David H. J. Bunka and Peter G. Stockley- Aptamers come of age – at last

8/3/2019 David H. J. Bunka and Peter G. Stockley- Aptamers come of age at last

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Since the iscovery in the ear y 1980s that RNA wasnot simp y a passive carrier of genetic information butcou participate irect y in cata ysis in iving ce s1,2,our un erstan ing of RNA structure an functionhas been in constant f ux. Another important insightemerge in 1990 when three separate groups use in vitro se ection approaches to iso ate RNA an DNAmo ecu es that bin tight y to severa nuc eic-acibin ing proteins, an to organic yes use for affinity chromatography 35.

The iso ation of nove RNAs that bin to smaorganic mo ecu es confirme the assumption baseon the abi ity of RNA to fo into comp ex, three-

imensiona shapes riva ing those of proteins thatin egenerate sequence ibraries of 1015 mo ecu es,which are easi y create an manipu ate in the

aboratory, there is probab y an RNA mo ecu e witha fo to bin a most any target5. A ogica extensionof this i ea was that aptamers that bin transition-state ana ogues that mimic the chemica structure of key interme iates in enzyme cata aysis wou a so becata ytica y active, ana ogous to cata ytic antibo -ies. This has turne out to be true even for comp exorganic reactions6,7. These resu ts provi e compe ingexperimenta evi ence in support of the RNA wor hypothesis, which postu ates that the bio ogica worevo ve from a se f-rep icating RNA mo ecu e that wasassemb e by chance8.

An iterative protoco for thein vitro se ection experi-ments iscusse above4 was intro uce an ca esystematic evo ution of igan s by exponentia enrich-ment (SELEX)(FIG. 1) . The pro ucts of the se ection5 were ca e aptamers (from the Latinaptus meaningfitting). By ana ogy with antibo ies the targets bounby aptamers were ca e apatopes, a though epitopeor target is the most frequent y use termino ogy. These ection protoco has subsequent y been re uce toan automate in vitro process911, opening the way for

high-throughput se ection against an a most infiniteof targets. The range of aptamer reagents iso ate canassesse by reference to anon ine atabase.

Given the bio ogica significance of aptamerapatopeinteractions, it seeme un ike y that Nature hneg ecte this fie of biomo ecu ar recognitioin ee over the past few years we have earne abnatura y occurring riboswitches12,13. These a ow regu-

ation of gene expression by changes in the conformtion of mRNA transcripts in response to a terations physio ogica con itions, me iate by the bin insma mo ecu ar-weight metabo ites within an ap

omain. These mRNA conformationa changes eftrans ationa or transcriptiona termination, or evse f-c eavage(BOX 1) .

Commercia exp oitation of the aptamer fieagge behin research iscoveries. However, ast

the first aptamer-base therapeutic agent (Macugenfor treating a form of macular degeneration entere c ini-ca use1416. So, on the sixteenth anniversary of the firpub ications one can argue that aptamers have finacome of age. Here we iscuss significant recent evements in the fie of aptamer research, an raw atttion to work aime at eve oping new aptamer-basanti-infective agents.

Structural lessons from aptamersAptamers se ecte against nuc eic-aci bin ing teins an sma mo ecu ar-weight igan s havuse to investigate protein an nuc eic-aci sequespecificity. The Go aboratory showe that a natRNA stem- oop sequence is one of two istinct bre ate sequences that can bin bacteriophage T4 Dpo ymerase with a most equa affinity 4. Therefore, thesequence space for such interactions contains severacombinations that give rise to mutua bin ing affinan potentia y there are combinations that have been exp oite uring evo ution.

Astbury Centre for Structural Molecular Biology,Universityof Leeds, Leeds LS2 9JT, UK.Correspondence to P.G.S.e-mail: [email protected]:10.1038/nrmicro1458

Macular degenerationA condition in which the light-sensing cells of the macula,which is in the centre of theretina, malfunction and ceaseto work, leading to reduction orloss of central vision. Thedisease can be caused by theleakage of newly forming bloodvessels into the retina and it isthis process that is susceptibleto treatment by Macugen.

Sequence spaceAll the possible sequencecombinations in a nucleic-acidlibrary used for SELEX. Asthere are many suchsequences, it is unlikely that allpossible combinations of sequence and function havebeen tried during evolution.

Aptamers come of age at lastDavid H. J. Bunka and Peter G. Stockley

Abstract | Nucleic-acid aptamers have the molecular recognition properties of antibodies, andcan be isolated robotically for high-throughput applications in diagnostics, research andtherapeutics. Unlike antibodies, however, they can be chemically derivatized easily to extendtheir lifetimes in biological fluids and their bioavailability in animals. The first aptamer-basedclinical drugs have recently entered service. Meanwhile, active research programmes haveidentified a wide range of anti-viral aptamers that could form the basis for future therapeutics.

