Review Article Nucleic Acid Aptamers: Research Tools in...

Review ArticleNucleic Acid Aptamers Research Tools in Disease Diagnosticsand Therapeutics

Baby Santosh and Pramod K Yadava

Applied Molecular Biology Laboratory School of Life Sciences Jawaharlal Nehru University New Delhi 110067 India

Correspondence should be addressed to Pramod K Yadava pkyadava1953gmailcom

Received 29 January 2014 Accepted 18 March 2014 Published 22 June 2014

Academic Editor Gary S Stein

Copyright copy 2014 B Santosh and P K Yadava This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognateligands with high affinity and specificity in a manner akin to antibody-antigen interactions It has been globally acknowledgedthat aptamers promise a plethora of diagnostic and therapeutic applications Although use of nucleic acid aptamers as targetedtherapeutics or mediators of targeted drug delivery is a relatively new avenue of research one aptamer-based drug ldquoMacugenrdquo isFDA approved and a series of aptamer-based drugs are in clinical pipelines The present review discusses the aspects of designunique properties applications and development of different aptamers to aid in cancer diagnosis prevention andor treatmentunder defined conditions

1 Introduction

Aptamers are relatively small biomolecules (typicallyoligonucleotides ranging from 40 to 180 nucleotides orpeptides with 10 to 30 amino acid residues) whose three-dimensional structure confers on them the ability to bindtheir cognate ligands [1 2] The term ldquoaptamerrdquo is derivedfrom a latin word ldquoaptusrdquo meaning ldquoto fitrdquo and introducedby Ellington and Szostak [1] Nucleic acid aptamers can bechemically modified on the sugar backbone (ie 21015840-fluro21015840-O-methyl phosphorothioate) to improve aptamer stabilityand functionality Such nucleic acid modifications help inachieving optimal pharmacokinetic properties of selectedaptamers towards chosen ligands During the past threedecades aptamers have been generated against hundredsof molecular targets Nucleic acid aptamers have beengenerated against various targets including organic dyesmetal ions drugs amino acids cofactors aminoglycosidesand other antibiotics base analogs nucleotides peptidesand numerous proteins of therapeutic interest like growthfactors enzymes immunoglobulins gene regulatory factorsand surface receptors [1ndash3] Beside all these aptamersare also selected against intact viral particles pathogenicbacteria and whole cancer cell as targets [3]

Nucleic acid aptamers selected from a library of randomsequences by systematic evolution of ligands by exponentialenrichment (SELEX) bind to the chosen ligands with highspecificity and affinity [1 2] The SELEX process allowsevolution or selection of molecules with highest affinityby their exponential enrichment among a population ofrandom sequence nucleic acid library It may be noted thatSELEX is applicable in the case of nucleic acids due tothe convenient intermittent amplification of affinity-selectedmolecules During the SELEX process nucleic acid moleculecan be amplified by RT-PCR or PCR Some limitations of theuse of antibodies can be overcome by the aptamers for exam-ple aptamers are generated in vitro and can be selected totarget virtually any protein even toxins or nonimmunogenicproteins within a relatively short period of time whereasantibody generation is limited by the need to use live animals[3] In addition to this aptamers are produced chemicallyin a readily scalable process and the selection process isnot prone to viral or bacterial contamination [3] Due tothe smaller size of the aptamer it may efficiently enter intobiological compartment of the chosen target inside cells [4]All these properties render aptamers superior for diagnosticapplication offering greater sensitivity reproducibility andeconomy [4] SELEX starts with a chemically synthesized

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 540451 13 pageshttpdxdoiorg1011552014540451

2 BioMed Research International

random oligonucleotide combinatorial library of large se-quence complexity typically consisting of about 1013 to 1015different variants of nucleic acid sequences and involves theselection for oligonucleotides able to efficiently bind desiredtarget molecules [4] For the selection of RNA aptamersbinding chosen target the RNA library is obtained by invitro transcription of a random DNA oligonucleotide libraryusing T7 RNA polymerase before starting the first roundof RNA SELEX process Target binding function of nucleicacid aptamers is mainly dependent on their unique three-dimensional folding The secondary structures of aptamersmainly consist of short helical arms and single stranded loopsdefined by intramolecular base complementarity whereastertiary structures of aptamers result from a combination ofthese secondary structures with pseudoknotting of segmentalsequence complementarity of loops and bulges and allowaptamers to bind target by noncovalent interactions likeVan der-Waals interactions hydrogen bonding topologicalcompatibility stacking of aromatic rings and electrostaticinteractions [5]

2 Designing Aptamer Library and BasicPrinciple Underlying SELEX

SELEX is started with a population of different randomsequences flanked by defined sequences The defined se-quences are placed to ensure amplification of all different se-quences in the selected population by polymerase chain reac-tion (PCR) The primers designed should anneal specificallyto the template without forming ldquoprimer dimerrdquo or secondarystructures Typically up to 20-nucleotide long primers areused for PCR and can be synthesized with good yield Forselection of RNA aptamers T7 RNA polymerase promotersequence is required 51015840 to the PCR template sequence withinthe primer design (Figure 2) In principle aptamer librariesup to 1020 oligonuclotides are technically feasible but arerarely used in practice [6] The major considerations madewhile designing libraries are summarized below

