Reagent 13, containing an additional protected hydroxylfunction, can be used for multiple rounds of deblockingand coupling to increase th e nu mbe r of linker groups(153).

Phosphoramidite derivatives of biotin(133), acridine(134), and anthraquinone(135) have been use similarlyand phosphate derivatives of acridine and tetramethyl-

rhodam ine have been used for triester synthesis both bysolid-support and solution-phase synthesis(131, 136).

During autom ated synthesis, reagents of general struc-ture 14 or 15 can be used to phosphorylate or thiophos-phorylate the 5’-hydroxyl group(118,137-140) and a num-ber of other reagents exist based on triester chemistry(141 and references therein). Th e phosphate can be usedfor modification at a later stageor it can be selectivelydeblocked before cleaving from the solid sup port an d con-densed, for example, with a n alcohol in the presence ofa sulfonyl triazolide(142).

As an alterna tive to reactions a t phosphorus, the 5‘-hy-droxyl group of sup port -bo und oligonucleotide can be acti-vated with carbonyldiimidazole for reaction with hexam-ethylenediamine to give carbamate16 (143). Described

16

in section C below is th e use of a vicinal diol as a linker.This can be introduced as a ribonucleotide joined 5’-5’during th e final coupling of the syn thesis(122).

Modifications a t the 3‘-end a re less common due t othe inaccessibility of th is site in solid-support synthesis.Th e usual way to solve thi s problem is to use a supportwith the modification already built in. For example, poly-amide or polypeptide chains synthesized on a supportwere used to initiate oligonucleotide synthe sis to give con-jugates with a 3’-polypeptide tail. Incorpora tion of lysinein the peptide furnished multiple amino linker sites forsubsequent derivatization(144, 145).

The spacer between the nucleoside and the supportcan be designedso tha t on cleavage,a reactive nucleo-phile is generated a t the en d of the oligonucleotide.Forexample, support17 gave a 3’-terminal thiol after amm o-

nia treat men t followed by reduction(146,147). To avoidthe need for four such suppo rts, a linker was developedto which t he first nucleoside was added during th e syn-thesis. On cleavage, this libera ted a 3’-terminal aminefrom a carbamate(148).

Reactions a t the 3’-end may be performed m ore readilyduring solution-phase synthesis. Examples are the con-

densatio n between a 3’-phosphate and th e hydroxyl groupof an acridine or phenanthroline derivative using tri-ester chemistry(136, 149, 150 ).

A site amenable to ready m odification during oligonu-cleotide synthesis is the internucleoside pho sphate, pa r-ticularly during the oxidation of interm ediate phosphitesor hydrogen phosph onates. Mu ch of the work on conju-gation here has been done by Letsinger an d his co-work-ers, who have reported several methods for linking throughphosphoramidates to prepare conjugates of phenanthri-dine and cholesterol(43, 61, 151). Other workers haveintroduced acridine by triesterificationof unprotectedinternucleoside phosphates(136). Of these approaches,the most routine and widely applicable would seem tobe generation of phosphoram idates d uring hydrogen phos-



Review Bioconjugate Chem., Vol. 1, No. 3, 1990 171

been used to label RNA w ith various reporter molecules(168).

Table V. Linker and Conjugate Groups IncorporatedEnzymatically into Oligonucleotides as Substituents onNucleotide Triphosphates

substituent refs

A t C(5) of (d)UTPnaphthalene derivative 102biotin 154-157(CHz)izNHz 157CH=CHCHZNHz 157

fluorescein 160SCH3' 161

At N(4) of (d)CTP(CHzhNHz 162

A t C(4) of U TPS 163,164

A t C(8)of ATPNH(CH&NHz 1572,4-dinitrobenzened 165

CHzCONH(CH2)sNHz 157

158, 159

At N(6) of ATP

a Used to cross-link with proteins. Used in site-specific cleav-age of DNA. c Used as a s ite for alkylation.d Used as a hapten.

phonate synthesis. Th is has been used to introduce aprotected amino linker for postsynthetic derivatization(152).

Another easily accessible phosphate derivative is thephosphorothioate described previously. T he sulfur atomreacts readily with alkylating reagents as will be dis-cussed in sectionc.

Finally, the versatile trifunctional phosph oramidite13can be used in place of a nucleotide during sy nthesis tointroduce oneor more amine linkers into the backbonea t any position(153).

B. Incorporation of Modified Nucleotides duringEnzymatic Synthesis of Oligonucleotides. The strat-egy of incorpo rating m odified nucleotid es can be appliedto enzymatic synthesis of oligo- or polynucleotides withor without a template. While the products are not sub-jected to th e conditions of chem ical synthesis and deblock-ing, this m etho d generally gives less control over the s itesof modification and is restricted by what is acceptableas a substrate for the enzyme.

A variety of DNA and RNA polymerases have beenused for this purpose, but those most commonly employedare Escherichia coli DNA polymeraseI and terminal deox-ynucleotidyl transferase. T he former is used with a tem-plate eithe r for interna l incorporation of a modified nucle-otide or for addition to th e end of a presynthesized oli-gonucleotide. Th e latter enzyme is used w ithout a templateto add one or more nucleotides t o the 3'-end.

Generally, the same positions on the nucleotides are

modified asfo r the chemical syntheses discussed previ-ously. Thes e are C(4) and C(5) of pyrimidines and C (8)of purines an d exam ples are given in TableV. Some ofthe more commonly used nucleoside triphosphates arecommercially available.

T4 RNA ligase has been used to introduce a single,modified nucleotide a t the 3'-end of RNAor DNA ter-minating with a ribonucleotide. Th e 3'-thiophosphorylderivative of pCp gave the s ame phosph orothioate func-tion whose chemical synthesis was described previously.Also incorporated was the fluorescent derivative withbimane attached t o sulfur(166).

In the absence of ATP, T4 RNA ligase transfer thenon-nucleo tide-bearing phospha te from the ADP deriv-ative 18 to the 3'-hydroxyl of an RNA(167). This has

0 0

0 0

R O - P - O - P - O A d o

1 8

C. Postsynthetic Modifications of Oligonucleo-tides. Conjugate groups are usually introduced after t he

synthesis a nd deblocking of the oligonucleotide. Th is nor-mally requires less effort than preparation of reagentsfor incorporation during synthesis but introduces otherproblems.

As oligonucleotides are polyionic, postsynthetic reac-tions are usually performed in water or an aqueous sol-vent in which the reagents must be sufficiently solubleand stab le. This, in itself, is restricting as few synthetic,organic reactions are inten ded t o be performed under the seconditions.