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588 | AUGUST 2006| VOLUME 4 www.nature.com/reviews/micro


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SELEX(Automated cycles)

a b

c

d

e

Nucleic-acid poolis incubated withtarget molecules(Toggle-SELEX,Photo-SELEX)

Nucleic acids are amplied(2-amino, 2 -uoro,2-methyl or 4 -thionucleotides, tailored-SELEX)

Partitioning o target-boundnucleic acids(SPR, CE)

Low afnity speciesare removed

Bound species are eluted

Surface plasmon resonance(SPR). A technique formonitoring the affinity betweenmolecules in solution (analytes)as they pass across animmobilized target on the SPRsensorchip. In aptamerresearch, this technique is usedto collect slowly dissociatingaptamer species that havehigher affinity than those thatelute early.

SpiegelmersA term derived from theGerman word for mirror. Theseare RNA aptamers synthesizedchemically with L-riboseinstead of the natural D-riboseand are therefore resistant tonuclease action.

A simi ar series of experiments were carrie outon the trans ationa repression comp ex that formsbetween a 19 nuc eoti e RNA stem- oop operator inthe genomic RNA an a imer of the coat protein in theRNA bacteriophage MS2. The trans ationa repres-sion comp ex is use as a mo e for un erstan ingsequence-specific RNAprotein interactions. SELEXshowe that the natura RNA stem- oop operatorbin s tight y to the phage coat protein17, a though anRNA aptamer that iffers from the consensus opera-tor by a sing e nuc eoti e emerge from this ana ysisas the tightest bin er. S ight y re ucing the se ectionpressures in such experiments, or intro ucing muta-tions into the RNA-bin ing site on the coat protein,yie e aptamers with sequence variants that seemeto break previous y i entifie consensus ru es forRNA stem- oopprotein bin ing18. However, X-ray structure etermination of these nove aptamer protein comp exes19,20showe that the aptamers stiboun at the natura RNA-bin ing site, an that thecrucia e ements in the aptamer consensus sequencesimp y nee e to be a juste to ref ect the structuraconstraints in this system, which are the number of nuc eoti es between a enines that are boun to theprotein(FIG. 2) .

Simi ar y, for DNA-bin ing proteins, the sequenof natura bin ing sites, such as operators, have bese ecte by evo ution in the context of many DNA-binproteins. To avoi acci enta cross-bin ing to the saregu atory site by ifferent transcription factors, se esites nee to be iscriminate efficient y, rather than bthe tightest bin ers. In ee , as such interactions neebe reversib e to a ow gene regu ation, the tightest pbin ing sequences are often not founin vivo but emergefromin vitro SELEX experiments21.

Interesting y, when aptamers are se ecte against smo ecu ar-weight igan s that bin natura RNA tasuch as aminog ycosi e antibiotics, a wi er range of binsequences is revea e22,23. Structura stu ies show that thesaptamers often present unusua bin ing surfaces, whthese igan s fit into; however, this oes not necessacorrespon to the bio ogica interaction24,25. Structuresof se ecte ribozymes, such as the Die sA er ribowhich cata yses the formation of two carboncarbon boin a reaction of genera importance in organic chemista so revea unusua ways in which reactants can be bto promote cata ysis26. Obvious y it is a so possib e to seaptamers against targets that o not norma y bin nucaci s, an this possibi ity exten s the potentia funcan app ications of nuc eic aci s into new rea ms.

Recent technical innovationsThe basic princip es of SELEX have remaine auna tere since its conception(FIG. 1) . Consi erab eimprovements have, however, been ma e to the se ectstep an to the properties of the resu ting aptamers.

Most a vances in aptamer iso ation have aimeimproving the efficiency of se ection, that is, re ucingnumber of cyc es or the time taken to iso ate high-affspecies. The E ington group was the first to automateprocess, re ucing the iso ation time from severa moto a few ays911. Other groups have focuse on improvinpartitioning efficiency. High-affinity aptamers have beiso ate usingsurface plasmon resonance (SPR) by fraction-ating the issociation phase of the ana yte-bin ing retion, thereby iso ating on y those species with the s ooff-rates(REFS 27,28) . High-affinity DNA aptamers havea so been iso ate in a sing e roun using capi atrophoresis to separate free protein or nuc eic aci s frcomp exe materia29. Other key areas of improvementare in aptamer biostabi ity an bioavai abi ity. The aof T7 RNA po ymerase to incorporate 2-amino-mo ifieor 2-f uoro-mo ifie pyrimi ines30,31a ows iso ationof aptamers with great y improve biostabi ity. Mrecent y, an evo ve RNA po ymerase was reportecou efficient y incorporate 2-methy pyrimi ines intotranscripts, which a so resu ts in increase biostaity 32. The 4-thio pyrimi ines have a so been a ethis fami y of RNA-stabi izing nuc eoti es33. An e egantapproach invo ves the use of l-ribose-base nuc eotto generate biostab eSpiegelmers 34 fo owing se ectioof natura d-ribose sequences against theenantiomer of the bio ogica target. A so, 33 inke inuc eoti e capan circu arization or isu phi e cross inking35 havebeen shown to improve aptamer stabi ity, a though thmo ifications are ess common y use .