21 Type of Randomization NA aptamer randomizationdepends upon sequence information of aptamer randomsequence regionThree types of randomization are employedin designing aptamer random sequence region that is par-tial segmental and complete Partially randomized (doped)library is used for selecting new functional molecules bytaking in consideration the motifs whose structure andfuctions are known to bind with the target molecule [7]Knowledge of structural and functional motifs suggests andsuffices for the use of partial random sequences in randomsequence region The extent of randomization and coverageof variant sequences flanking the motif are the basis ofdesigning and synthesis of the partial randomized pool Itcorresponds to mutation at constant rate for each nucleotidein sequence resulting in an array of point mutations similarto nature but at much higher frequencies [7] In segmentalrandomization a compromise between partial and completerandomization is made in longer oligonucleotide sequencesCompletely random library design allows launching aptamer

Number of nucleotides

Max

num

ber o

f va

riant

s rep

rese

nted

per

mg

and

max

num

ber o

f var

iant

s (lo

g sc

ale)

35

30

25

20

15

10

5

00 5 10 15 20 25 30 35 40 45 50

Maximum number of variants represented per mgMaximum number of variants

Figure 1Maximumnumber of variants (filled squares) and numberof variants per milligram (solid circles) of oligonucleotides as afunction of the number of nucleotides in the nucleic acid sequencein aptamer library

selection with minimal or no prior knowledge of structure ofeither the target or the aptamer [7]

22 Length of RandomSequence Region (AnAptamerUnit) Itis very important to choose the length of the random regionwhile selecting for an aptamer It is a wishful assumptionthat libraries with a longer random region may facilitateselection ofmore efficient aptamers because longer sequencesare able to form a wider range of different three-dimensionalstructures [7] The longer sequences offer a large numberof short structural motifs in addition to diverse and largemotifs [8] If the length of this region is 119873 nucleotideslong it will give a maximum of 4119873 possible sequencevariants which means a theoretical maximum of 4119873 probableconformations Short randomized nucleic acid segmentsof 20 to 50 nucleotides provide a vast sequence space fornovel nucleic acid structures and functions [8] If a targethas intrinsic nonspecific and weak affinity for nucleic acidsthen a partial or segmental random sequence pool of 30 to60 nucleotides based on natural ligands will yield aptamersSince the total mass of oligonucleotides used for startingSELEX is limited it puts a natural as well as a practical limiton the total number of variants that can be taken irrespectiveof the size of the oligonucleotides and theoretical maximumnumber of variants it may potentially form Therefore it isstatistically impossible and practically nonfeasible (presentedin Figure 1) to achieve the synthesis of a library with themaximum theoretical diversity For the practical reasons invitro selections of nucleic acids are typically limited to 430(approx 1020) different sequences

23 Chemistry of Aptamer Library Chemistry of the oligonu-cleotide pool also affects the selectionmethod to be employed

BioMed Research International 3

dsDNA library

RNA library

Constant defined sequence Oligo2

PCR

In vitrotranscription (IVT)

Matrix unbound RNA pool pass through ligand immobilized matrix

Negativeselection

Pass through immobilized matrix

Elution of binding aptamer

RT-PCR

IVT

of SELEX

RNA Pool Cloning followed by sequencingand characterization of aptamer

cDNA library

Step 1

Step 2

Step 3

Step 4

Oligo1+ T7 promoter

8ndash15 cycles

Random region (30ndash50 nucleotides)

Figure 2 Schematic representation of SELEX technology A suspen-sion of ligand coated matrix is often used instead of column

for the development of aptamers with affinity for chosenligands With increasing size of the DNA molecule a largermass would be needed to represent each possible variant inthe pool As the number of nucleotides increases that is thelength of random nucleotide sequence maximum numberof variants per unit mass (mg) shows saturation and in factdeclines for larger aptamers (Figure 1) So larger amounts ofDNA are required for including maximum possible numberof structural variants In vitro selection can also make useof modified nucleotides (NTPs) which further confers thedesired pharmacokinetic properties on the aptamers Themodified nucleotides can affect the chemistry which in turncan bias the course and outcome of SELEX [7] The T7 RNApolymerase can incorporate many modified ribonucleotidessuch as 21015840-flouro and 21015840-amino rNTPs or phosphorothioatenucleotides [6 7]

The basic approach behind the SELEX protocol is tominimize the total library pool size towards relatively smallnumber of binding partners This also includes enrichmentof high affinity and high specificity binding motifs by repet-itive cycles of affinity selection and enzymatic amplificationsimultaneously This process is mainly divided in four stepsnamely in vitro transcription affinity binding and elutionand reverse transcription (Figure 2) [1 2] as follows

(1) synthesis of library of random oligonucleotidesflanked by defined oligonucleotide sequences and

incubation with cognate ligands after negative selec-tion

(2) binding the cognate aptamers and washing off thenonbinding molecules from random oligonucleotidesequences

