Use of Aptamers as Diagnostics Tools and Antiviral Agents

34 Pages·2016·2.01 MB·English

pharmaceuticals
Review
Use of Aptamers as Diagnostics Tools and Antiviral
Agents for Human Viruses
VíctorM.González1,*,M.ElenaMartín1,GerónimoFernández2andAnaGarcía-Sacristán2
1 DepartamentodeBioquímica-Investigación,InstitutoRamónyCajaldeInvestigación
Sanitaria(IRYCIS)-HospitalRamónyCajal,28034Madrid,Spain;[email protected]
2 AptusBiotechSL,c/Faraday,7,ParqueCientíficodeMadrid,CampusdeCantoblanco,28049Madrid,Spain;
[email protected](G.F.);[email protected](A.G.-S.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditors:CristinaRomero-LópezandAlfredoBerzal-Herranz
Received:13October2016;Accepted:13December2016;Published:16December2016
Abstract: Appropriatediagnosisisthekeyfactorfortreatmentofviraldiseases. Timeisthemost
importantfactorinrapidlydevelopingandepidemiologicallydangerousdiseases,suchasinfluenza,
EbolaandSARS.ChronicviraldiseasessuchasHIV-1orHCVareasymptomaticoroligosymptomatic
andthetherapeuticsuccessmainlydependsonearlydetectionoftheinfectiveagent. Overthelast
years,aptamertechnologyhasbeenusedinawiderangeofdiagnosticandtherapeuticapplications
and,concretely,severalstrategiesarecurrentlybeingexploredusingaptamersagainstvirusproteins.
From a diagnostics point of view, aptamers are being designed as a bio-recognition element in
diagnostic systems to detect viral proteins either in the blood (serum or plasma) or into infected
cells. Another potential use of aptamers is for therapeutics of viral infections, interfering in the
interaction between the virus and the host using aptamers targeting host-cell matrix receptors,
orattackingthevirusintracellularly,targetingproteinsimplicatedintheviralreplicationcycle. Inthis
paper, we review how aptamers working against viral proteins are discovered, with a focus on
recent advances that improve the aptamers’ properties as a real tool for viral infection detection
andtreatment.
Keywords: aptamer;diagnosis;ebola;HBV;HCV;HIV;influenza;SELEX;therapeutic;virus
1. Introduction
Virusesareinfectiousagentsthatenterandreplicateonlyinsidethelivingcellsofotherorganisms.
Oncethevirusreplicatesinsidethecell,itmayremaindormantforlongperiodsoftimeorbereleased
immediatelyandattachtootherhealthycellstobegintheinfectionprocessagain. Manydiseasesare
causedbyvirusessuchastheinfluenza,hepatitis,humanimmunodeficiencyvirus(HIV)oremerging
viraldiseases. Whiletheydifferinsymptomssuchasfeverandweakness,somepresentnosymptoms
atall. Rapidandsecurediagnosisofviralinfectionsisakeyfactorfortreatmentofthesediseases
avoidingnewspread.
Virusescausedifferenttypesofdamagetothebodywhich,ifleftuntreated,canleadtodeath.
Therearemanyantiviraldrugsthatblocktheinfectionprocessatdifferentstages. Somedrugsprevent
thevirusfrominteractingwiththehealthycellbyblockingareceptorthathelpsinternalizethevirus
intothecell. Otherdrugsinhibittheproliferationoftheviruswithinthecell. Thesimultaneously
useofseveraldrugsaffectingdifferentprocessesincreasestheprobabilityofrecoveryofthepatient.
AlthoughsomeviralinfectionssuchashepatitisorHIVremainlatentforalongtimecurrenttreatments
controlthevirusandpreventfurtherdamagetothebody.
Viralinfectionsusuallyproduceanimmuneresponseinthehostthateliminatestheinfectingvirus.
Thesameprotectiveeffectisproducedbyvaccines,whichconferanartificiallyacquiredimmunityto
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theviralinfection. However,somevirusesincludingthosethatcauseacquiredimmunedeficiency
syndrome(AIDS)andviralhepatitisevadetheseimmuneresponsesandresultinchronicinfections.
Antibioticshavenoeffectonviruses,butseveralantiviraldrugshavebeendeveloped. Becauseviruses
usevitalmetabolicpathwayswithinhostcellstoreplicate,theyaredifficulttoeliminatewithoutusing
drugsthatcausetoxiceffectstohostcellsingeneral. Themosteffectivemedicalapproachestoviral
diseasesarevaccinationstoprovideimmunitytoinfection,andantiviraldrugsthatselectivelyinterfere
withviralreplication.
Most of the antiviral drugs are nucleoside analogues which lack the hydroxyl groups.
Viruses mistakenly incorporate these analogues into their genomes during replication and,
inconsequence,thenewlysynthesizedDNAisinactiveandthelife-cycleofthevirusisthenhalted.
Some of the most frequently prescribed antiviral nucleoside analogues based-drugs are aciclovir
for Herpes simplex virus infections and lamivudine for HIV and Hepatitis B virus infections [1].
During the last years, the nucleoside analogue drug ribavirin combined with interferon has been
usedforhepatitisCtreatment[2],althoughcurrentlythereisamoreeffectivetreatmentthatincludes
simepreviravailableforpatientswithgenotype1andgenotype4[3]. Thetreatmentofchroniccarriers
ofthehepatitisBvirusbymeansofasimilarstrategyusinglamivudinehasbeendeveloped[4]. Today,
thefirstlinetreatmentofchoiceincludesoneofthreedrugs: Peg-IFN,entecavirortenofovirbecause
oftheirgreaterpowerandbecausetheyproduceaverylowrateofresistance.