Figure 1 | Basic outline o a single SELEX round. a | A degenerate nucleic-acidsequence library is incubated with the target molecule under defined solutionconditions. b | Target-bound nucleic acids are partitioned. ce | Species withlower binding affinity are removed and the bound species are eluted, allowingpreferential amplification of higher affinity species. This enriched pool is then used as thestarting point in subsequent cycles. Typically, 10 to 20 cycles are carried out beforeaptamer characterization. In early rounds, species with no affinity are competed out ofthe pool. In later rounds, molecules with affinity compete for binding sites on the target.Such competition results in enhancement of the pool binding-affinity in a manner similarto Darwinian evolution. Recent technical developments described in the text are listedalongside each step in brackets. CE, capillary electrophoresis; SELEX, systematicevolution of ligands by exponential enrichment; SPR, surface plasmon resonance.

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NATURE REVIEWS| MICROBIOLOGY VOLUME 4| AUGUST 2006| 589


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UU

U

U U GG

G

GG G G G

GA

AA

A

A A A

A AA

A

AA

U A A U C GC C C

CC

C

CC

U U

UACUUCGG

U

U U

UU

UU U U

Translationalrepressor

O

120

81

100

AUU

U

U

UU

UUU

U

UUUU

U

U

U

U

C

CC

CC

C

C G

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SD

337

32

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47 52

57

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22

15

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A AAAA

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U AC GG U

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CC

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Translationalactivator

On

37

32

42

47 52

57

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62

120

22

1005

3

15 81

U20

A76U49

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C53G46

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b

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SD

EnantiomerTwo molecular structures thathave identical chemicalcompositions but are non-superimposable in threedimensions they are mirrorimages of each other. Foramino acids and ribose sugarsthese are known as the D- andL-forms.

The bioavai abi ity of aptamers is a significant issuethat affects their app ication asin vivo iagnostic antherapeutic too s. The most common approachesto improve bioavai abi ity inc u e surroun ing theaptamer with ipoproteins36 or the attachment of bu ky groups, such as po yethy ene-g yco3638, cho estero39 or biotinstreptavi in40, to the 5 or 3 en s, resu tingin re uce rena c earance an therefore an increasep asma ha f- ife. Aptamers that bin intrace u ar

targets require either targete e ivery or recombinexpression, each of which has associate technicprob ems.

Variations of the SELEX protoco have a oiso ation of aptamers with specific esire propties, expan ing the repertoire of aptamer functions. Togg e-SELEX, for examp e, a ows isoof aptamers with a broa er range of specificities bse ecting against re ate targets in a ternating cyc41.

Box 1 | Riboswitches Natures aptamers

Applications of artificial RNA ligand-bindingdomains were fairly advanced 90 before it wasrealized that such interactions are biologicallyrelevant and are used to regulate geneexpression. Riboswitches are rapidly joininganti-sense and small interfering RNAs as

important biological tools13,91

. It is now knownthat regulated conformational changes inmRNAs in response to temperature 92 or ligandbinding lead to translational or transcriptionalregulation. Ligand binding can even driveRNA to fold into an auto-cleaving ribozyme 93.

Discoveries about riboswitches are nowbeing exploited actively to develop newgene-expression control and selectionsystems, including applications ofrecombinant ribozymes 94 and aptazymes 95.Aptazymes are allosterically regulatedribozymes. Novel engineered ribo-regulatory elements that allow artificialcombinations of ligand-binding domains

and allosterically regulated RNAconformational change to control gene expression have also been described 96,as have bioinformatics approaches to identify functional ribo-domains in naturaltranscripts 97. These approaches will allow development of controllable geneknockouts, which could provide information about the temporal nature of agenes activity more readily than traditional knockouts 98.