As linkers are usually nucleophiles, unwanted reac-tions may occur at many internal sites in oligonucle-otides. T he separa tion of oligonucleotides with differ-ent numbers of conjugate groups is difficult as is theircharacterization , which is usually not attem pted in anyrigorous way and often not at all. Th e structur e of theproduct is frequently assumed from the nature of thestarting materials, particularly with conjugates of large,multifunctional molecules, whereit is generally not pos-sible to apply the more rigorous standar ds of organic chem-istry. However, th e reasons for making these com-pounds are usually for practical applications and, pro-vided that they function as intended, then the precisenumb er and location of the conjugate groups may not becrucial.

Postsy nthetic reactions on oligonucleotides may be usedto generate or modify linkers or to introd uce th e conju-gate group. Th is section describes, in rather broad term s,the types of reactions tha t have been employed with var-ious linker functions. T he em phasis is on the nature ofthe bond-m aking reactions rath er tha n particulars of indi-vidual cases. Subsections are devoted to coupling reac-tions used for different linker groups.

(i) Reactions o f Primary Alkylamines. Primary alk-ylamines (and hydrazines) are among the most com-monly used linkers because of their affinity for electro-philes. Usually, these are some activated form of a car-boxylic acid such as the ester of N-hydroxysuccinimidethat gives an amide (99, 00 , 1 2 1 , 122, 124, 30, 169-171). Other related species tha t have been used includenitrophenyl(126,151,172) nd pentachlorophenyl esters(115)) n acid anhydrid e(102,173,174)) nd sulfonyl chlo-rides (99,102). lternatively, the w ater-soluble l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide ondensing

reagent (EDC) can be used for amide formation with acarboxylic acid( I 70,175). ormally, this reagent accom-plishes a deh ydration reaction in w ater.

It was found with EDC th at as many as 35-45% of theamide bonds formed were with the amines of the basesrather than with the intended linker. Th e N-hydrox-ysuccinimide ester gave less reaction a t the bases but wasalso less efficien t overall(170). This stud y wasof reac-tions between oligonucleotides and carboxyl groupsattach ed t o solid matrixes, bu t extensive base modifica-tion was found also on EDC treatment of an oligonucle-otide bearing a 5'-carboxyl group(127).

Chief among the reagents giving non-amide productsare isothiocyanates tha t give thioureas(99,113,122,130).Other reactions that have been used are hydrazone for-



172 Bioconjugate Chem., Vol. 1, No. 3, 1990

Scheme I1R

Goodchild

minal phosphates may be introduced during chemicalsyn-thesis, by enzymic phosphorylationor by &eliminationof a terminal ribonucleoside following periodate oxida-tion. Naturally derived material may already have a ter-minal phosphate. Th e only reactions used to form con-jugates postsynthetically at phosphate are condensa-tions with amines or alcohols to give phosphoramidatesand esters. Thiopho sphates are used in a different classof reactions with alkylating agents.

An early method from Khorana’s laboratory for reac-

tion a t terminal phospha tes was the formation of a phos-phoram idate from aniline using dicyclohexylcarbodiim-ide (DCC ) as the condensing reagent in a mixed solventof water, dimethylformamide, and butanol(183). Thisis still basically the method used to modify oligonucle-otides except tha t DCC has been replaced by water-sol-uble EDC (67, 184-186). Recently, cyanogen bromidehas been used for the same purpose. This reagent mayalso be used to generate a variety of unusual internucle-oside phosphate bridges by coupling contiguous oligonu-cleotides on a template in a form of chemical ligation(67, 185, 186).

Although they were not the first to use EDC w ith oli-gonucleotides, Chu et al. developed the protocol most com-monly used of first preparing the phosphorimidazolidewith EDC and reacting this with the amineor other nucleo-phile (187). This interm ediate, used previously in chem-ical ligation (188) , s faster th an direct coupling of sim-ple amines and perm its the use of nucleophiles th at wouldthemselves react with EDC. Although side reactions a tthe ba ses do occur, they were considered to be minor un derthe conditions used. Oth er workers have reported tha tN-hydroxybenzotriazole is superior to imidazole for th ispurpose (67).

This approach has been used to attach a variety of link-ers and conjugate groups to oligonucleotides(169, 170,173, 179). Direct coupling to the amine without goingthrough the interm ediate phospho rimidazolide has beenused also (67 ,189) . Th e reaction has been extended to

nucleophiles other than amines and has been studied insome detail t o minimize base m odification(184).Thiophosphate may be introduced during chemicalsyn-

thesis or with polynucleotide kinase(190). The sulfuratoms a t both terminal and internucleotide sites react a tleast l o 3 times faster than the other groups in nucleicacids with 2-chloroethylamines andso can be used forselective coupling(190). A variety of alkylating agentshave been used t o form conjugates in this way(118 ,140,

( i u ) E l ec t r o p h il i c L i n k e r s . Most examples of conju-gate form ation use a nucleophilic linker to react with someelectrophilic reagent. Unwan ted reactions may occur a tothe r sites in nucleic acids which are predo mina ntly nucleo-philic in nature, particularly the bases. An electrophiliclinker may be used providing that it does not undergointramolecular coupling a t these sites. The re are a fewexamples of this approach, the electrophiles being car-bonyl or activated carboxyl groups.

One of the earliest me thodsfor labeling RNA with biotinused period ate to oxidize the 3’-terminal ribonucleosideto a dialdehyde that was reacted with a primary amineand reduced w ith borohydride(195). This has been usedto label RNA with a num ber of fluorescent dyes and othe ragents (196-198). In the case of oligodeoxynucleotides,a terminal ribonucleotide can be introduced by5’-5’ cou-pling as the last step in synthesis(122). Steric hinder-ance around the reacting amino group may be trouble-some in some cases with this method.

150, 166,191-194).

Scheme I11

1

mation with an aldehyde(176) and a less general reac-tion with nitrobenzodiazole fluoride(113,122,130). T h eunusual reaction in Scheme I1 brought about by horse-radish peroxidase in the presence of hydrogen peroxidewas used to introduce an intercalating agent(1 77).

A molecular adapter has been designed t ha t will replacean amino linker with a thiol(121) while bromopyruvatewas used to couple an amino linker with a thiol in a pro-tein (1 78).

(ii) Re a c t i o n s of T h i o l s . The chemistry of sulfur ismore complex than that of nitrogen, and thiol linkersmay be used in a greater variety of reactions than amines.As well as their nucleophilic propertie s, they bo nd readilyto mercury and form disulfides with other thiols. In fact,disulfide formation can be a problem on storage(127).