(3) amplification of selected aptamers by RT-PCR (RNAaptamers) or PCR (DNA aptamers)

(4) cloning and characterization of selected aptamersand further enrichment of high affinity aptamers byiterative rounds of selection

Negative selection for subtraction is a crucial step to mini-mize enrichment of nonspecifically bound oligonucleotideswith the matrix absent of ligand [3 4] For this reason therandom pool of RNA library is passed through an affinitymatrixbeads without immobilizing ligand Further beadsare washed with appropriate buffer and unbound RNA poolsare collected and the above process is repeated with ligandimmobilized affinity matrixbeads Next binding aptamersare eluted with competitor in elution buffer after washingand finally amplified by PCR or RT-PCR The sequences soselected are used as template for the next round of RT-PCRor PCR The same cycle is repeated 10 to 12 or more timesdepending on nature and concentration of ligand design ofthe starting random DNA oligonucleotide library selectioncondition ratio of ligandmolecules to oligonucleotide or theefficiency of the partitioning method to get an enriched poolof RNA molecules which specifically bind the desired ligandwith high affinity [9] RNA aptamers have been selectedagainst numerous targets listed in Table 1

3 Salient Features of Therapeutic Aptamers

Aptamers are increasingly being used as a replacement optionfor antibody mainly because of the special features describedin the following sections

31 High Specificity and Efficiency The method for nucleicacid aptamer selection is quite simple and yields highlyspecific aptamers within a short time [4 6] It has been shownthat the aptamer often possesses high affinity towards theselected molecule as compared to antibodies and often yieldsKd value in the nanomolar range [4 5]

32 Nonimmunogenic Nontoxic and Nonrecalcitrant NatureThere are no reports demonstrating toxicity or immuno-genicity of aptamers It is observed by clinical cellular orbiochemical measures that high doses of aptamers (10mgkgdaily for 90 days) are not toxic in rats or woodchucks [44]While the efficacy of many monoclonal antibodies can beseverely limited by immune responses against antibodiesthemselves it is quite difficult to raise antibody responseagainst aptamers (most likely because aptamers cannot bepresented by immune cells (T cells) via the major histo-compatibility complex and the immune response is generallytrained not to recognize nucleic acid fragments as nonselfentities [45]


Table 1 A list of aptamers generated against various ligands

Nature Aptamer name Ligandtarget Kd (nM) Disease Reference

RNA aptamer RIG1 (1-925aa) le372 Antiviral activity via modulatingIFN120572120573 production [10]

AFP aptamer AFP (fetal protein) 33 Hepatocellular carcinoma [11]

Class III GSH aptamer 817 Glutathione 418 Breast cancer (human breastadenocarcinoma cell line MCF-7) [12]

MDA aptamer M1 MDA(441015840-methylenedianiline) 450 Carcinogenic and DNA damaging

agent [13]

RNA aptamer Rev peptide 19ndash36 HIV [14]RNA aptamer S66A-C6RNA aptamer S69A-C15 V3 loop of gp120 HIV-1RT 406 637 HIV [15]

Anti-gp120 aptamerchimera Glycoprotein 120 (gp120) na HIV [16]

Class I and Class IIAChR aptamer

Acetylcholine receptor(AChR)

2 and 12respectively Neuromuscular disorder [17]

RNAapt TH14 Beta-SecretaseBACE1 (B1-CT) 280 Alzheimerrsquos disease [18]

Anti-EGFR aptamer (E07) Epidermal growth factorreceptor 24 Breast and lung cancer [19]

RNA DBL1120572-specificRNA aptamer

Erythrocyte membraneprotein 1 (PfEMP1)

33 inhibitoryconc Malaria [20]

21015840F RNA and 21015840NH2aptamer

Human keratinocytegrowth factor

00003ndash0003and 04 Cancer [21]

RNA aptamer(10th rounds 4∘C) L-selectin 17 Inflammation [22]

RNA aptamer NF-kB p50 homodimer 54 plusmn 22 Cancer and inflammation [23]

Anti-PSMA chimera Prostate specific membraneantigen na Prostate cancer [24]

Aptamer number 5 T-cell factor 1 100 Colon cancer [25]TTA1 Tenascin-C 5 Glioblastoma breast cancer [26 27]RNA aptamer TGF120573 type III receptor 1 Ovarian cancer [28]

Pegaptanib sodiumNX1838

VEGF 165 (vascularendothelial growth factor165)

005 Age related macular degeneration [29]

RNA aptamer 22 WT1 700 Wilmrsquos tumor [30]RNA aptamer 120573-Catenin 5 Colon cancer [31]RNA aptamer G4 K Ras-derived peptide 139 plusmn 12 Oncogenic protein [32]

RNA aptamer Amyloid beta-peptide A4(1-40) 29 to 48 Alzheimerrsquos disease [33]

Aptamer BC15(ssDNA aptamer)

Heterogeneous nuclearribonucleoprotein A1(hnRNPA1)

1110 plusmn 369 Breast cancers and liver cancer [34]

DNA aptamer HB5 Her 2 (human epidermalgrowth factor receptor 2) 189 Her 2 positive breast cancer [35]