An exciting development is the use of synthetic riboswitches to detect bindingof small molecular-weight ligands in vivo . For instance, the introduction of theanti-theophylline aptamer into the 5 UTR of a -galactosidase reporter in Escherichiacoli induced increased expression of -galactosidase on addition of theophylline, butnot the closely related caffeine, to the culture medium 99. The authors had anticipatedthe opposite effect they had assumed that theophylline binding would ablate-galactosidase expression by obstructing ribosome binding. However, the effect washighly specific and relatively unaffected by changes to the position of the aptamersequence. Modification of this approach allows a range of applications for molecular

evolution studies in vivo .The molecular basis for these remarkable RNA-based regulatory systems is now

beginning to be understood at the atomic level, as a result of several recent X-raycrystal structures 26,100 . The figure, part a , shows a summary of the results from X-raycrystallography of the adenine-bound form of the Bacillus subtilis add A -riboswitch.The added base (shown in red) is recognized at every hydrogen-bonding position byinteractions with three bases from the riboswitch. This explains the discriminationagainst guanine binding. Hydrogen bonds are shown as dotted lines, and the bluecircle denotes a water molecule. The structure of a DielsAlder ribozyme with andwithout bound product has also been solved. The -shaped ribozyme creates a three-dimensional fold that closely matches the shape of the transition state of the reaction,explaining the catalysis 26.

The B. subtilis add A-riboswitch is converted by addition of the base adenine from atranslationally repressed state, in which the ShineDalgarno (SD) sequence (shaded red)and the initiation codon (shaded yellow) are sequestered by pairing interactions, to anactivated state, where these segments are free to interact with the ribosomal and tRNA(see figure, part b). Figure modified with permission from REF. 100 (2004) Elsevier.

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O

O

O

O

O

O

O

O

O

O

O

OH

OH

P

P

P

P

P

P

P

G8

G9

G11

U12

A13

C3

C2

C1

A+1

U+2

A7A4

U5

U6

A10

P

P

P

P

P

P

P

P

O

O

OH O

O

LysB61

ArgA49

SerB47

ArgB49

SerB52

AsnB55

LysB57

TyrA85

LysA43

AsnA87GluA63

O

O

O

O

O

O

O

O

O

O

O

OH

OH

P

P

P

P

P

G7

A8

C9

C11

C12

C3.5

U3

G2

G1

G+1

A4

U6

A10

P

P

P

P

P

P

P

P

OO

OH

O

LysB61

ArgB49

SerB52SerB51

AsnB55

LysB57

TyrA85

ThrB91

P

C5

ThrA59

ThrA45

SerA47ThrA59

ThrA45

SerA47

SerB47

ThrB45

hrB45

MS2stem-loop operator MS2F6

Tai ore -SELEX invo ves igation an c eavage of primer sites before an after amp ification42, a owingthe iso ation of shorter aptamer sequences that aremore rea i y synthesize chemica y. Photo-SELEXinvo ves UV-in uce cross inking of aptamers con-taining ight-sensitive nuc eoti es to their targets,great y increasing bin ing affinity 43.

Applications of aptamersThe app ications of aptamers are so numerous thstu ies escribing their use appear in the iteratuon a week y basis. The versati ity of this techno oref ecte in the fact that there are few areas of reseato which aptamers cannot be app ie . Some examptheir expan ing use are out ine be ow.

Figure 2 | Structural lessons rom RNA aptamerprotein complexes. The X-ray structures of the translationalrepression complex formed by the MS2 coat-protein dimer and the natural stem-loop operator of 19 nucleotides (top left),and the equivalent complex with the consensus aptamer sequence (F6) from a SELEX experiment (top right) 18,20 areillustrated. The coat-protein polypeptide backbones are shown as gold and blue ribbons with the RNAs as orange stickmodels covered by space-filling surface representations. Note the overall similarity of the complexes formed in each case,including the fact that the aptamer binds to the natural stem-loop interface on the protein. Below are representations ofthe intermolecular contacts made in each case. Hydrogen bonds between the MS2 protein and RNA are shown as dotted

lines, the two adenosines that are tightly bound by the protein (A10

and A4

) are indicted in bold. It was believed that a 4base loop was an essential feature of the operator consensus but F6 has a 3 base loop. The loops are highlighted in red ineach case. The number of nucleotide steps between the adenosines at positions 10 and 4 (numbers relative to thereplicase start codon) seem to be more important and these bases have been coloured yellow in each case. Watermolecules are shown as blue ovals.

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CapillaryelectrochromatographyA collection of separationtechniques, which involve theapplication of high voltagesacross buffer-filled capillariesto achieve separations basedon a range of different physicalproperties.