In order t o generate conjugates from two thiols by mixeddisulfide formation, competing reactions to give sym met-rical products mus t be suppressed. This is usually doneby first forming a mixed disulfide between o ne thiol and2-thiopyridine followed by an exchange reaction with thesecond thiol (127, 146, 179, 180). Alternatively, one ofthe thiols may be used in large excess(181).

A useful feature of disulfide formation is tha tit is readilyreversed by treatmen t w ith dithiothreitol (D TT )or othermerca ptans. Thi s has been used to remove one-half ofthe conjugate after it has fulfilled its function(181) orto reversibly bind oligonuc leotides to a solid matri x(127).Reversal of disulfide form ation was used t o generate thiollinkers from blocked disulfide precursors(121,146,179).

Th e most com monly used electrophilic groups for con-jugation with thiol linkers are iodo- or bromoacetates andmaleiimides (121, 123, 130, 131), but some fluorescentmarkers proved troublesome by this approach(130) .Immobilization on a solid su pport was claimed to be farsuperior with thiol as opposed t o carboxyl or amine link-ers (127).

By a type of M ichael addition, the thiol on an oligo-nucleotide was linked with an amine in a one-pot reac-tion using the bifunctional reagent in Scheme I11(182).

Th e affinity of thiols for mercury com pounds was usedfor immobilizationof an oligonucleotide on c hloromer-curibenzoate-derivatized agarose (127).

Another finding reflecting the more complex chemis-try of sulfur is tha t a two-carbon spacer between sulfurand phosphate was unstable at pH8 while longer oneswere not (123, 146).

( i i i )Reactions of Ph o s p h a t e s a n d T h i o p h o s p h a t e s . Ter-



Review Bioconjugate Chem., Vol. 1, No . 3, 1990 173

Table VI. GrouDs Used in Conjugates of Olieonucleotidessingle aldehyde function can be gene rated a t the 5'-endby periodate oxidation of a vicinal diol introduce d du r-ing synthesis. Reaction with an amineor hydrazine fol-lowed by reduction under very mild conditions did notshow any competing side reactions a nd was particularlygood for immobilization on a solid supp ort(125).

Another electrophilic species was generated a t the 5'-ter-minus by treatm ent of a carboxyl group with EDC in thepresence of imidazole. Th is was used for amide forma-tion (125) .

( u ) Reactions a t Na tura l ly Occurr ing Si tesin NucleicAcids. Unmodified oligonucleotides contain a numberof reactive sites that are generally less potent than thesynthetic linkers bu t which have been usedfo r conjuga-tion. For the m ost part , these are nucleophiles and includenitrogen functions on the bases,C(5) of pyrim idines, C(8)of purines, prim ary a nd secondary hydroxyl groups, andphosphomono- and diesters. Electrophilic substitutioncan also occur at some positions on the bases. Reactionsa t these sites are likely to lead t o complex mixtures unlessthey can be controlled in some way. This may be accept-able for labeling of large DNAor RNA, where modifica-tion can be restricted to a very small fraction of nucle-otides and the level of heterogeneity in the product isundetec table and irrelevant. Th e approac h is less satis-factory for oligonucleotides where incomplete m odifica-tion gives produc ts with a spectrum of properties. Fourof these reactions th at ar e better defined chemically aregiven below.

The first uses a photoactivatable derivative of biotincarrying a phenyl azide th at rea cts with nucleic acid bases(199). Thi s is used t o label about only1 in 50 bases inDNA so as not to impair hybridization and may there-fore not be applicable t o oligonucleotides.

In a second method, mercury at C(5) of pyrimidines,introduced by reaction with mercuric nitrate, was cou-pled with haptens bearing thiol groups(200) .

Bisulfite adds across the 4,5-double bond of cytidinean d encourages nucleophilic displacement of the am inogroup at C(4). This reaction is selective for single-stranded nucleic acids and has been used to introduce anumber of substituents(178, 201, 202).

Finally, an electrophilic substitution occurs at C(8) ofguanine in nucleic acids on reaction with N-acetoxy-2-aminofluorene to give a label for immunological detec-tion (203, 204).

None of the above reactions can be used to modify anoligonucleotide in a controlled way othe r th an by restrict-ing to one or two the number of reacting nucleotides,thereby severely limiting th e sequence. The re are fewexamples where a more controlled approach is possible.

4-Thiouridine is a naturally occurring nucleoside thatcan be incorporated into oligonucleotides. It was shown

th at alkylation with a-haloacetamido derivativesor phen-acyl bromides could be direc ted exclusively to th e sulfurof this base to introduce a nu mber of different conjugategroups (163, 164).

An example of a reaction restricted to a particularsequence involves introduc tion of psoralen on to thymi-dine only wheni t is flanked on th e 3'-side by adenosine(205). This was possible because, on irradiation, psor-alens preferentially cross-link double-stranded oligonu-cleotides a t thymid ine in the sequence 5'-TpA-3'. Th ereaction can be partially reversed on irradiatio n a t a dif-ferent wavelength to give, after stra nd separation , a sin-gle strand with the psoralen monoadduct of thymidine.This serves as a reactive probe that cross-links with itscomplementary sequence on irradiation.

groups refsFluorescent Dyes

fluoresceins

tetramethylrhodamineTexas red 99, 113pyrene 109, 210bimane 130, 166, 192mansyl 102dansyl 109, 126, 191

proflavine 197130, 163osin

naphthalene derivatives 123, 130coumarin derivatives 130, 207

acridine

99, 103, 113, 114, 146, 157, 160,

99, 113, 131, 168, 209, 211163, 168, 198, 200, 208-211

Intercalating Agents48, 49, 118, 134, 136, 149,

212-217oxazolouvridocarbazole 177. 218

I

anthraquinone 135phenanthridine 151phenazine 219

peroxidasesIgG 179alkaline phosphatases

nucleases 147, 180, 228

Cross-Linking Agents

Proteins99, 140, 176, 179, 220

99, 103, 121, 170polylysine 130, 221-227

alkylating agents 106, 119, 229-233azidobenzenespsoraleniodoacetamide 101azidoproflavin 194azidouracil 158, 159platinum(I1) 227, 234