DNA DNA aptamer 0mpC 20 Food borne disease [36]

DNA aptamer A and CTTF1 (member of the NKhomeodomaintranscription factors)

336 and 325 Primary lung adenocarcinomas [37]

SYL3 DNA aptamer EpCAM (epithelial celladhesion molecule)

38 plusmn 9

67 plusmn 8

Breast cancer (MDA-MB-231)gastric cancer (Kato III) and solidcancer

[38]

MUC1 DNA aptamer MUC1 peptide (Mucin-1) 0135 to 3338 Tumor marker in neoplastic cell [39]


Table 1 Continued


Peptide

Peptide aptamer RAD 51 (pY315) na Leukemia [40]Peptide aptamerA8 and A17 HSP70 na Cancer [41]

Peptide aptamer E6 HPV16 E6 oncoprotein na HPV positive tumor cells [42]

Peptide aptamer AII-7ErbB2 (avian erythroblasticleukemia viral oncogenehomolog 2)

na Breast cancer [43]

na not available

33 Route of Administration Nucleic acid aptamers can beadministered by either intravenous or subcutaneous injec-tion whereas most antibody therapeutics are administeredvia intravenous infusion Due to low solubility large vol-umes are necessary for therapeutic monoclonal antibodiesWith good solubility (gt150mgmL) and comparatively lowmolecular weight (aptamer 10ndash50 kDa antibody 150 kDa)aptamers are advantageous for tumor penetration and bloodclearance Aptamer bioavailability via subcutaneous adminis-tration is gt80 in monkeys [46] Therefore for the systemicdelivery of nucleic acid based therapeutics subcutaneousadministration has been suggested as an effective strategy[46]

34 Optimal Pharmacokinetics and Clearance from the LivingSystem For improving the stability of aptamers pyrimidineas well as purine nucleotides can be modified at their21015840 positions and tested for their ability to accommodatestabilizing modifications like 21015840-O-methyl groups [47]Thesemodified nucleotides can be introduced either chemicallyor enzymatically Protection against exonucleases can beprovided by modifications at the 51015840 caps (such as PEGadducts) and 31015840 ends (eg by addition of a 31015840-31015840-linkedthymidine cap) to increase aptamer retention time in theblood [47] Such modifications have a profound effect onaptamer retention in animals extending aptamer half-lifefrom minutes (no PEG) to several hours (40 kDPEG) [45]Conjugation with 40 kDPEG at its 51015840 terminus increases thein vivo half-life of the thrombin aptamer from 24min to 6 h inrats with little or no effect on thrombin binding affinity [48]However these stable forms also are eventually cleared fromthe system [48]

35 Possibility of Economic Scale-Up and Stability Therapeu-tic aptamers are chemically synthesized and consequentlycan be readily scaled as needed to meet production demandTherapeutic aptamers are chemically robust They are intrin-sically adapted to regain activity following exposure to heatdenaturants and so forth and can be stored for extendedperiods (more than 1 year) at room temperature as lyophilizedpowders [45]

4 Application of AptamersThe above-mentioned properties of aptamer specificitytoward target ligands make them an ideal tool for diag-nostics and therapeutics Aptamers have been extensively

Disease diagnosis

Gene expression modulators

Biosensorsbiochips

Riboswitches

Prophylaxis

Therapeutics

Research tool

Combating infectious agent

Molecular mimics

Nucleic acid aptamer

applications

Figure 3 A summary showing potential application of nucleic acidaptamers

used in analytical applications Nucleic acid aptamers havewide applicability as exemplified in the following sections(Figure 3)

41 Aptamers in Prophylaxis The applicability of aptamer isnot limited to targeted delivery to their cognate moleculesbut they can function for prophylactic help as well Thesemolecules could either be intracellular (eg beta-cateninthyroid transcription factor etc) extracellular (eg VEGFTenascin-C or small molecules such as ATP AMP andtheophylline) or cell-surface targets (eg PSMA Mucin-1 gp120 EGFR etc) Nucleic acid aptamers have beenprojected as convenient molecular mimics of antibody toprovide the basis for developing prophylactic idiotypes inmuch the sameway asmonoclonal antibodies have beenusedNowadays increased chances of catheter associated urinarytract infections (CAUTIs) are imposing a big challenge[49] Proteus mirabilis (causative agent of CAUTIs) blocksthe flexible tube connecting body cavity which disallowsfluids to pass into or out of it by forming a crystallinebiofilm [49] Blockage is often not detected until majorcomplications arise in the patients such as pyelonephritissepticaemia and shock Early diagnosis and prophylacticaid are needed to deal with many such diseases Thereforehigh affinity DNA aptamers (PmA102 PmA109) for theimprovement against Proteus mirabilis were selected usingcell-SELEX in combination with in silico maturation (ISM)These aptamers would be utilized for biosensor development


to get an insight into pathogen associatedmembrane proteins[50] In another example of prophylactic aptamer CD28aptamers (CD28Apt7-dimer CD28Apt2)were also developedthat boost antitumor immune response induced by idiotypicvaccination as well as by blocking B7-CD28 interaction bybinding to it [51] TCR-mediated signaling and engagementsof CD28 are basically required for efficient induction of theT-cell response This costimulatory CD28 molecule playsimportant roles in induction and maintenance of antiviral T-cell responses The efficacy of promoting strong cellular andhumoral response by CD28Apt7-dimer is key to promisingapproach in cancer immunotherapyThe same aptamerworksas an inhibitor for CD28 functions and after dimerization itfunctions as a potent adjuvant [51]