2. Aptamers: APotentialDiagnosticandTherapeuticAlternative
Novel anti-viral agents have emerged from the field of invitro selection of nucleic acid
aptamers[5,6]. Aptamersaresingle-strandedfoldednucleicacids(RNAorssDNA)abletospecifically
recognizeatargetmoleculewithhighaffinity. Thetermaptamer,derivedfromtheLatinword“aptus”
whichmeanstofit,wasfirstlyintroducedbytheNobellaureateSzostakandEllington[7]whenthey
describedtheinvitroselectionofRNAmoleculesthatbindspecificallytoavarietyoforganicdyes.
This invitro process, called SELEX (Systematic Evolution of Ligands by EXponential enrichment),
wasdevelopedbyGoldandTuerk[8].
The SELEX process begins with the synthesis of an oligonucleotide library consisting of a
central region with random sequence flanked by constant 5(cid:48) and 3(cid:48) ends that serve as primers
(Figure 1). Every member of the library is a linear oligonucleotide with a unique sequence that
acquirethree-dimensionalstructuredependingontheexperimentalconditions(pH,ionicstrength,
temperature,etc.) orthepresenceofaligand[9]. Thesehighlystructuredaptamersarecapableof
binding to the target with high affinity and specificity. The diversity of an oligonucleotide library
dependsonthenumberofrandomnucleotidesthatcontainseacholigonucleotidemolecule. Thus,
anoligonucleotidelibrarywhosemoleculescontainarandomsequenceof40nucleotides(440)would
berepresentedby1.2×1024 differentsequences. However,inpracticalterms,thecomplexityofa
combinatoriallibraryofoligonucleotidesislimitedto1012–1018differentindividualsequences[10].
Thefirstselectionstepimpliestheincubationofthetargetmoleculewiththeinitiallibraryin
theoptimizedconditions(temperature,pH,saltconcentration)forthefinaluseoftheaptamer[9].
During the selection step, a subpopulation of individual sequences specifically interacts with the
targetmoleculeandthisenrichedsubpopulationisisolatedbyphysicochemicaltechniques[11–13].
Subsequently,boundsequencesareelutedandamplifiedforanextroundofselectioninwhichthe
stringency of the selection conditions may be increased to identify the tightest-binding sequences.
Iterative rounds are performed until the population is enriched with sequences that display high
affinityandspecificityforthetarget(Figure1).
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Figure 1. Scheme showing characteristics of each aptamer molecule in the population and the basic
Figure1.Schemeshowingcharacteristicsofeachaptamermoleculeinthepopulationandthebasic
steps of a SELEX process. Each aptamer contains a central region with random sequence flanked by
stepsofaSELEXprocess.Eachaptamercontainsacentralregionwithrandomsequenceflankedby
constant 5′ and 3′ ends that serve as primers. The SELEX method consists in iterative cycles of in vitro
constant5(cid:48)and3(cid:48)endsthatserveasprimers.TheSELEXmethodconsistsiniterativecyclesofinvitro
selection with several critical steps: (1) incubation with the target; (2) removal of unbound sequences;
selectionwithseveralcriticalsteps:(1)incubationwiththetarget;(2)removalofunboundsequences;
(3) amplification of bound sequences; (4) reconstitution of single stranded DNA or RNA. Nucleotides
(3)amplificationofboundsequences;(4)reconstitutionofsinglestrandedDNAorRNA.Nucleotides
(N), Polymerase chain reaction (PCR); Reverse transcription polymerase chain reaction (RT-PCR).
(N),Polymerasechainreaction(PCR);Reversetranscriptionpolymerasechainreaction(RT-PCR).
During the last 25 years, several modified SELEX processes have arisen that allow adapting the
Duringthelast25years,severalmodifiedSELEXprocesseshavearisenthatallowadaptingthe
technology to different targets (whole cells, small molecules, organic dyes, peptides, etc.) or to
technologytodifferenttargets(wholecells,smallmolecules,organicdyes,peptides,etc.) ortodifferent
different final applications. As a final point, the selected aptamers are produced by chemical synthesis
finalapplications. Asafinalpoint, theselectedaptamersareproducedbychemicalsynthesisand
and purified to a very high degree and, therefore, eliminating the batch-to-batch variation found
purifiedtoaveryhighdegreeand,therefore,eliminatingthebatch-to-batchvariationfoundwhen
when using antibodies. The main advantages of aptamers in relation to antibodies are summarized
usingantibodies. ThemainadvantagesofaptamersinrelationtoantibodiesaresummarizedinTable1.
in Table 1.
Table1.Mainadvantagesofaptamersoverantibodies.
Table 1. Main advantages of aptamers over antibodies.