Purification and biotechnology. One of the most obvioususes for high y specific, high-affinity, reusab e mo ecu esis as an affinity purification me ium. An a vantage of aptamers over most other so i me ia erivatize withthe affinity target is that the pure protein can be obtainein fewer steps, owing to the abi ity of aptamers to is-criminate between c ose y re ate igan s. Furthermore,aptamers can be se ecte to bin to the natura form of the protein, e iminating mo ifications with tags (suchas g utathione S-transferase (GST) an His) which cana verse y affect protein fo ing, structure an function.Subsequent tag-c eavage steps that often re uce yie sare a so no onger necessary. An ear y examp e of thisapproach use an immobi ize anti-se ectin aptamer topurify a se ectin-receptor g obu in expresse in Chinesehamster ovary ce s44. A 15,000-fo purification of thefusion protein in a sing e step with 83% recovery wasreporte . More recent y, the iscriminatory abi ity of aptamers was emonstrate by the purification of d-arginine-vasopressin ( issociation constant (K ) of ~1M) from its l-enantiomer, for which no bin ing wasobserve 45. Aptamers can a so function as the station-ary phase incapillary electrochromatography 46. The powerof these affinity app ications is shown by a stu y thatgenerate an RNA affinity tag to a ow the purificationof sp iceosomes. Anti-tobramycin sequences were incor-porate into pre-mRNA, which a owe arge-sca e puri-fication of native sp iceosomes through an interactionwith tobramycin immobi ize on a resin47.

These same properties make aptamers i ea for usein assays eve ope previous y for antibo ies, such asWestern b ots or chromatin immunoprecipitation (ChIP)assays48. Aptamers have istinct a vantages over antibo -ies in such assays as they usua y have higher affinity anspecificity, giving much better signa -to-noise ratios,an they can theoretica y be raise against any protein,inc u ing those which are poor y immunogenic or toxic.They are usua y sma er than antibo ies a owing themto bin to apatopes that are sterica y inaccessib e toimmunog obu ins (see be ow for anti-HIV g ycoprotein120 (gp120) aptamers). They are a so chemica y mo ifieeasi y by routine processes.

Aptamers can be use to monitor the phosphor-y ation state of proteins, giving information about thetempora activity of proteins in signa ing casca es anbiochemica pathways49. Using aptamers to a ter theactivity of proteins in these pathways cou be usefu for

issecting protein functionsin vivo . However, it woube of greater use if this effect were reversib e so that theprotein cou be activate an eactivate at wi . Suchregu atab e aptamers can be iso ate by inc u ing a smamo ecu e effector that issociates the aptamer from itstarget protein at the e ution step of the SELEX protoco .The resu ting aptamers bin an inhibit their targets but

issociate from them when cha enge with the sameeffector, thereby restoring activity 50. Another way toregu ate aptamers is by the WatsonCrick base-pairingpotentia of comp imentary RNA mo ecu es (anti-senseRNAs). The function of an aptamer se ecte against theb oo coagu ation factor IXa(REF. 51) has been regu ateby a ition of the anti-sense RNA, potentia y provi ing

an a ternative metho for contro ing coagu atipatients who are into erant of heparin. It a so provia metho for generating regu atab e aptamer-batherapeutic an iagnostic reagents.

An a ternative metho of aptamer regu ation wrecent y reporte using cage , photo abi e th

ine resi ues in the we -characterize anti-thromaptamer52,53. These mo ifie resi ues were p acefunctiona y important positions in the aptamer, cop ete y inhibiting its bin ing. On UV irra iation the tecting groups were remove , restoring fu activity

emonstrating one route for precise spatia an temporegu ation of aptamer activity.

Diagnostics and biosensors. The high affinity anspecificity of aptamers make them i ea iagnosreagents. Most iagnostic app ications of aptamers ron igan -in uce conformationa changes. Thesebe etecte by ifferentia ye bin ing, f uorescquenching or f uorescence resonance energy transfSo ca e aptamer beacons have many uses, wrange from etecting environmenta contaminants monitoring carcinogen or rug eve s in the b oo54.

The escription of mo u ar aptameric sensors reresents another step in the use of aptamers as biosensors55. In these systems, a recognition aptamer for t

igan of interest is coup e to a signa ing aptby irect fusion of their nuc eic-aci sequences.theory, the tan em aptamers cou be incubate wa samp e of interest, a owing the recognition omto bin . The aptamers an any comp exes they haforme cou then be washe with a ye so utionbin s to the signa ing omain on y when the igachoice is boun , high ighting samp es containing scific igan s. This simp e system has a major a vanin that the recognition omain oes not require anmo ifications that might a verse y affect its structor function, a owing faci e coup ing of the m

igan -bin ing an ye-bin ing aptamers a recharacterize . In proof-of-princip e experiments, tanti-theophy ine aptamer was coup e to the ama achite-green aptamer. Bin ing of theophyresu te in conformationa changes in the two- omaptamer, which a owe ma achite-green bin ingextension of this technique to a mu tip exe arraobvious, a though this has yet to become commonp

espite the fact that the use of aptamers in arge scaiagnostic arrays was escribe as ong ago as 1