42 Aptamers as Riboswitches Riboswitches are naturallyoccurring RNA sequences with specific structural recogni-tion of cellular metabolites to modulate gene expressionThe untranslated regions of many mRNAs are known toact as sensors of metabolic pool and reorganize themselvesresulting in altered efficacy of translation [52] This helps thecells switch their expression platforms Such noncoding RNAsequences are naturally occurring aptamers and referred toas riboswitches [52] These are cis-acting genetic control ele-ments that regulate metabolic genes via structured sequencespresent in 51015840-untranslated region of mRNA Binding ofmetabolites to aptamer domain is independent of proteinfactors [52] Discovery of riboswitches illustrates biologicalrelevance of molecular recognition by natural RNA aptamers[53] It has been speculated that we should find new aptamersin hitherto unexplored regions of the genomes [54] Thesestructured RNA domains are widespread in bacteria and helpin modulation of many fundamental biochemical pathwaysby sensing the metabolites such as adenine guanine FMNglycine and lysine [52] Guanine sensing riboswitches arequite similar to adenine riboswitch binding of adeninepromotes mRNA transcription by preventing formation ofa terminator stem [53] Both belong to the same class ofriboswitch that is purine riboswitch [55] A riboswitch hasbeen reported in plants and fungi that bind to TPP (TPPriboswitch) [53] Generally riboswitches have two domainsan aptamer domain that binds the effector ligand and anexpression platform that transduces the binding event intoa change in gene expression [55] Riboswitches are alsoinvolved in a form of feedback inhibition via binding ofmetabolite further decreasing the production of relatedgene products and resulting in transcriptional attenuation bypreventing formation of full length mRNA or inhibition oftranslation initiation

43 Aptamers in Disease Diagnosis Clinical diagnosis ofdisease with the help of aptamers is attractive due to theirsmall size stable folding structure and economy There hasbeen rapid advancement in application of such aptamers indisease diagnosis imaging and new biomarker discoveryAptamers can detect very low amounts of diseased or tumorcells for which they are selected For example immobilizedanti-EGFR RNA aptamer on a chemically modified glasssurface can determine the presence andor extent of GBM

(glioblastoma) tumor cells [56] EGFR is the most commononcogene in glioblastoma [56] It is barely detectable with asmall number of diseased cells By using this novel approachit has become possible to make early diagnosis and monitorresidual diseased cells after surgical removal of tumor [56]Aptamers can also be used in lieu of antibodies in flowcytometry to detect a wide variety of cells such as humancancer cells For example newly developed CD30 aptamerprobe acts as an antibody free replacement option for thediagnosis of CD30 expressing lymphomas [57] Furtherdevelopment is needed to push this technology to makeit preferred option in the clinic The future of the useof aptamer-based probe in noninvasive imaging of manypotential clinical applications such as lesion detection andmonitoring of treatment is becoming increasingly promisingand needs further validation studies to benefit patients Theprinciple of aptamer-based probe designing is similar tothat of developing aptasensor Such aptamer-based probeusually consists of two domains one is sensing domainwhichrecognizes the target molecule and the other is signalingdomain which gives signal through a reporter molecule Thereporter molecule can be fluorescent or radionuclide Anaptamer-based diagnostic approach was developed to detectmalachite green (MG) a suspected carcinogen in fish foodThis MGRNA aptamer is used as alternative recognition toolover conventional antibody (Figure 4) [58] Leucomalachitegreen (LMG) is a primary metabolite of MG and persistslonger in fish tissues However this MG aptamer shows neg-ligible affinity towards LMG and is specific for MG Hencethe detection of malachite green by using this RNA aptameris preceded by an oxidation step that ensures conversion of allLMG to MG [58]

Biomarkers are important and crucial in diagnosis andtreatment of cancer [60] They can be expressed in differentforms such as proteins unique to cancer type and sub-type It could be soluble (secretory) or membrane attachedImportantly cell-SELEX for novel aptamer selection is beingexploited for biomarker discovery without prior knowledgeof the cell markers [60] Protein tyrosine kinases (PTK7) likemolecules were found to be highly expressed in a series ofleukaemia cell lines by using whole cell-SELEX in a two-step process that is aptamer selection followed by biomarkerdiscovery [61] Emerging application of aptamers includesdetection of protein biomarker at picomolar concentrationand in biomarker discovery For example SPRI (surfaceplasmon resonance imaging) methodology can sense humanthrombin at a concentration of 500 fM and VEGF protein atbiologically relevant concentration of 1 pM in biological sam-ples by forming a sandwich structure (RNA aptamer-proteinbiomarker-antibody-HRP) [62]This technique is utilized forfinding protein biomarkers at very low concentrations [62]Formation of surface aptamer-protein-antibody complex isdetected by adsorption of proteins on to immobilized RNAaptamermicroarrays After binding of protein SPRI responsesignal is further amplified by HRP conjugated antibodiesby forming localized precipitationcolour reactions [62] Byusing SPR imaging assay Li et al first immobilized an arrayof three thrombin binding RNA aptamers to enrich serumthrombin followed by signal amplification by horseradish