100, 163, 164, 190, 193, 196, 215112, 181, 182, 189, 205, 249

Chain-Cleaving AgentsEDTA/FeII 108, 173, 174, 236-238phenanthroline/CuII 120, 150, 239, 240porphyrin/FeII 241, 242

biotinOthers

107, 109, 110, 122, 124, 126, 130,133, 142, 143, 145, 148,

195, 199, 200, 202, 210, 243125, 127, 128, 140, 170, 175, 244,

245

154-157, 162, 168, 169, 171,

solid matrixes

dinitrophenyl 109, 165trinitrophenyl 200proxy1 spin-lab el 191fluorene 203, 204isoluminol 99digoxigenin 246puromycin 247DTPA (chelating agent) 182phospholipid 248cholesterol 61, 307

Finally, attention is drawn to an old reaction tha t has

not been used for conjugation but which is unusual inthat i t permits selective reaction a t oligonucleotidehydroxylgroups when more oftenit is the bases tha t react mostreadily. This is the reaction of acetic anhyd ride in water(206). N o reaction was found at the bases and mixed-anhydride formation at phosphates is readily reversed.Th us , in the case of 5'-phosphorylated oligodeoxynucle-otides, reaction occurred only on th e 3'-hydroxyl group.

D. Conjugate Groups. Many of the conjugate groupsused with oligonucleotides are given in Tab leVI. Thisis not an exhaustive list of the use of these co mpounds,particularly of probes where th e literature is extensive.Rather, it is intended to indicate the nature of conju-gates studied a nd to serve as a source of the metho dsforcoupling different groups.



17 4 Bioconjugate Chem., Vol. 1, No. 3, 1990

Included are some rece nt, more specific ways for immo-bilizing oligonucleotides on solid supports, but the olderli terature on this subjectis not covered and may beobtained from th e references cited.

There is a very large amount of literature on chemi-cally reactive oligonucleotides, particula rly on tho se bear-ing nitrogen m ustard ty pe alkylating groups. Referencehas been given to a review plus a few more recent exam-ples. No att em pt is made here to discuss the chemicalreactions of these compounds.

A choice tha t has to be ma de for each conjugate groupis the length of the sp acer used to linkit to th e oligonu-cleotide. Thi s is particularly im porta nt in the case of anintercalating agent that has to interact with the helix.Here, a very small change in spacer length can influencethe o utcom e, bu t cha ins of five or six carbon a tom s werebest ( 6 4 , 1 7 7 , 2 12 , 2 1 3 ) . For psoralen to cross-link withth e opposite stran d, a shorter spacer of two carbons wasrequired, and yields fell six-foldif this were increased( 2 4 9 ) . For biotin or fluorescent tags that do not inter-act with the helix, a longer spacer of eleven or twelveatoms was preferable to minimize steric inhibition ofhybridization ( 1 0 3 , 1 2 7 ) .

4. P R O P E RT I E SOF MODIFIED OLIGONUCLEOTIDES

AND CONJUGATESThe development of conjugates with new properties

should improve existing tec hniques and lead to th e devel-opm ent of new uses and ideas. Advances are being madein the area s of autom ated sequencing( 11 3 , 2 0 8 )and non-radioactive probes( 2 5 0 )an d in accu rate chemical cleav-ing of DNA and RNA( 1 0 1 ,251-253) . Chemically reac-tive probes can be used to place a tag on a particularbase within an RNA( 1 8 1 ) and have been used t o dem-onstrate parallel helix formation( 1 2 0 , 1 9 4 ) . Nonradia-tive energy transf er between oligonucleotides can be usedto indicate their separation in solution( 2 0 9 , 2 2 0 ) . Thisseems a particularly promising technique that could beapplied in many d ifferent ways and ha s already been usedto investigate the struc ture of the Holiday junction( 2 11 ) .Th e same principle has been used to investigate interac-tions of oligonucleotides with proteins( 2 0 7 ) .

I t is beyond the scope of the present review to covera l l the uses and developments involving ol igonucle-otides. Rat her, the em phasis here will be on the biolog-ical properties of modified oligonucleotides, in particu-lar the effect of modification on hybridiz ation, stability,an d cell uptake . Th e final section discusses antisenseinhibition, whichis the most deman ding use of oligonu-cleotides, requiring tha t they find an d hybridize with theircomple mentary sequences inside cells. Again, th e approachtaken here is to examine the general consequences of mod-ification rathe r th an the pa rticulars of individual cases.

A. The Effect of Modification on Oligonucle-

otide Hybridization. Modifications to the internucle-oside phosphates can affect hybridization in a numberof different ways, butit is important that they shouldnot prevent base pairing. Redu ction in charge densitylessens electrostatic repulsion between the strands andshould facilitate their association. Th is effect will be great-est a t low salt concentratio ns, where th e shielding of thecharges is least. Steric interactions of substit uen ts willnormally destabilize the helix as,it has been suggested,will their electronic and other effects( 2 5 4 ) . These mightinclude disrupt ion of hyd ratio n of th e helix. However,the grooves of the hybrid might also provide a morelipophilic environment for the sequestrationof hydro-phobic substituents, thereby promoting hybridization.Cases where stronger hybridization resulted on increas-

Goodchild

ing the lipophilicity of th e substitu ent m ay be examplesof such a n effect( 4 3 , 6 2 ) .

Th e relative contribution s of all these factors are gov-erned by external conditions such as sa lt concentrationas well as intrinsic facto rs such as the length of th e oli-gonucleotide, the degree of modification, the localiza-tion of a given modification relative to the ends or mid-dle of the helix, and th e sequence of bases arou ndit ( 2 5 4 ) .Th us, the consequences of a partic ular m odification willvary from case to case. T he complexity of this situatio n

has prevented a clear understanding of th e effects of phos-pha te modification on hybridization.A complicating feature is the chirality of the phospho-

rus ato m following modification. Absolute stereochem-istry has been assigned by X-ray crystallography( 2 5 5 )and NMR( 2 5 6 , 2 5 7 ) nd by enzymic(258-261)and chem-ical metho ds( 3 3 , 4 4 , 7 6 ) . A molecule withn chiral phos-phorus atom s will consist of2" isomers, and one of themost challenging areas of oligonucleotide che mistry is th edevelopment of diastereospecific synthesis( 2 6 2 - 2 6 7 ) .Attempts to use diastereomerically pure starting mate-rials in the usual synthetic ap proach es resulted in race-mization (53 , 73 , 268 , 269 ) .