(REF. 56) . However, with the eve opment of automathigh-throughput aptamer iso ation, mo ification anscreening, aptamer-base microarrays are now bein

eve ope57, 58 (FIG. 3) .The recent eve opment of quantum- ot aptam

beacons cou a so he p to estab ish a ro e for apin biosensors59. Quantum ots are nove f uorophoreeach having a istinct sharp emission profi e, but thcan a be excite at the same wave ength. In quantumaptamer beacons, mu tip e copies of an aptamer are boto a sing e quantum ot. Each aptamer is base pairea comp imentary stran carrying a quencher. The comp ement is isp ace on igan bin ing, resu ting i

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cDNA microarray Aptamer microarray

UnmodiedcDNA oligos

Fluorescent cDNA

Oligooligohybridization

Aptamer capture of protein

Biotinylatedaptamers

Polylysine coating Streptavidin coating

PseudoknotA common three-dimensionalfeature of RNA, in which basesin a single-stranded loop basepair with complementary basesoutside that loop. Pseudoknotsare commonly usedrecognition and controlelements in vivo but oftenstabilize selected aptamers.

increases in f uorescence emission. If ifferent aptamersare immobi ize onto each quantum ot, mu tip e igan scan be etecte in a sing e assay. Such high y specifican sensitive aptamer beacons have great potentia asear y warning etection systems by bin ing ce -surfaceapatopes specific for amage or isease ce s.

An interesting eve opment in the iagnostic app ica-tion of aptamers was recent y reporte in a simp e assay base on igan interactions with aptamers boun to thesurface of go nanopartic es60. Ligan bin ing in uces aconformationa change in the aptamer ea ing to isas-semb y of the aggregate nanopartic es. This isassemb y affects the absorbance properties of the nanopartic es,resu ting in a visib e co our change. These stu ies coube exten e to eve op aptamers use for the simp e,rapi etection of i ega rugs or substances associatewith terrorism.

Therapeutics. Aptamers have enormous potentia asiagnostic reagents, which is on y a stones throw away from targeting aptamers for the treatment of isease.Therapeutic agents, such as erythromycin an Tamif u,are tra itiona y sma organic mo ecu es that fitsnug y into c efts on the surface of their target macro-mo ecu e, forming an intricate network of stabi izinginteractions61,62. Aptamers can a so fit into crevices onmacromo ecu es an can fo to form c efts into whichprotru ing parts of the target protein can bin . Thisincreases the potentia number of contacts ma e withthe target, a owing aptamers to form tighter, morespecific interactions than sma er mo ecu es.

Potentia therapeutic targets can be ivi e into tc asses, intrace u ar targets, such as transcriptiontors, an extrace u ar targets, such as inva ing viAptamers against extrace u ar targets can be a mtere intravenous y or subcutaneous y. Pharmacokinstu ies in humans confirm that RNAs e iverethese routes are rea i y istribute throughout tbo y an are easi y taken up by ce s63. The simp ic-ity of this approach is one of its main a vantages. Taptamers can be prepare in their stab e functiona san injecte irect y into the patient. However, R

egra ation an c earance is inevitab e, an repea ministration is require unti treatment is compThe e ivery of aptamers to efine anatomica tions is a rea y an approach in c inica use (see bean topica app ications, for instance to prevent patgens from interacting with their receptors on mucossurfaces, show potentia .

De ivery of aptamers to intrace u ar targets hasmost y by incorporation into iposome vesic es oexpression from vira -base vector systems. A techniusing a fusigenic vira iposome vector to e iver aptamers to their target ce s showe that DNA ethat sequestere the pro iferogenic transcription factE2F e to a re uction in the abnorma vascu ar tigrowth that is typica y seen after angiop asty 64. By con-trast, other groups suggest that whereas conjugation carrier mo ecu es to RNA might be require for upby ce s cu turein vitro , it is not necessary in vivo anthat c inica formu ations require on y simp e so utions.65

A though these approaches show promise for eery of aptamers to their intrace u ar targets, sogroups have begun to shift from stu ying the e ivof aptamers to stu ying their expression in ce s. Sintramers are intro uce by transfecting ce s wretrovira vector enco ing the aptamer. The transfectce s then continuous y pro uce the aptamer, in thprovi ing ife- ong treatment. An examp e of this tenique is the transient expression of a chimeric transcriconsisting of a human initiator tRNAMet sequence anthe anti-HIV reverse transcriptasepseudoknot aptamerun er the contro of an RNA po ymerase III promin human 293 T ce s. The chimeric RNA resu te>75% re uction in vira rep ication. Simi ar resu tseen with stab y transfecte Jurkat ce s66. Obvious y,intramers o not re y on mo ifie nuc eoti esstabi ity. However, it has been shown that f ankingaptamer sequence with stab e stem- oop structures increase resistance to 35 exonuc ease attack67, thereby achieving a simi ar resu t.