AC

B

5998400

3998400

(a)

AC

B G-24

C-28

G-29

A-30C-7

G-8

(b)

AC

B

G-24

C-28

C-7

G-8

(c)

Figure 4 Solution structure (NMR) ofmalachite greenRNAaptamer (a) showsmalachite green ligand bound to RNAaptamer backbone (b)shows residues involved in malachite stacking and (c) shows ball and stick model of RNA residues involved in malachite stacking interaction(yellow colour ring structure represents malachite green as ligand) adapted from Flinders et al [59]

peroxidise conjugated antibody against human thrombin[62] It is recommended and required for prediagnosis ofdisease and tracking followup procedure during therapeuticapplication In addition to this it is a powerful method tokeep rapid detection and profiling of protein biomarkers inblood or serum This method is dependent on the use oftwo different binding sites on target by both RNA aptamerand an HRP conjugated antibody In the future aptamerswould possibly eliminate the use of antibodies from thedetection approach by replacement with second-generationbiotinylated nucleic acids or peptide aptamers

44 Combating Infectious Disease Aptamer technology of-fers feasible alternative options for addressing the challengeof early diagnosis in human health [63] A single-strandedDNA aptamer was developed against botulinum toxin [64]This DNA aptamer was selected against aldehyde inactivatedtoxins and a short peptide fragment of 1177 to 1195 aa ofthe heavy chain in a single microbead based selection assayThe DNA aptamer exhibits affinity with toxoid in nanomolar

range (Kd 3 nM) Further progress in this field was markedby the selection of DNA aptamers against cholera toxin andStaphylococcal enterotoxin B (SEB) [65] The cytotoxin ricinis derived from castor bean plant that disrupts translationby depurinating a conserved site in eukaryotic rRNA [63]RNA aptamer was developed against the Ricin A chain withKd 73 nM [66] Selected RNA aptamers that recognize andpotentially inhibit ricin might be used as prophylactic ortherapeutic agent or function as a biosensor for the detectionof aerosolized ricin contaminated by the toxin [66] DNAaptamer (SSRA1) against ricin B chain can also serve as adiagnostic and preanalytical tool for direct ricin detection[67] Biotoxins from pathogenic bacterial strains in humansare solely responsible for severe health problems that demandinnovative technologies with greater sensitivity and specifici-ties [63] Although traditional antibody based methods arequite sensitive in some cases the demand for robust aptamertechnology is quite high RNase resistant RNA aptamer wasselected against OmpC protein of Salmonella typhimuriumwith high affinity (Kd 20 nM) and specificity [36] It did


not show binding with any other Gram-negative or Gram-positive bacteria Thus this RNA aptamer can diagnose foodborne sickness caused by Salmonella typhimuriumThesemayfind use in prophylaxis or targeted delivery vehicles

45 Aptamers as BiosensorsChips Aptamers are also used inbiosensors due to the behaviour of ligand induced conforma-tional changes Such aptamerswork asmolecular beacons andmay be used for the detection of environmental contaminantand monitoring carcinogen or to check the level of drugsin the blood [3] Over the past decade the dependence andapplicability of aptamers have steadily increased Aptamersare used as fascinating tools for biosensor and biosecurityapplications In the novel approach aptamer magnetic beadelectrochemiluminescence (AM-ECL) sandwich assay wasused for the selection and development of ssDNA aptamersagainst autoclaved anthrax spores [68] This cost effectivessDNA aptamer acts as in vitro-generated receptor for use as abiosensor for molecules used in biological warfare Anothersensory system (RNA aptamer) for opium alkaloid codeinea class of benzylisoquinoline alkaloids (BIAs) and metabolitein opium alkaloid biosynthesis pathway can precisely mea-sure the codeine concentration Two best codeine bindingRNA aptamers (FC5 and FC45) with Kd 25 and 40 120583Mrespectively are good enough to discriminate codeine fromits structural analog [69] Another example of aptamer-basedsensor is utilized in detection of cocaine at concentration of 1ndash100 120583M in human blood serum by a ldquosplit aptamerrdquo [70] Thesplit aptamer is utilized as recognition element for sensingsmall molecules that is cocaine metal ions and adenosine[71] Designing of such aptamer-based strategies involves theengineering of an aptamer into two fragments of ssDNA andinteraction with target causes ligand induced conformationalchange resulting in assembling of both nucleic acid frag-ments by forming junction trimer complex [70]

Aptamers can be used as chip based biosensor array byimmobilizing fluorescent labelled nucleic acid aptamer on aglass slide where they can still rotate at a rate that correspondsto their apparent volume and mass [72] The binding impliesa change in the mass and consequently in the rotationrate which conditions the fluorescence polarisation Thistechnique has been proven to be efficient even with complexbiological matrices such as human serum or cell extracts [72]This aptamer biosensor array provides an easy detection ofmultiplex analyte in complex biological mixtures