When small num bers of isomers are involved, resolu-tion is possible by chroma tography , and t he pure d ia-stereomers have been used for block conde nsation. Thisapproa ch has been largely limited to t he co nstruction ofshort backbones with alternating unmodified an d chiralphosphates or longer molecules with a single modifica-tion ( 4 6 , 4 8 , 2 7 0 - 2 7 2 ) . Enzym atic synthesis of phospho-rothioates gives theR , isomer exclusively( 5 0 , 5 1 , 2 7 3 )and has enabled studies of polynucleotides with exten-sively modified, stereopure backbones( 2 7 4 ) . It is notyet possible to synthesize by chemical means diastereo-merically pure chains of the length necessary for antisenseinhibition.

In their early studies of uncharged methylphospho-nates and triesters, Miller and Ts'o found t ha t racemicdi- to tetramers hybridized to unmodified strands with

greater affinity than t he pa rent phosphodiesters( 2 7 5 , 2 7 6 ) .While diastereoisomers of dimers differed from each o ther ,both formed more stable hybrids than th e natur al, chargedcompounds an d were less effected by sal t concentratio n.Thi s was attrib uted to the lack of charge-charge repul-sion between the strandsof the complex ( 1 7 , 3 2 ) . Anadverse effect on hybridization as the size of the subst it-uent increased from PCH3 to POCHB to POCHL!Hsseemed to be due to steric interactions.

Similar improvements in hybridization were reportedfor other low molecular w eight triesters a nd pho sphora-midates ( 3 4 , 43 , 6 2 ) . With substituted amidates, theseincluded a positively charged backbone. In some cases,however, bulkier substituents improved rath er tha n dimin-ished hybridization.

When a single, uncharged group is incorporated intoan oligonucleotide, a different effect is seen an d th e sta-bility of the helix is unchanged( 1 9 1 ,2 7 1 ) or reduced inmost situations( 7 5 , 2 5 4 , 2 5 6 , 2 7 7 ) .Possibly th e removalof a single charge out of many makes little difference tothe overall electrostatic repulsion between the strandsand the other destabilizing effects of substitution gainin relative importance. Th e difference between the dias-tereomers is more pronounced with greater destabiliza-tion when the substituent points into the major grooverather than away from the helix( 7 5 ,2 5 4 , 2 5 6 ) .

With longer, extensively modified oligonucleotides, thecomplexity of the m ixture becomes much greater a nd aseach diastereomer in a pair is slightly different, some het-



Review Bioconjugate Chem., Vol. 1, No. 3, 1990 175

In an attem pt t o stabilize hybridization with very shortoligonucleotides, Letsinger an d S chott attac hed a n inter-calating agent t o the phosp hate group of T p T(151) . Thisproved successful an d, as discussed late r, permits th e useof sh orter oligonucleotides than would otherwise be pos-sible for antisense studies.The physical chemistry ofthese interactions has been investigated(165, 212, 213,285) and i t has been shown tha t an intercalating agenta t th e end of an oligonucleotide, especially th e 3'-end, ismore beneficial than on an internucleotide phosphate and

th at a second intercalating group offers no further advan-tage ( 136, 21 3) .a-Oligonucleotides and others with m ethylphospho-

nate or phosphotriester linkages also benefited from th eaddition of an intercalator( 4 8 , 6 4 , 1 7 7 , 2 1 5 , 2 1 8 ) .n theseconjugates, two different m echanisms for stabilizing theduplex are combined. Th e only intercalator tha t has beencombined with a phosph orothioa te backbone was of some-what uncertain efficacy( 1 3 4 ) . No stabilization wasobserved and , if confirmed in othe r cases, this m ay reflectsteric effects of the larger sulfur ato m a t the site of inte r-calation.

B. The Effect of Modification on Nuclease Re-sistance. A number of studies have demonstrated deg-radation of unm odified oligonucleotides a t greatly vary-ing rates in different cellsor in the serum -containing mediaused for cell culture. Survival times vary from minutesto days (56,286-293) . Consequently, it has been a goalin many approaches, particularly in the antisense field,to develop nuclease-resistant derivatives on the assump-tion th at these will be more pote nt. Of course, the re isthe ch ance tha t they will also be more toxic because theysurvive longer.

It was recognized early on th at the rate of degrad ationof RNA by exonucleases from sna ke venom and spleenwas slowed considerably by phosphorothioate groups(294) .The former enzyme can cleave theR , diastereomer butnot the S , or adjacentR,S, or S,S, groups (53 , 262 , 295-297) . In contrast, nucleasesS1 and P1 are specific for

the S , isomer (53, 297, 298) while ribonucleases A andT2 do not distinguish between different configurations(296) . With DNAseI, sequenceor base com position alsoeffect the rate of digestion ofphosphorothioate-substi-tuted polynucleotides(274) .

As a result, oligodeoxynucleotides with a high propor-tion of unresolved phosphorothioate groups are almosttotally resistant to snake venom phosphodiesterase, aredegraded 2-45 times more slowly than no rmal byS1 andP1 nucleases, and survive many times longer tha n un sub-stituted oligonucleotides in human serum( 5 6 ) .

All other m odifications to internucleoside pho sphatesthat have been investigated also inhibit the action ofnucleases. Most reports suggest complete resistance ofphosphoramidate( 4 3 , 4 5 , 5 8 , 6 2 ) , hosphonate ( 1 8 , 2 7 0 ) ,or phosphotriester (32 , 43 , 299 ) linkages toward thenucleases tha t have been tested. An exception is the veryslow rate of cleavage by snak e venomor spleen phospho-diesterases of unsubstituted phosphoramidate observedfor the sequence d(ApA) but not for Tp T( 4 3 , 6 5 ) . AllN-substituted phosphoramidates were resistant. An earlyreport of slow cleavage of one of the diastereomers ofphosphonates by snake venom phosphodiesterase couldnot be confirmed(18 , 270) .

While the modified linkages themselves m ay be resis-tant, in oligonucleotides containing mixtures of modi-fied and natural linkages, exonucleases that work pro-gressively from one end of the chain can som etimes skipover an isolated phosphonateor triester linkage to cleave

erogeneity in overall properties is expected. An unchargedoctamer of thymidine with an ethy l phosphotriester back-bone could be separate d into fractions with differen t affin-ities for poly(dA) (4 0 ) . The real test of these com-pounds, however, is the effect of extensive substitutio non the properties of a longer heterosequence.