The i ea of using nuc eic aci s as therapeutic ageis not a new one. In 1990, RNA ecoys with the sasequence as the TAR RNA of HIV were shown to p vent HIV rep ication in ce s by sequestering aab e Tat protein68. However, aptamer techno ogy waon y use for the first time in c inica therapy 15

ater, in ear y 2005. The Foo an Drug A ministratapprova of Eyetech/Pfizers aptamer (Macugen) the treatment of age-re ate macu ar egeneratioa mi estone in the app ications of aptamer techno

Figure 3 | Schematic showing the use o aptamers in array ormats. The figurecompares the formats of complimentary DNA (cDNA) and aptamer microarrays.Immobilization in the cDNA microarray is by a charge interaction between the DNAand a polylysine-coated glass slide. In the aptamer microarray, the aptamers can

easily be biotinylated at one end allowing capture on a streptavidin-coated surface.In the cDNA microarray each spot is hybridized to differentially labelled fluorescentcDNAs prepared under the different test conditions and the result is analysed for therelative amounts of each dye, which indicates the relative amount of cDNA in eachsample. A similar approach can be used with anti-protein aptamers if the proteinsare differentially labelled as shown. Figure reproduced with permission from REF 57

(2005) Elsevier.

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Macugen is targete against the angiogenic cytokine vascu ar en othe ia growth factor, an bin ing pre- vents choroi a neovascu arization16,69. This new c assof therapeutics is expecte to be joine by inhibitory aptamers against severa other growth factors (inc u -ing p ate et- erive growth factor an basic fibrob astgrowth factor) for the treatment of cancers (for a review,seeREF. 70 ). There are a so severa groups eve opingaptamers against amy oi ogenic proteins such as theA-pepti e associate with A zheimer isease71, anagainst abnorma proteins foun in prion iseases72,73 such as scrapie an Creutzfe tJakob isease.

Combating infectious agents. Aptamers a so have thepotentia to be use as anti-infectious agents. RNAscan function as antibiotics if se ecte to inhibit acrucia bacteria protein or to isrupt ce membraneformation. Aptamers cou a so be use as a targetingsystem, specifica y bin ing an carrying an antibioticagent to the pathogen. An examp e of this approachuse an aptamer to e iver a ow-affinity inhibitor toits target74.Two active areas of interest are the use of aptamersas inhibitors of the chronic vira infections HIV (for areview, seeREF. 75 ) an hepatitis C. There are severakey stages in the HIV ife cyc e that can be targete by aptamers, a though so far inhibiting HIV rep icationhas been the main approach for treating patients. Mostrecent y avai ab e HIV treatments inc u e nuc eosi eana ogues, which ack the 3OH group an therefore

ea to premature termination of DNA e ongation by the HIV reverse transcriptase (RT). Unfortunate y,these ana ogues a so inhibit norma ce u ar DNArep ication, resu ting in severe si e effects. In a ition,resistance can arise through sing e point mutations inthe RT active site. Severa groups have iso ate anti-RTaptamers76,77an one such pseu oknot aptamer bin sRT with ow-nanomo ar affinity. It has been shownto cover ~2,600 2 of the RT surface, inc u ing theDNA-bin ing region. It is thought that resistance tosuch an inhibitor is un ike y to eve op because of theextensive protein a terations that wou be require toprevent aptamer bin ing.

Other anti-HIV rugs target the HIV protease.Protease- eficient mutants cannot c eave the GagPopo yprotein to re ease functiona vira proteins an aretherefore non-infectious. The abso ute requirementof HIV for functiona protease makes it an i ea rugtarget. Simi ar to RT inhibitors, however, a sing e pointmutation can make the protease resistant to current

rug therapies. Coup e with the fact that HIV proteaseinhibitors a so affect en ogenous proteases ea ing tosevere si e effects, it is c ear that the search for moreeffective treatments such as aptamer-base therapy mustcontinue.

HIV integrase cata yses the insertion of the nascentsDNA into the host genome. As the protease integrase

is crucia for vira rep ication but there is no humanequiva ent of this enzyme, si e effects from inhibitorsshou be minima . Severa groups are focusing on

eve oping anti-integrase aptamers an one set of RNA

aptamers with aK of ~2 nMin vitro has been reporte .However their efficacy in ce -base assays has yet estab ishe78.