Characteristic Advantage
Characteristic Advantage
Selectionconditionscanbemanipulatedtoobtainaptamersstableinawide
Aptamersareidentifiedthrough Selection conditions can be manipulated to obtain aptamers
rangeofenvironmentalconditions
Aanptinamvietrrso aprreo cidesesn(tSifEiLedE Xth)rough an in stable in a wide range of environmental conditions
Aptamersmaybeobtainedagainstnon-immunogenicproteinsandtoxins
vitro process (SELEX) Aptamers may be obtained against non-immunogenic proteins
Littleornobatchtobatchvariation
and toxins
Aptamersareproducedby Aptamerscanbemodifiedincreasingtheirstability
Little or no batch to batch variation
chemicalsynthesis
Reportermoleculescanbeattachedtoaptamersatpreciselocationsnot
Aptamers are produced by chemical Aptamers can be modified increasing their stability
involvedinbinding
synthesis Reporter molecules can be attached to aptamers at precise
Theycanbeamplifiedtobeeasilydetected
locations not involved in binding
Denaturedaptamerscanberegeneratedwithinminutes
They can be amplified to be easily detected
AptamDeresnaaretusrteadbl eaptotalmonegrst ecramn sbteo rraeggeeannedractaend bweittrhainns mpoirntuedteast
Aptamersareoligonucleotides ambienttemperature
Aptamers are stable to long term storage and can be transported
Theyarenotimmunogenic
Aptamers are oligonucleotides at ambient temperature
TheirsmThaellys iazree anllootw imsfmorumnoogreenefific ciententryintothecelland
itscompartments
Their small size allows for more efficient entry into the cell and
its compartments
RNA and DNA aptamers have specific characteristics that would give them advantages for
different applications. For instance, the process of selection of DNA aptamers is shorter and the
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chemicalsynthesisofDNAoligomersischeaperandeasierthantheRNAoligomers. Inaddition,
DNAaptamersusuallyhavegreaterchemicalstabilityandlongershelflifethanRNAoligomersand
aremoreresistanttonucleasesactivityinsidelivingorganisms. Thesecharacteristicswouldseem
to confer to DNA aptamers important advantages to be used. However, RNA aptamers seem to
havegreaterflexibilitywhenitcomestofoldingandacquiringtertiarystructuresandtheycanbe
geneticallyencoded,andsotheycanbeexpresseddirectlybythetargetcellsororganisms. Although
itiscommonlyacceptedthatRNAaptamersarepreferablyusedfortherapy,whileDNAaptamersare
preferentiallyusedasdiagnostictools,therearesomeexceptionstothisnon-wroterule[14].
Individualaptamerscanbefurthermodifiedinordertoimprovetheirapproachability[12,15].
For instance, chemical modifications may be introduced rising the aptamer invivo stability.
Additionally,morevariationsmaybeneededforintendedapplicationslikebiotinand/ordigoxigenin
labelling, fluorescent reporters, or any other chemical group (thiols, amines, etc.) required for
biosensing or diagnostic purposes. Moreover, aptamers can be further truncated to eliminate the
oligonucleotide sequences which are not important for the interaction with the target or for the
correctthree-dimensionalaptamerstructure[16,17]. Identificationoftruncatedaptamerslimitedto
theminimumdomainofinteractionrequiresconsiderableeffortseventhoughaptamersoflessthan
40nucleotidesinlengthhavebeenalreadyreported[18–20].
3. AptamersforVirusDiagnosisandTreatment
3.1. AptamersagainstHumanImmunodeficiencyVirus(HIV)
Humanimmunodeficiencyvirusisalentivirus(asubgroupofretrovirus)thatcausesHIVinfection
andovertimeAIDS[21]. HIVinfectsessentialcellsinthehumanimmunesystemsuchasCD4+ T
cells, macrophages, and dendritic cells. When CD4+ T cell numbers decline below a critical level,
cell-mediatedimmunityislost,andthebodybecomesprogressivelymoresusceptibletoopportunistic
infections. HIV enters cells by endocytosis through the interaction of gp120 viral surface protein
(SU) with CD4 host cell. The subsequent interaction of this complex with chemokine coreceptors
produces a conformational change in viral protein gp41 that promotes the fusion of virion and
target cell membranes leading to the release of HIV particles into the cell. Once inside the cell,
viraluncoatinggeneratestheviralreversetranscriptioncomplex,andthereversetranscriptiongives
theHIVpreintegrationcomplex(PIC).ThePICgetsintothenucleusandtheHIVDNA(provirus)is
integratedintothecellularchromosome. Integrationcanleadtoeitherlatentortranscriptionallyactive
formsofinfection. Thelatentformgivesthevirallatencyincellsthatcanreplicateinnewinfected
cells;thisproviruscanremainhiddenduringyearsorreplicateandformnewviralparticlesinany
moment. Thetranscriptionalactiveformsaretranscriptandtranslatedformingnewviralparticlesthat
deadthehostcellandgoestoinfectnewcells. Greateffortsarebeingmadetogetsensitive,fastand
simplediagnosticmethodsandeffectivetherapies.
3.1.1. AptamersHIVinDiagnostics
At present, the initial clinical testing for HIV in human is made with an antigen/antibody
combination immunoassay that detects HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen
to screen for established infection with HIV-1 or HIV-2 and for acute HIV-1 infection [22].
Enzyme-linkedimmunosorbentassay(ELISA)andrealtimePCRarethemethodsusedandaccepted
inclinicalsamples.
Aptamers that recognize HIV proteins, like Tat and Rev, have been mainly used in several
biosensor systems based in Surface Plasmon Resonance (SPR) and quartz crystal microbalance
(QCM)techniques. Thus, Tombellietal. generatedbiosensorsbasedonSPRandQCMtechnique.
They introduced aptamers capable of detecting the HIV-1 Tat protein, using biotin-streptavidin
interaction. This approach allows for high specificity distinction between Tat and Rev (other HIV
protein,structurallysimilartoTat)[23]. OthersystemtargetTatproteinusingbiosensorbasedonthe
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diamondfield-effecttransistor(FET)technique[24]. These“apta-biosensors”havehighsensibilityand
specificitybutthedevicesarecomplexandexpensive.
diamond field-effect transistor (FET) technique [24]. These “apta-biosensors” have high sensibility
and specificity but the devices are complex and expensive.