T he hybrid of a 20-mer containing18 methylphospho-nates melted only4 "C below the fully charged duplexwith little broadening of th e melting curve in 0.1 M sa lt(278) . In another study, a 15-mer containing11 phos-phonate linkages melted 5"C igher than the diester in0.015 M salt a nd8 "C ower in 0.15 M salt( 6 4 ) . Heter-ogeneity in hybridization w ould be reflected in a b road-ening of th e melting curve which was not appreciable ineither of these examples. From the limited data pres-ently available, it would appe ar th at, in practice, the com-plexity of the diastereomeric mixture does not greatlyreduce or broaden the spectrum of affinity for the tar-get sequence of phosphonates w hen compared to tha t ofphosphodiesters. Th at is not to say th at higher meltingwould not result from the optimum, stereopure back-bone.

A t variance with these findings is the suggestion thatmethylphosphonates are inherently unsuitable for adopt-ing a right-handed helical conformationso that meltingtem pera ture decrease to below 20 "C with chain lengthsgreater than four. Me thyl triesters were not found toshare this property an d were proposed a s superior antisenseagents with high melting temperatures th at are not influ-enced by the chirality of the phosphorus( 3 6 , 2 7 9 ) .How-ever, they hybridize poorly with RN A, which is the usualtarget for antisense inhibition(280) .

Extensive subs titutio n of longer oligonucleotides withvarious phosphoramidate linkages gave som ewhat less sta-ble hybrids than the phosphonates. Those derived fromprimary amines were superior to those from secondaryamines ( 6 4 ) . Cationic amidates showed a reversal in sal tdependence and formed hybrids that were more stablein low salt then phosphodiesters but less stable in high

salt ( 6 3 ) .A comm on finding in many studie s of noncharged phos-

phate modifications is th at hybridization to RNA is lessefficient than that to DNA(40, 43 48, 63, 86, 174, 270,280) . It has been suggested in the case of ethyl phos-photriesters that this results from the inability to forman A type helix, where the loss of rotational freedom ofthe ethyl group is greater( 4 0 ) .

With a single phosphorothioate linkage, only theR ,isomer with sulfur pointing into th e major groove desta-bilized the helix( 2 8 1 ) . Multiple phosphorothioate link-ages, either allR , or a racemic mixture, lowered the melt-ing temperature by an am ount tha t depended on the basecomposition of the oligonucleotide but was a t least7 O C

for a 15-mer(56, 134, 274) .Modifications other than at internucleoside phos-

pha te may also affect hybridization. a-Oligonucle-otides, for example, hybridize with @RN A and DNA byforming the usual Watson-Crick base pairs. In fact, thesehybrids are considerably more stable than when bothstrands are p ( 2 4 3 ) . Unlike natural duplexes, however,those with an a-chain have parallel strands(215, 216,282, 283). An exception is the complex between a-olig-othymidylic acid an d P -poly(rA),which is antiparallel(239) .This particular a-oligonucleotide is also unusual in thatit can form a double-stranded helix with itself contain-ing T.T base pairs (284) .Sim ilar parallel self-pairing wasobserved on neutralizing the phosphates of 6-oligo-thymidylate as methyl triesters( 4 2 ) .




the adjacent phosphodiester a t a reduced rate(27, 270,300). This may be more difficultif the phosphonate isnear th e end of the chain an d the presence of two adja-cent interna l methylph ospho nates in oligothymidylic acidblocked the progress of the enzyme much more effec-tively than just one(27, 300). Similarly, blocks of con-tiguous phosphorothioates a t the ends of the chain gaveresistance to exonucleases while conserving desirable p rop-erties of the u nmodified backb one in between(56) . Resis-tance t o endonucleases may be improved by reducing runsof contiguous phosphodiesters, particularly below four(300).

Modifications a t groups other th an phospha te may alsoinduce resistance to nucleases. a-Oligodeo xynucle-otides, for example, proved far more stable than P-oli-godeoxynucleotides in a number of different biologicalenvironments(291, 301). Also, the presence of a bulkygroup such as an intercalating agent or even methyl-thiophosphate at the appropriate end of an oligonucle-otide can preempt exonuclease attack(134, 237, 291).

RNA was found to be more stable than DNA in nuclearcell extracts(302). Methylation of the 2’-hydroxyl groupincreases its resistance to nucleases(303).

Nucleases are not the only enzymes involved in the

catab olism of oligonucleotides inside cells. In culture dfibroblasts, oligonucleoside ethyl phosphotriesters werebroken down qu ite rapidly, probably following deethyl-ation. Methylpho sphonates survived much better but wereslowly degra ded by a pathwa y th at m ay begin with degly-cosylation (304).

C. Cellular Uptake of Modified Oligonucleo-tides. A problem common to uptake studies is the dif-ficulty in distinguishing ma terial inside the cell from tha tbound to the outer membrane. Few studies have man-aged to do this convincingly enough to eliminate do ubt.Conseque ntly, confidence in the lite rature concerning thi ssubject is not as high as is desirable.

Despite the ir high charge density , oligonucleotides aretaken u p reasonably well by mammalian cells. Thi sappea rs to be a n energy-requiring process and may involvereceptor proteins on the cell surface. Intracellular con-centration s may rise to abou t 1 0% of those outside thecell within 15 min to2 h (286, 290, 305, 306). Shorteroligonucleotides are taken up som ewhat more rapidly andphosphorothioates are taken u p more slowly than unmod-ified oligonucleotides(134). Lipophilic substituents suchas intercalating ag ents or cholesterol facilitate uptake(216,237, 307).

Uptake of uncharged methylphosphonates appears tobe quite different. Intracellular levels of dimers to non-amers reached extracellular concentrations within1.5 h(276). Th is would app ear t o be passive diffusion acrossthe cell mem brane. Prokary otes have not been investi-

gated extensively, but in co ntrast,E . coli cells were imp er-meable to chain lengths greater than four(308).D. Modified Antisense Oligonucleotides. Oligo-

nucleotides complem entary t o strategic regions of viralor messenger RNA’s were first shown by Zamecnik a ndStephe nson to inhibit viral replication(309). The s t ruc-tures of these highly specific, biologically active com-pounds can be predicted from the sequence of the tar-get RN A an d are therefore useful for genetic analysis andattractive candidates for therapeutic agents. As they gen-erally preven t expression of the sense strand, they havebecome known as antisense oligonucleotides. Th e objectof this section is not to review th e antisen se approa ch orto discuss strategies for its use, as this has been doneelsewhere (310, 311). Rather, attention will be focused

Goodchild

on the effects of chemical modification on activity.Comparatively few true conjugates have been used for

antisense studiesso far although modifications t o the back-bone have been used extensively. The se will play anincreasingly imp ortan t role in designing th e next gener-ation of compounds where components with particularproperties will be required.