The inhibition of vira invasion using aptameis another important area of HIV research. This haa vantages over the inhibition of vira rep icatiothe aptamers function extrace u ar y, obviatingnee to enter or be expresse in infecte ce s. invasion by HIV is a two-step process me iate by t vira g ycoproteinsgp120an gp41. Fo owing an inter-action between gp120 an CD4 receptors on the csurface, conformationa changes a ow gp41 to intewith the chemokine receptors CCR5 or CXCR4. Thin turn in uces a series of structura rearrangementsthe gp41 he ica regions (HR) 1 an 2, resu ting iformation of a six-he ix bun e. This rearrangempu s the two membranes into c ose proximity afusion an therefore infection. The first FDA-approvHIV-invasion inhibitor was a pepti e correspon into HR2 (amino aci s 127162) of gp41. This peptibin s to HR1 an inhibits the formation of the six-hebun e. Severa groups have targete aptamers agathe fusion process with the aim of inhibiting vira insion of the host ce . Anti-gp120 aptamers that brecombinant gp120 with aK of ~5100 nM have beenreporte an one of these, B4, inhibits HIV ce fuby up to 10,000-fo in ce cu ture79,80. Interesting y these aptamers seem to bin to a cryptic site that is onexpose when gp120 has boun to CD4 an is stericainaccessib e to antibo ies.

Severa groups have recent y reporte aptamers gete against proteins of hepatitis C virus (HCV), thcausative agent of chronic hepatitis, iver cirrhosis ahepatoce u ar carcinoma. The HCV non-structuratein NS3 is a crucia mu tifunctiona component of HIt has three known enzymatic activities: serine proteasnuc eosi e triphosphatase an he icase activity. Ntherefore an attractive target forin vitro se ection, anthe iso ation of omain-specific inhibitory aptamers recent y been reporte8185. Another important HCVprotein target is the RNA- epen ent RNA po ymerNS5b(REF. 86) . Aptamers that target NS5b have beenshown to inhibit RNA po ymerase activity in vitro .Aptamers have a so been se ecte that function as inhtors of the high y conserve interna ribosome entry(IRES)87, an others irecte against the apica oopIRES omain III have been shown to inhibit trans aof the vira genome bothin vitro an in vivo 88.

A though HIV an HCV are current y the main tarfor anti-vira aptamers, the iso ation of aptamers againrange of vira targets, such as human inf uenza viruscytomega ovirus has a so been escribe89. Presumab y,simi ar approaches wou a ow the eve opmaptamers that target avian inf uenza.

Future prospectsThe resu ts an eve opments escribe above showaptamer techno ogy is rapi y maturing from a simresearch too into a major techno ogy with commerpotentia . Given that aptamers mimic an exten maof the features of monoc ona antibo y reagents, we m

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expect a simi ar eve opment of commercia app icationsover the next few years. In a ition, the rapi expan-sion in our un erstan ing of the function an mo ecu armechanisms of natura riboswitches are ike y to spurnewer formu ations of the basic aptamer concept. TheE ington aboratory has recent y iso ate an aptamer

omain that wi e iver mo ecu ar cargoes into ce s(A. E ington, persona communication). The Harrisan Stonehouse aboratories have a so se ecte aptam-ers against HIVNef an the foot an mouth isease virus (FMDV) po ymerase, respective y (M. Harris anN. Stonehouse, persona communications). A though theanti-Nef aptamers are on y just being ana yse , it is c ear

that some of the anti-FMDV po ymerase aptamers funtion as non-competitive inhibitors, bin ing a jacent the enzyme active site. In co aboration with the von group in Frankfurt, we have se ecte aptamers agaithe HR2 region of HIV gp41 an have shown that tresu tant aptamer poo s are inhibitory across many vstrains (D.H.J.B. an P.G.S., unpub ishe observatioIt is therefore possib e that a range of anti-vira aptamcan be generate easi y an that these might show sergistic activity, opening up new prospects for anti-virprophy axis or therapy. It is c ear that no one workin the fie of mo ecu ar microbio ogy can ignopotentia of aptamer techno ogy any onger.

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AcknowledgementsWe would like to thank D. Burke, A. Ellington, M. Famulokand W. James for helpful comments during the preparationof this manuscript and for sharing unpublished or recent workfrom their laboratories. We thank W. Horn for providing fig-ure 2. Aptamer research in the P.G.S. laboratory is supportedby the UK Medical Research Council and the Biotechnologyand Biological Sciences Research Council, and by TheWellcome Trust and The Leverhulme Trust.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe ollowing terms in this article are linked online to:Entrez Genome Project: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprjFMDVUniProtKB: http://ca.expasy.org/sprotE2F | gp41 | gp120 | Nef

FURTHER INFORMATIONPeter Stockleys homepage: http://www.astbury.leeds.ac.ukThe Ellington Laboratory Aptamer Database: http://aptamer.icmb.utexas.eduAccess to this links box is available online.

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