3.1.2. AptamerstoHIVasAntiviralAgents
3.1.2. Aptamers to HIV as Antiviral Agents
Atpresent,thetreatmentHIV-1/AIDSisbyacombinationofseveralantiretroviraldrugs(cART),
whichcanAstl porwestehnet,p throe gtrreeastsmoefntth HeIdVi-s1e/aAsIeDaSn isd brye da ucocmetbhineartiisokn ooff sdeevaetrhala anndtirdeitsreoavsireacl odmrupglsi c(caAtiRoTn),,b utit
isnotwchuircaht civane. sMlowor tehoev perro,gmreasns yofp tahtei ednistesadseo annodt rteodleurcaet tehceA riRskT obf edceaauths eanitdh daissesaesve ecroemspidliecaetfiofenc, tbsu,ta ndit
istooite ixsp neont sciuvreatfiovre.p Matoierenotvseirn, mdeavneyl oppatiinegntcs oduon ntroite tso.leInratthe icsArRegTa bredc,aaupseta imt hearss sheavvereeb seideen ecfofencstisd, ered
and it is too expensive for patients in developing countries. In this regard, aptamers have been
an alternative or adjuvant to the chemical antiviral agents in cART to overcome these limitations.
considered an alternative or adjuvant to the chemical antiviral agents in cART to overcome these
Todate,highlyspecific,nucleicacid-basedaptamersthattargetvariouspartsofHIV-1genomes,HIV-1
limitations. To date, highly specific, nucleic acid-based aptamers that target various parts of HIV-1
proteins(includingHIV-1protease(PR),reversetranscriptase(RT),nucleocapsid,gp120,andGag)and
genomes, HIV-1 proteins (including HIV-1 protease (PR), reverse transcriptase (RT), nucleocapsid,
cellularproteins(nucleolin,CD4orCCR5)havebeenisolatedandshowntoeffectivelysuppressviral
gp120, and Gag) and cellular proteins (nucleolin, CD4 or CCR5) have been isolated and shown to
repliceaffteioctnivteolya spupplpyrienssH viIrValt rheeprlaicpaytio[n5, t2o5 a–p2p7]ly( Finig HuIrVe t2h)e.rapy [5,25–27] (Figure 2).

Figure 2. Scheme showing the targets for aptamers in HIV-1. (1) The HIV viral particle has an inner
Figure 2. Scheme showing the targets for aptamers in HIV-1. (1) The HIV viral particle has an
capside containing ssRNA viral genome and integrase and retro-transcriptase proteins, mainly; (2)
innerTchaep osuidteer ceonvnetaloipnein hgass sgRp1N2A0 avnidr aglpg4e0n pormoteeinasn idnvinoltveegdr ainse inatnerdacrteiotrno w-tritahn cseclrluiplatra sreecpeprototresi n(Cs,Dm4,a inly;
(2)ThCeCoRu5,t NerLeSn) avnedlo fpuseiohna tsog cpel1lu2l0ara mndemgbpr4an0ep; (r3o)t Ieni nthsei cnyvtooplvlaesdmi tnhein stseRrNaAct ivoinralw gietnhomceel lius lraerleraescedep tors
(CD4a,nCdC thRe5 ,reNtrLo-St)raannsdcrifputsioionn stteopc iesl pluroladrumceedm; (4b)r aTnhee ;in(3te)gIrnasteh eprcoytetoinp blainsdms ttoh edssDsRNNAA vivrairl aglegnoemnoem eis
releasbeyd LaTnRd enthdes sreeqturoe-ntcreasn asncdri optthioern csetleluplairs pprrootediunsc efodr;m(4in)gT thhee ipnrtee-ignrtaesgeraptiroont ecoinmbpilnexd (sPtIoC)d; s(5D) NThAe viral
genomPIeC bgyoeLsT Rinteon dnuscsleequus etnhrcoeusgahn dthoe thnuerclceealrl uploarrep arnodte iisn sinftoergmraitnegd tihne tphere c-ienlltuelgarr agtieonnomcoem bpyl ethxe( PIC);
integrase protein activity (provirus); (6) The viral RNAs are transcripted from proviral DNA and
(5)ThePICgoesintonucleusthroughthenuclearporeandisintegratedinthecellulargenomeby
exported to cytoplasm to translate viral proteins as protease and a big pre-protein that are assembled
theintegraseproteinactivity(provirus);(6)TheviralRNAsaretranscriptedfromproviralDNAand
to new RNA viral genomes and leave the cell with outer envelope from the cellular membrane; (7)
exportedtocytoplasmtotranslateviralproteinsasproteaseandabigpre-proteinthatareassembledto
After the budding viral particle, the proteases process the big pre-protein and get a mature viral
newRNAviralgenomesandleavethecellwithouterenvelopefromthecellularmembrane;(7)Afterthe
particle.
buddingviralparticle,theproteasesprocessthebigpre-proteinandgetamatureviralparticle.
Aptamers to HIV Genome
AptamerstoHIVGenome
Long terminal repeats (LTRs) are sequences necessary for proper expression of viral genes.
LInotenrgferteenrmce inwaitlhr ethpee afutsnc(tLioTnR osf) tahreeses eRqNuAen dcoemsaninecs eesisthaeryr bfyo rdipsrruoppteinrge xthperier ssstirounctuorfesv iorra lbyg enes.