The factors that are usually assumed to l imit the activ-ity of antisense oligon ucleotides are cellular upta ke, resis-tance to nucleases, and t he stability of th e hybrid formed.Modifications are usually chosen to improve oneor moreof these prope rties, as discussed in the previous sections.Overall activity results from the interplay of these andother factors whose relative importance is generally notknown. However, a steady improveme nt in activity hasbeen achieved by using this rational approach that isencouraging for the futu re of designing con jugates to meetspecified requirements.

The modifications to th e backbone th at have been usedmost extensively are those discussed in previous con-texts: phosphorothioates, methylphosphonates, phospho-ramida tes, and phosphotriesters. Chimeric oligonucle-otides with several modified linkages at each end havebeen more successful with phosphorothioates th an m eth-

ylphosphonates(28, 60, 278).A fundam ental difference between negatively chargedphosphodiesters or phosphorothioates and th e unchargedderivatives is their acceptance by ribonuclease H. Th isenzyme degrades the RNA stra nd of an RNA /DN A duplexand has been shown to be an important factor for theactivity of antisense oligonucleo tides in a numb er of sys-tems. In these cases, binding of oligonucleotide at anysite on the RNA should lead to cleavage an d irreversibleinactivation. In situations where this enzyme is no t avail-able, oligonucleotides are thought to inh ibit expressionby passive steric blocking of translation or other events(hybridization arrest). Th e particular binding site is thenof great importance. With mRN A, for example,it appearsthat oligonucleotides can be readily displaced by ribo-somes and were only effective when bound to th e 5’-cappedend o r, to a lesser extent, across the AUG initiator(312).

RibonucleaseH recognizes the charged, unmodified orphosphorothioate backbone in oligodeoxynucleotides bu tnot th e uncharged m ethylphosphonate or phosphorami-dates (56,249,252,300,313-315). Th us , passive hybrid-ization arrest is the only known mechanism open t o thelatter compounds and the target site may be of primeimporta nce with these modifications. Ther e is a sugges-tion, however, tha t m ethylphosphonates may be more resis-tant to displacement from the RNA by cellular factorswhich could be a property of the uncharged backboneand could improve activity by the passive hybridizationmechanism (316).

Th e importance of the binding site is well illustratedby the a-deoxyoligonucleotides which, despite the ir goodhybridizing ability, do not activate ribonucleaseH andwere found inactive as antisen se agents exce pt when com-plem entary t o the 5’-capped end of mRNA(223,314,317-320).

With chain lengths of 20, methylphosphonates, phos-phorothioates, and phosphonam idates were found to reducethe concentration of oligonucleotide necessary for goodviral inhibition from over 20 pM to5 pM or less(58,278).Perhaps because of their somewhat weaker hybridiza-tion, reducing their chain len gth to 15 reduced activityfar more than with the unmodified series. [In anotherstudy , however, 15 was found to be t he optimu m chainlength for phosphonates(3311.1 Methylphosphonates with



Review

chain lengths of 10 or less may have to be used a t con-centrations of 100pM or higher to achieve good results.Even so, they ar e often more active tha n th e unmodifiedcompounds(276,308,316,321-326) .Inappropriate chainlength or binding site might be among the reasons forpoor or no activity found in other examples(28 , 224 , 314 ,315, 327 ).

In the rib0 series, both methylation of the 2’-hydroxylgroup and phosphorothioate substitution were neces-sary for antiviral activity in one study( 6 0 ) . The 2’-0-

methyl substituent alone does not permit ribonucleaseH digestion of the complementary RNA(328) .With chemical modification, th ere is always the possi-

bility of introducing unwan ted biological properties. Th iswas seen with the pho sphorothioates, which can bind ratherwell to a num ber of proteins an d inhibit certain enzymes(314, 329, 330) . As a result, in some antiviral and o therassays, inhibition was not restricted to antisense seque nces(58, 61, 327, 331 ). In further studies, antisense effectswere separated from other effects of phosphorothioates(332 ,333) .

Among the true conjugates are derivaties with acri-dine linked at the 3‘-end to stabilize hybridization. Th ismodification increases activity and permits the use ofunusually sho rt oligonucleotides(334-337).

Conjugates of polylysine were reported to lower theconcentration necessary for good antiviral activity to1pM or below (59 , 221, 224-226). This more than com-pensates for the increase in mass of material requireddue to the doubling of m olecular weight (th e preferredmolecular weight for the peptide is about the same asth at of a 20-base oligonucleotide). Th is approach doesnot work in all cells and polylysine is toxic at higher con-centrations (225) .

Another group that, like polylysine, is intended toincrease upta ke is cholesterol. Th is has beneficial effectsfor both unmodified an d phosphorothioate backbones aswell as for alkylating derivatives(61, 307) .

In 1967, Belikova e t al. mad e a dinucleotide carrying

an alkylating reagent for the m odification of complemen-tary sequences ( 119) . This was probably the first exam-ple of what would now be called an antisense oligonucle-otide. Reactive compounds of this type have been inves-tigated extensively by groups in th eUSSR (229). In recentyears, attentio n in the W est also has turned to these andother derivatives in Ta ble VI th at can cross-linkor cleavethe target RNA or DNA. This has come about with theneed to develop more potent derivatives to improve thepotential for therapeutic applications.If hybridizationis reversible, so one argument goes, then a higher con-centration of the oligonucleotide is required in the cellto maintain the complex thanif the process were notreversible. Hence oligonucleotides th at irreversibly changethe target may be more potent.

This was demonstrated using antisense oligonucle-otides to inhibit replication of a single-stranded DNA byE . coli DNA polymerase I(227) . The enzyme was notinhibited by an unmodified oligonucleotide hybridizedto the temp late unless th e two were cross-linked. Pre -sumably, as the enzyme reads t he DNA, i t can displacehybridized oligonucleotide in its p ath but becomes stalledwhen th e oligonucleotide is irreversibly bound t o th e tem-plate.

Despite th e large body of chemical work in this area,there are only a few examples of the use of chemicallyreactive oligonucleotidesas antisense agents e ither in vitroor in vivo. Th ese includ e an early inhibition of IgG syn-thesis in cells(229) . More recently, the photoactivated


cross-linker psoralenwa sattached to oligonucleosidemeth-ylphosphonates and found to increase their potency by20-40-fold in antiviralor inhibition of translation assays(249, 326) .