46 Aptamers as Modulators of Gene Expression ChimericRNAmolecules have been constructed by fusion of aptamerswith targeted ribozyme [73] Such chimeric constructs actas ligand responsive ldquoaptazymesrdquo Aptazymes can serve asextremely sensitive molecular sensors in which the aptamerdomain recognizes the ligand and the catalytic domaincleaves and splices the target These are engineered nucleicacid whose activity can be regulated by small molecules orcofactors A group 1 aptazyme (allosteric enzyme) engineeredby inserting thymidylate synthase intron in tandem withtheophylline aptamer has been projected as a genetic reg-ulatory switch in gene therapy [73] This aptazyme showsdependence over theophylline both in vitro and in vivo for its

activity of ligand dependent splicing Insertion of aptamersinto the 51015840-untranslated region of mRNA provides a handlefor translational control of expression of specific genes inliving cells Translation of such aptamer RNA constructs canbe regulated by reversible ligand dependent conformationalchange of aptamer domain [74]

Cancer cells show altered expression of signaling path-ways due to mutation in one or more than one geneThere are upregulation of oncogenes and downregulationof tumor suppressor genes Cancer cells exhibit tendencyfor uncontrolled cell division and successfully escape fromconventional apoptosis pathways The Wnt (beta-catenin)signaling plays an important role in controlling humantumorigenesis Beta-catenin serves as an effective anticancerdrug target In vitro selected TCF RNA aptamer binds withT-cell factor and inhibits the tumorigenic function of beta-catenin in colon cancer cells by modulating beta-catenintarget genes such as cyclinD1 andmatrixmetalloproteinase-7[75] CD28 is constitutively expressed onnaiveT lymphocytesand acts as a potent costimulatory signal for T-cell activationAfter signaling from CD28 in conjunction with other T-cellsurface molecules T cells produce and secrete interleukinsExpression of B7 the ligand for CD28 is upregulated onthe surface of antigen presenting cells (APCs) after gettingsignals from ligand of Toll-like receptor This costimulatorysignal is very important for the production of interleukinslack of this signal causes stage of anergy that is impairedresponse to its antigen CD28 aptamers modulate immuneresponse as agonist and also as antagonist This aptameracts as an antagonist by blocking the interaction of B7 withCD28 and functions as an agonist via elevating vaccineinduced immune responses in cancer immunotherapy [51]This aptamer-vaccine combination has shown preliminarysuccess in mice [51]

47 Aptamers as Therapeutics Due to their merits as cog-nate molecules aptamers are quite attractive for therapeuticapplications too As therapeutic agent RNA aptamers haveadvantages over other classes of RNA tool like short hairpinRNA (shRNA) small interfering RNA (siRNA) ribozymeor antisense oligonucleotides (AS OGNs) [76] in terms ofintracellular targeting capabilities direct binding to extra-cellular targets and sequesteringinhibiting their functionAptamers can be improved by labeling or conjugation withsiRNA or toxins in therapeutic applications [77 78] It hasalso been found to be a powerful tool for delivery of varietyof therapeutic agents such as small molecules peptides ortoxins for the treatment of human diseases [79] Currentlya number of such aptamers are available in clinical andpreclinical trials for treatment of cancer and other humandiseases (Tables 1 and 2)

The inhibition of viral invasion and replication usingnucleic acid aptamer is another important area of researchAn RNA aptamer ldquoMacugenrdquo is targeted against the angio-genic cytokine VEGF and its binding prevents choroidal neo-vascularization In 2004 US Food and Drug Administration(FDA) approved the first aptamer-based drug Macugen orPegaptanib sodium (aptamer against VEGF Eyetech Pharma-ceuticsPfizer) [80] This was a milestone in the application


Table 2 A list of aptamers in clinical pipeline

Name of aptamer Company Target Indication Current phase ReferencesMacugen PfizerEyetech VEGF AMD Approved [29 80]AS1411 Antisoma Nucleolin AML Phase II [81]

REG1 Regado Coagulationfactor IXa ACS Phase III [82]

ARC1779 Archemix vWF TTP Phase II [83]NU172 ARCA Thrombin CABG Phase II [84 85]E10030 Ophthotech PDGF AMD Phase II [86]ARC1905 Ophthotech C5 AMD Phase I [84 85]

NOX-E36 NOXXON MCP-1(CCL2) Type2DN Phase II [87]

NOX-A12 NOXXON SDF-1(CXCL12) Cancer Phase I [87]

NOX-H94 NOXXON Hepcidin Anemia Phase I [88]BAX499 orARC19499 BaxterArchemix TFPI Hemophilia Phase I [84 89]