Interfbelroecnkcinegw tihtehirt hinetfeuranccttiioonn wofitthh evsireaRl oNrA cedlloumlaar ifnascteoirtsh emrabyy sdeirsioruupsltyi ncgomthperiormstirsuec HtuIrVe-s1o vriabbyilbitlyo.c king
theirSinritsearwacatt iaonndw Einthgevlkirea sleolerccteedll uRlNarAf aacpttoarmsemrsa ythsaet rciaonu sbliyndc otom tphreo LmTiRses HofI HV-I1V-v1i aDbNilAity [.2S8r].i sTahweya tand
Engelke selected RNA aptamers that can bind to the LTRs of HIV-1 DNA [28]. They showed that

conserved segments present in several of the aptamers could form duplexes via Watson-Crick
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base-pairing with preferred sequences in one strand of the DNA, assuming the aptamer invaded
the duplex and, in consequence, these specific aptamers would inhibit the transcription process.
Meanwhile, Sanchez-Luqueetal. reportedtheinvitroselectionofspecificRNAaptamersagainst
the 5(cid:48)-untranslated region of HIV-1 genome. Those aptamers inhibited more than 75% of HIV-1
productioninahumancellline. Theanalysisoftheselectedsequencesandstructuresallowedfor
theidentificationofahighlyconserved16nt-longstem-loopmotifcontainingacommon8nt-long
apicalloop(RNApt16;5(cid:48)-CCCCGGCAAGGAGGGG-3(cid:48))thatproducedanHIV-1inhibitioncloseto
85%,thusconstitutingtheshortestRNAmoleculesofardescribedthatefficientlyinterfereswithHIV-1
replication [29]. The reason to use RNA aptamer is to go into the cell as DNA plasmid and to get
intracellularexpressionofRNAaptamertoblockthetarget.
AptamertoHIVProteins
AptamerstoProtease(PR)
Duringitslifecycle,HIVmustsuccessfullycompleteseveralkeystepsinordertoreplicateinthe
hostenvironment. OnesuchcriticalstepinvolvestheprocessingofitsGagandGag-Polpolyprotein
precursorsintotheirmaturefunctionalcomponentsduringviralmaturation. Thisfunctionisprovided
bythevirallyencodedaspartylprotease(PR)andisthoughttooccureitherduringorimmediatelyafter
budding[30,31]. Initsactiveform,thisproteinisa22kDahomodimerconsistingoftwo99-aminoacid
longsubunitseachofwhichcontributesacatalyticaspartateresiduetotheactivesite. Inhibitionthe
proteolyticactivityofPRleadstotheproductionofimmatureandnon-infectiousparticles.Duclairetal.
havedevelopedRNAaptamersagainstviralproteasethatinhibitedHIVreplicationinvitro[32].
AptamerstoIntegrase(IN)
Several small aptamers containing G-quadruplex selected against HIV proteins have
demonstrated antiviral activity [33]. The viral enzyme integrase (IN) is essential for retroviral
replication,catalyzingtheintegrationofthenewlysynthesizeddouble-strandedviralDNAgenome
intothehostgenomic[34]. Ojwangetal. obtainedamodifiedaptamer,namedT30177,abletoinhibit
integraseactivitywithIC50valuesinthenanomolarrange[35]. T30177wasthefirstINinhibitortested
inclinicaltrials(Zintevir™,developedbyAronexPharmaceuticals,TheWoodlands,TX,USA)[36].
DerivativesofT30177,morestablethantheparentalmoleculeandalsocapableofefficientlyinhibiting
HIV-1replicationincellculturehavebeenlaterdeveloped[37–39].
AptamerstoReverseTranscriptase(RT)
Reversetranscriptasehastwoenzymaticactivities,aDNApolymeraseactivitythatcancopyeither
aDNAoranRNAtemplate,andanRNaseHthatcleavesRNAonlyiftheRNAispartofanRNA/DNA
duplex. ThetwoenzymaticfunctionsofRT,polymeraseandRNaseH,cooperatetoconverttheRNA
intoadouble-strandedlinearDNA[40]. DeStefanoandNairconfirmedinvitroeffectivenessofDNA
aptamer,named37NT,directedagainstthereversetranscriptaseofHIVHXB2strain. Theaptamer
competed with the natural template for the binding site in the enzyme, subsequently producing
inhibition of the viral replication [41]. In parallel, Michalowski et al. identified three aptamers
(RT5,RT6andRT47)whichcontainedabimodularstructurecomprisinga5(cid:48)-stem-loopmodulelinked
toa3(cid:48)-G-quadruplex. Inaddition,theauthorsdemonstratedthatthisDNAaptamersinhibitedRT
fromdiverseprimatelentiviruseswithlownMIC values[42].
50
Another interesting approach is to inhibit the RNase H activity associated with RT. With this
purpose, Andreola et al. identified two DNA aptamers with G-rich sequences, named ODNs 93
and112,capableofformingG4structures. TheseaptamersinhibitedtheRNaseHactivityofHIV-1
RTinvitrowithIC valuesinthesub-micromolarrange,whilenoeffectwasobservedoncellular
50
RNase H [43]. Shorter DNA aptamers derived from ODNs 93 and 112, named 93del and 112del,
whichmaintainedthecapabilitytoformstableG4structures,wereabletoinhibitalsoHIV-1integrase
inthenanomolarrange[44,45].
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AptamerstoNucleocapsidProtein
Thenucleocapsid(NC)proteinofHIV-1playsanimportantroleintheencapsidationofviralRNA
andassemblyofviralparticleandtheprogressofsomeviralinfectionscanbepreventedbyinhibition
ofnucleocapsidsynthesis[46]. SincetheNCproteinisresistantformutation,itmightbeanexcellent
targetfortheanti-viraltherapy. Kimetal. isolatedRNAaptamersthatbindtothematureformofthe
NCproteinwithhighaffinityandcompeteforthepackagingelement(psi)RNAbindingtotheNC
protein. AuthorssuggestedthatstabilizedRNAaptamerisexpectedtoactasaninhibitorfortheviral
packaging[47].