A concern in using reactive conjugates of this type isthe possibility of nonspecific reaction with oth er c ell com-ponents leading to toxicityor, alternatively, self-inacti-vation. Th e latter has been found to limit the use ofEDTA attached to m ethylphosphonates due to autocleav-age of the conjugate group by the free radicalsit gener-

ates (174) .5. CONCLUDING COM MENTS

Much curren t research a t the interface of biology andchemistry is directed a t understan ding and predictingthe effect of molecular structure on biological activity.Oligonucleotides are particularly well-suited for th is typeof activity. Th is is because of th e nat ure of their s ite ofaction. Unlike most active compounds, this is not somehydrophobic pocket on a protein with u nique an d unpre-dictable properties. Rath er it is a nucleic acid whose pre-cise sequence can be determined and whose interactionwith th e oligonucleotide can be predicted with some con-fidence.

Th e encouraging finding from th e work reviewed hereis just how robust this mechanism for base pairing is towardchemical modification. Th e natu re of the backbone canbe changed from anionic to unchargedor cationic, fromhydrophilic to lipophilic without s eriously interfering withhybridization. Atte mp ts to improve antisense inhibi-tion in a rational way by altering specific features of themolecule have successfully increased activity.This is prom-ising for prospects of tailoring molecules to particularpurposes. These include improving performance an d util-ity in areas where oligonucleotides have already foundapplication such as diagnosis, genetic analysis, auto-mated sequencing, and many others. Italso includes meet-ing the pharmacological requirements for possible futuredrug development.

Whether or not modified oligonucleotides and their con-jugates have the necessary attri but es for pharmaceuticaluse, it is clear th at th ey provide an unusual opp ortun ityfor the rational design of useful molecules with specificproperties.

ACKNOWLEDGMENT

I wish to than k P aul Zamecnik for his enthusiastic sup-port and encouragement and the referees for valuablesuggestions. Th is work was supp orted by a grant fromthe G. Harold and LeilaY. Mathers Foundation, byNational Cooperative Drug Discovery Group for th e treat-ment of AIDS Grant U01 A124846 from the NationalCancer Institute and the National Institute of Allergy

and Infectious Diseases, and by Cancer Center SupportGr ant P30 C 12708-18 from the National Cancer Insti-tute .

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TEAC HING EDITORIAL

A Brief Guide to Nucleic Acid Chemistry

Paul S. Miller

Departm ent of B iochemistry, School of H ygiene and Public Health, Th e Johns Hopkins University, 615 NorthWolfe Street, Baltimore, Maryland 21205. Received January 19, 1990

STRUCTUREOF NUCLEIC ACIDS

Nucleic acids encode t he genetic info rmatio n of all liv-ing organisms an d are intimately involved in the conver-sion of this informa tion into cellular proteins an d enzymes.Cellular nucleic acids are polymeric molecules which arecomposed of thr ee basic units: a base, a sugar, and aphosphodiester group. Th e arran gem ent of these threegroups to form either DNAor RNA is shown in Figure1.

T he nitrogenous, heterocyclic bases are derivatives ofpurine or pyrimidine. Th e four bases commonly foundin DNA are adenine, guanine, cytosine, and thymine. Thesesame bases are also found in RNA with the exceptionth at thym ine is replaced by uracil. In addition, a vari-ety of modified bases, such asN 4,N -dimethyladenineand N ‘-methylguanine are found in messenger RNA,transfer RNA, an d ribosomal RNA.

T he bases are linked to 2’-deoxyribose in DNA or ribosein RNA via an N-glycosyl bond t o form a nucleoside. T henucleosides are na me d according to the heterocyclic basewhich they contain. Th e nucleosides found in DNA are2’-deoxyadenosine, 2’-deoxyguanosine, 2’-deoxycytidine,and thymidine. Th e nucleosides commonly found in RNAare adenosine, guanosine, cytidine, and uridine.2’-0-Methylribosyl nucleosides are also found in RNA, par-ticularly in messenger RNA and ribosomal RNA.

Th e nucleosides ar e linked together via phosphate estergroups to form th e sugar phosphate backbone of the nucleicacid. Th us esterification of the 3’-hydroxylof one nucle-oside and the 5’-hydroxyl of t he next nucleoside unit resultsin the formation of a3‘-5‘ internucleotide bond. Th istype of linkage is found in both DNA and RNA. In thecase of RNA th e internucleotide bond could also extendfrom the 2’-hydroxyl to form a 2‘-5‘ internu cleotid e bond.Such 2’--5’ linkages are found in certain oligoadenylateswhich are synthesized in mamm alian cells in response tointerferon ( I ) .

A single phosphorlyated nucleoside unit is called a nucle-otide. T he sequence of nucleotides within the nucleicacid chain determ ines th e genetic information encoded

nine, can form hydrogen bonds w ith the pyrimidine bases,thym ine (uracil in RNA) a nd cytosine, respectively (seeFigure 2 ). Th e base pairs formed between these so-calledcomplementary bases enable separate chains of nucleicacids to interact with one another. In DNA, sepa ratenucleic acid strands form a double-helical structure inwhich the sugar phosphate backbones run in an antipa r-allel direction. Double-helical DNA , which usually existsin a right-handed, B-type conformation, can exist in avariety of conformational forms including left-handed heli-ces (2) . Th e particular conformation depends upon th enucleotide sequence and the environment of the DNA.DNA can also exist in a triple-stranded form in whichthree bases form a triad via hydrogen-bonding interac-tions as shown in Figure 2(2, 3 ) .

Although RN A is o f ten tho ugh t of as a s ing le -s t randed molecu le , se l f -complementa ry nuc leo t idesequences present within the single strand give rise toth e forma tion of intram olecula r helical regions. Th eseintramolecular interactions can produce a trem endous vari-ety of helical and looped structur al regions and accountfor the secondary structure within RNA molecules. Inaddition to these secondary structural features, furtherfolding an d hydrogen-bonding interactio ns between basesin remote parts of the molecule give rise to a tertiarystruc ture. Th e combination of these interactions resultsin overall three dimensional structure, whose complex-ity approaches th at found in proteins. Th is complexityhas been most clearly revealed in the stru cture of trans-fer RNA ( 4 ) .

Nucleic acid structure has been elucidated a t the atomiclevel of resolution by nuclear magnetic resonance spec-troscopy and X-ray diffraction techniques. In additionto studying nucleic acid structure, recent X-ray experi-ments have been used to examine th e interactions of pro-teins with nucleic acids. For example, the structu res ofthe complex formed between th e restriction enzyme EcoRI and a deoxyribonucleotide duplex, and of th e complexformed by glutaminyl tRNA with its cognate aminoacylsynthetase have been determined( 5 , 6). Such studies

conjugates of oligonucleotide

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