AptamerstoSurfaceGlycoprotein(gp120)
Gp120 is essential for virus entry into cells as it plays a vital role in attachment to specific
cell surface receptors mainly on helper T-cells. Several small aptamers containing G-quadruplex
selectedagainstgp120havedemonstratedantiviralactivity[33]. Thefirstofthesemoleculeswasthe
phosphorothioate8-merd(TTGGGGTT),namedISIS5320,whichformsatetramericG-quadruplex
structure that binds the V3 loop of gp120 inhibiting virus entry [48]. Later on, Koizumi et al.
synthesizedasetofG-richoligonucleotidesandidentifiedthehexadeosyribonucleotided(TGGGAG),
known as Hotoda’s sequence, [49,50]. Several authors have used the Hotoda’s sequence as a lead
sequencetomakeaseriesofmodificationsatthe5(cid:48) and3(cid:48) endsofthemoleculeormutationsinthe
sequencethatallowedtofindmoleculeswithhighanti-HIVactivity[50–56].
Khatietal.in2003andDeyetal.twoyearslaterdescribedtheisolationof2(cid:48)-fluoropyrimidine-
substitutedRNAaptamersthatbindspecificallytothesurfaceglycoprotein(gp120)ofHIV-1that
potentlyneutralizedHIV-1infectivityinhumanperipheralbloodmononuclearcells[57,58]. Oneof
these aptamers (B40) and its truncated form (B40t77) were further structural characterized [59].
Shortly later, these authors showed that the aptamer binds to the CCR5-binding site on gp120
in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing
potency[58,60].
AptamerstoGagProtein
OthertypicaltherapeutictargetisGagpolyprotein,becauseofitslowvariability,ascomparedto
othersequencesofHIV-1genome[61]. Twoaptamershavebeendevelopedagainstthisprotein. Thus,
Lochrieetal. isolatedRNAligandsthatbindtotheHIV-1gagproteinintheregionscorrespondingto
thematrix(MA)proteinortothenucleocapsid(NC)protein. Interestingly,althoughthesequenceof
theaptamerwasdifferenttotheHIV-1RNApackagingelement(psi),itboundtoNCregioninterfered
withbindingofpsi[62]. Yearslater,Ramalingametal. selectedanewRNAaptameragainstHIV-1Gag
protein,namedDP6-12. Thisaptamerdisplayed20-foldinhibitionintheextracellularcapsidlevels
andreducedcellularlevelsofmRNAforGag,probablybyperturbationofspecificGag-genomicRNA
interactions[63]. TheseresultssuggestthatRNAaptamersmayprovideanovelmethodforinhibiting
HIVreplication.
AptamerstoCellProteins
Another strategy to block HIV infection is to target human proteins in host cells, like the cell
co-receptorthatinhibitsHIVentranceintothehostcell,whichmayhavelesssideeffects.
AptamerstoCCR5
HIV-1commonlyusesC–Cchemokinereceptortype5(CCR5)orC–X–Cchemokinereceptor
type4(CXCR-4)asco-receptorsalongwithCD4toentertargetcells. HumanCCR5isanimportant
co-receptorformacrophage-tropicvirusexpressedbyT-cellsandmacrophages. DifferencesinCCR5
areassociatedwithresistanceorsusceptibilitytoHIV-1. Asanessentialfactorforviralentry,CCR5has
representedanattractivecellulartargetforthetreatmentofHIV-1. Thus,Zhouetal. havereportedthe
selectionofRNAaptamersagainstCCR5usinghighthroughputsequencing(HTS)toanalyzetheRNA
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poolsfromselectionrounds5to9. Theindividualsequenceswereclassifiedintosixmajorgroups
(Group1–6). Group2,4and5sharedaconservedsequence,whichiscomprisedof10nucleotides
UUCGUCUG(U/G)G,namedG3. TheG3activitywasstudiedbya“prophylactic”HIV-1experiment
determiningwhethertheaptamerwouldblockHIVinfectivityofR5virusesincellculture. Theresults
showedthattheG3aptamerefficientlyneutralizedHIV-1infectivityofR5strainswithIC about
50
170~350nM[25].
AptamerstoNucleolin(NCL)
Nucleolin (NCL) is a multifunctional cellular protein that is overexpressed in cancer cell
membranes. NCL is involved in the very initial step of HIV-1 virion-cell recognition. NCL is in
cellularfractionscontainingtheHIVgenome,viralmatrixandreversetranscriptaseinadditionto
incomplexeswithCD4andCXCR4/CCR5atthecellmembrane. Thissupportsthepotentialrole
of this protein in viral entry. In the absence of the cellular receptors as CD4 and CXCR4/CCR5,
HIV-1attachmentcanoccurthroughcoordinatedinteractionswithheparansulfateproteoglycansand
cell-surface-expressedNCL.
AS1411isaG-richaptamerthatformastableG-quadruplexstructureanddisplaysantineoplastic
propertiesbothinvitroandinvivo[14]. ThemajormoleculartargetofAS1411isNCL.Perroneetal.
testedwhethertheaptamerAS1411wasabletointerferewithHIV-1cellularentryusingdifferentHIV-1
strains,hostcellsandatvarioustimespost-infection[26].TheresultsdemonstratedthatAS1411efficiently
inhibitedHIV-1attachment/entryintothehostcellintheabsenceofcytotoxicityatthetesteddoses.
UseofAptamersforDeliveryofTherapeuticMolecules
Aptamerscanalsoserveaselementsthatselectivelyrecognizeandbindtodefinedcelltypes
ortissues. Byattachingdrugmolecules,theaptamerscanbeusedtodelivercargomoleculestoor
intospecificcellsortissuesofinterest[64]. InordertoreachefficientRNAiactivity,aptamer-siRNA
conjugatesmustbesuccessfullyinternalizedandreleasedintothecytoplasmwheretheycanmeet
the RNAi machinery [65]. To improve the Dicer entry and processing of the siRNA, one pair of
complementary guanosine and cytosine (GC)-rich “sticky bridge” sequences can be chemically
appendedtothe3(cid:48) endoftheaptamerandoneofthesiRNAstrands,respectively. Boththeaptamer
andsiRNAportionsarechemicallysynthesizedandsubsequentlyannealedvia“stickybridge”[66].
Througheithercovalentconjugationorphysicalassembly,differentsiRNAmoleculeshavebeen
successfully functionalized with aptamers against HIV-1 gp120 and the CD4 receptor to achieve
targeted RNAi efficacy which relies on specific interactions between the aptamer and its receptor
expressedonthetargetedcellsortissue. Zhouetal. isolatedRNAaptamersagainsttheHIV-1(BaL)
gp120proteinthatwereusedtocreateaseriesofdualinhibitoryfunctionanti-gp120aptamer-siRNA
chimeras[67]. Theauthorsalsodemonstratedthatoneoftheseanti-gp120aptamer-siRNAchimerais
specificallytakenupbycellsexpressingHIV-1gp120,andthattheattachedsiRNAisprocessedby
Dicer,resultinginspecificinhibitionofHIV-1replicationandinfectivityinculturedCEMT-cellsand
primarybloodmononuclearcells(PBMCs). Interestingly,boththeaptamerandthesiRNAportions
intheaptamer-siRNAchimerahavepotentanti-HIVactivities[68]. Finally, theauthorstestedthe
antiviralactivitiesofthesechimericRNAsinahumanizedmousemodelwithmultilineagehuman
hematopoiesis showing that treatment with either the anti-gp120 aptamer or the aptamer-siRNA
chimerasuppressedHIV-1replicationbyseveralordersofmagnitudeandpreventedtheviral-induced
helper CD4+ T cell decline [69]. In order to improve the utility of aptamers as siRNA delivery
vehicles, thesameauthorschemicallysynthesizedamodifiedgp120aptamerthatwascomplexed
with three different siRNAs (HIV-1 tat/rev and two HIV-1 host cell proteins, CD4 and TNPO3),
resultinginaneffectivedeliveryofsiRNAsinvivo,knockdownoftargetmRNAsandpotentinhibition
ofHIV-1replicationinahumanizedmousemodel[70]. Veryinterestingly,followingendingofthe
aptamer-siRNAcocktailtreatment,HIVlevelsreboundedfacilitatingafollow-uptreatmentwiththe
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aptamercocktailofsiRNAsthatresultedincompletesuppressionofHIV-1viralloadsthatextended
severalweeksbeyondthefinalinjection.
Another interesting approach to targeted delivery was developed by Zhu et al. [27]. In this
work,theauthorsshowthataDNAaptamerobtainedfromtheconversionofapreviouslyreported
RNA aptamer could be used to deliver siRNA into CD4+ T cells specifically. This DNA aptamer
wascovalentlyconjugatedtothesensestrandofthesiRNAtargetingHIV-1protease(HIV-PR)and
the resulting DNA aptamer-siRNA chimera specifically entered into CD4+ T cells and efficiently
knockdowntheexpressionofexogenousHIV-PRgene. ThisstudydemonstratedthatDNAaptamers
withintrinsicstabilityhadagreaterpotentialtobeusedforsiRNAdelivery.
Table2showsinformationontheaptamersdescribedagainstHIV.
Table2.SummarydataontheaptamersselectedagainstHIVdiscussedherein.
Virus Name DNA/RNA Target References
n.d. DNA Tat,Revviralprotein [23,24]
ASn,ALn,BSn,BLn RNA LTRviralsequence [28]
RNApt16 RNA 5(cid:48)-untranslatedregionofHIV-1genome [29]
PR10.1,PR10.9,PR10.13,PR10.18 RNA Proteaseviralprotein [32]
T30177,T30695 DNA Integraseviralprotein [35,37–39]
37NT DNA ReverseTranscriptaseviralprotein [41]
RT5,RT6andRT47 DNA ReverseTranscriptaseviralprotein [42]
HIV ODNs93and11293del,112del DNA RNAseactivityassociatedtoRTviralprotein [43–45]
aptamers8–6,8–10,8–13 RNA Nucleocapsidviralprotein [47]
Hotoda’ssequenceandmodifications DNA gp120viralprotein [48–56]
B4,B40,B40t77 RNA gp120viralprotein [55–59]
ML6.8t33-82,ML8.20t14-75.DP6-12 RNA Gagviralprotein [62,63]
G3 RNA CCR5cellularreceptor [25]
AS1411 DNA NLCcellularprotein [14]
n.d. DNA CD4cellularreceptor [27]
n.d.=notdetermined.
Adirectapplicationoftheuseofaptamer-siRNAchimerahasbeenproposedbyWheeleretal.[71].
TheydemonstratedthatCD4aptamer-siRNAchimeras(CD4-AsiCs)specificallyblockgeneexpression
in CD4+ T cells and macrophages invitro, in polarized cervicovaginal tissue explants, and in the
femalegenitaltractofhumanizedmice. Thus,CD4-AsiCscouldbeusedastheactiveelementofa
microbicidetoavoidHIVsexualtransmission.
Insummary,alltheseinterestingresultspointouttheuseofaptamersfordevelopmentofnovel
anti-HIV-1therapies.
3.2. AptamersagainstHBV
HepatitisBvirus(HBV)isapartiallydouble-strandedDNAvirusoftheHepadnaviridaefamily
classifiedintoeightgenotypesfromAtoH.ThemainelementoftheviralparticleofHBVvirusand
alsothemostcharacterizedcomponentisthehepatitisBsurfaceantigen(HBsAg)[72].
3.2.1. AptamersHBVinDiagnostics
OneofthecurrentobjectivesinthediagnosticofHBVistodevelopadailyscreeningassaywitha
shortperiodofdetectionbetweeninfectionandrecognition. Therefore,Suhetal. havedevelopedafast
andlowcostdetectiontestbasedoncompetitivebindingassaycombinedwithfluorescenceresonance
energy transfer (FRET) [73]. The assay was built with an aptamer selected against the hepatitis B
virus surface antigen (HBsAg), the best characterize and most frequently used HBV marker [74].
Thedescribedaptasensorwasapproximately40-foldmoresensitivethantheconventionalmethod.
In 2015, a new set of three different DNA aptamers was selected against HBsAg and applied to
developachemiluminescenceplatform. Thenewaptasensorwasdesignedwithaptamers-conjugated
tomagneticnanoparticlesreachingadetectionlimitfive-foldbetterthanthecurrentenzyme-linked
immunosorbentassay(ELISA)kitsusedinhospitals[75].
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3.2.2. AptamerstoHBVasAntiviralAgents
During the last years, different strategies to inhibit HBV activity have been described using
aptamers selected against diverse viral target proteins. First, in 2010, Liu et al. proposed as a
therapeutictoolRNAaptamersselectedagainstHBsAgproteinthatareabletodiscriminatebetween
HbsAg-expressinglivercellsandHBsAg-negativecells[74]. Later,Fengetal. describedaninnovative
strategytofightagainstHBVinfectionsuppressingtheribonucleoproteincomplexinteractionbetween
thereversetranscriptase(namedPprotein)andtheRNAstem-loop(ε).Withthispurpose,theyisolated
individualRNAaptamerswithhighaffinityfortherecombinanttruncatedHBVPproteinthatcompete
withtheεRNAinteraction,withoutcytotoxiceffectsinculturecells,suggestingapotentialantiviral
activityofdescribedaptamers[76]. Ontheotherhand,Zhangetal. havechosenHBVcoreprotein
(HBVc) as a target to isolate DNA aptamers to inhibit nucleocapsid formation. They have shown
thatselectedaptamerApt.No.28preventsnucleocapsidassemblyandviralreplicationinthehepatic
cellularlineHepG2.2.15[77]. Finally,Orabietal. haveisolatedDNAaptamerstorecognizespecifically
thematrixbindingdomain(MBD)butnotthedomainmutatedintheresidue126(I126A).Oneof
theisolatedaptamers,AO-01,hasbeenproposedasatherapymoleculebyinhibitionofthevirion
productioninterferingtheinteractionbetweenthematrixdomainandtheMBD[78].
Table3showsinformationontheaptamersdescribedagainstHBV.
Table3.SummarydataontheaptamersselectedagainstHBVdiscussedherein.
Virus Name DNA/RNA Target References
anti-HBsAgRNAaptamer RNA HBsAg [73]
HBs-A22 RNA HBsAg [74]
HO1,HO2,HO3 DNA HBsAg [75]
HBV
ClassI,An&ClassII,Sn RNA MiniPprotein [76]
AptNo28 DNA Core [77]
AO-01 DNA Capsid [78]
3.3. AptamersagainstHCV
HCVviralgenomeisapositive-strandRNAthatcontainsapproximately9.6kilobasesandcodified
forapolyproteinofaround3000aminoacids. TheHCVpolyproteinmaturationprocessmediated
byproteasesgivesriseto10viralproteins,fourstructural(C,E1,E2andp7)andsixnon-structural
(NS2,NS3,NS4A,NS4B,NS5AandNS5B)[79]. Todate,onlythreeproteins(p7,NS4AandNS4B)
havenotbeenusedasantiviraltargetforisolatespecificaptamers.
3.3.1. AptamersHCVinDiagnostics
Aptamer-basedbiosensorsareapromisingdiagnosticplatformtoallowHCVinfectiondetection
in early stages or in immunosuppressed patients. Thus, different groups have developed diverse
aptasensorstoimprovediagnosticassayofHCVinfection. First, Leeetal. developedabiosensor
prototype that specifically recognizes the HCV core protein from sera of an infected patient using
selected RNA aptamers against core antigen. The HCV viral particles were retained by the 2(cid:48)-F
aptamers immobilized in a 96-well plate and detected by sequential steps with anti-core and
Cy3-labeled secondary antibodies [80]. Later on, Chen et al. developed an early diagnostic assay
based on sandwich-ELISA to recognize HCV viral proteins using biotin-labelled DNA aptamers
againstHCVEnvelopeglycoproteinE2. Theobtainedresultsfrominfectedpatientsshowedagood
correlationbetweenviralgenomequantificationassay,HCVantibodydetectionandsandwich-ELISA
aptasensor[81]. Afterwards,Shietal. developedasimilarplatformforearlydetection,coatingthe
bottomofthewellwithC7,aDNAaptameragainstHCVcorelabelledwithbiotin,andHCV-core
antibodyconjugatedwithhorseradishperoxidase(HRP)isappliedoverthesurface. Theplatformwas
appliedtothedetectionoftheproteininserafromHCV-infectedpatientsandshowedaproportional