AMD age-related macular degeneration AML acute myeloid leukemia ACS acute coronary syndrome vWF A1 domain of von willebrand factor TTPthrombotic thrombocytopenic purpurea CABG coronary artery bypass grafting DN diabetic nephropathy CCL2 chemokine (C-C motif) ligand 2 alsoknown as MCP1 CXCL12 chemokine (C-X-C motif) ligand 12 also known as SDF-1120572 and TFP1 tissue factor pathway inhibitor

of aptamer technology The VEGF protein has a centralrole in physiological and pathological angiogenesis throughbinding to its receptors (VEGFR1 and VEGFR2) that acti-vate downstream signaling The VEGF gene expresses fourmajor isoforms of 121 165 189 and 206 aa proteins throughalternative splicing Out of the four VEGF

165is the dom-

inant isoform primarily responsible for pathological neo-vascularization in age-related macular degeneration (AMD)and diabetic macular edema (DME) [80] Researchers haveselected RNA aptamers against this VEGF

165selectively

leaving other isoforms unaffected and modified them toincrease their stability in serum by fluorination methylationand addition of a 51015840-poly ethylene glycol moiety [29 80]ThisRNA aptamer showed no toxicity and has very long half-life in pharmacokinetic studies and was developed as a drugapproved for treatment of AMD and DME [80]

Similarly EGFR protein family consists of four closelyrelated receptor tyrosine kinases namely EGFR or ErbB-1 and its three homologs ErbB-2 or HER2neu ErbB-3 orHER3 and ErbB-4 or HER4 that can form homo- andheterodimers after receiving signals from EGF TGF-120573 andheregulin ligands to activate downstream signaling cascades[19] Mutation in expression or activity of EGFR familyprotein leads to a wide variety of cancers such as glioblastomaand breast ovarian stomach bladder salivary gland andlung cancer It has been shown that EGFR binding aptamers(E07) could be a promising candidate for cancer therapyby blocking receptor activation and inhibiting cancer cellprogression [19] Further DNA aptamer (HB5) against HER2was isolated and characterized for its target binding [35]HER2 and HER3 (homologs of EGFR family) both sharehomology between the extracellular domains (ECD) In spiteof this A30 (RNA aptamer) shows no binding to the ECDof HER2 even at concentrations far above those used ininhibition studies [90] A30 RNA aptamer is specific and

shows high affinity for HER3ECD and inhibits the heregulin-induced activation of HER3 receptor following inhibition ofgrowth of MCF7 cells [90] Further functional validation isstill needed to confirm the antitumorigenic properties of theabove-mentioned nucleic acid aptamers

Prostate specific membrane antigen (PSMA) is a uniquemarker for prostate cancer cells PSMA specific RNAaptamers (xPSM-A9 and xPSM-A10) were isolated and usedas intracellular delivery tools in conjugation with siRNAsor shRNAs for specifically targeting cancer cells instead ofnormal cells [91] In this aptamer siRNA chimera RNAaptamer (A9 A10) when conjugated with different siRNAssuch as lamin AC polio-like kinase 1 (PLK1) cancer survivalgene and BCL2 gene caused more pronounced regressionof PSMA specific tumors in vivo [24 92] Radiotherapies aswell as chemotherapies are powerful conventional tools forcancer therapy but the disadvantage of thesemethods is theirnonspecificity for target cells as it kills normal cells as wellHowever there are DNA repair genes and signaling cascadesthat play role in overcoming this disaster throughDNA repairmechanisms up to a certain level beyond this thresholdcells undergo apoptosis RNA aptamer shRNA chimera (A10-DNA-PK) has been used to target such vital genes as DNAactivated protein kinase to increase the radiosensitivity ofprostate cancer cells [93] This approach allows low level ofionizing radiation to inactivate cancer cells and decreasesthe necessity of exposure to surrounding tissues such as thebladder

The surface glycoprotein gp120 of the human immunod-eficiency virus interacts with host CD4 receptors leading toinitiation of membrane fusion and delivery of viral RNA andenzymes to host cells Aptamer siRNA chimeras have beendeveloped to silence virus replication and propagation in Tcells In this chimera siRNA is used against the HIV-1 tatrevregion [16] In combination with pRNA a dual functional


RNA nanoparticle was developed for targeted inhibition ofHIV [94] Modified pRNA-aptamer-siRNA chimeras canbe functionally processed by dicer to silence target geneexpression and provide a novel tool for cancer therapeutics

48 Aptamers as Research Tools Aptamers have wide appli-cability in pathway elucidation through their effect as specificinhibitors of intracellular signaling pathways One exampleof such an inhibitory aptamer is the mitogen activatedprotein kinase (MAPK) RNA aptamer [95] MAPK path-way is involved in various physiological responses such ascell proliferation apoptosis and differentiation Mutationsin MAPK lead to inappropriate activation of the MAPKsignaling cascade and ultimately promote diseased states suchas cancer in humans [95] Inhibitory RNA aptamers havebeen used as novel anticancerous therapeutic tools eitheralone or in conjunction with siRNA as mentioned in Table 1[16 24] Applicability of aptamers is extended to studies inmolecular biology and immunology as targeted modulatorof various functions [57]The advancing aptamer technologyholds promise to develop novel therapeutic molecules whichmay soon find their own way to routine laboratory andclinical procedures

49 Aptamers as Molecular Mimics In vitro selected nucleicacid aptamers serve as molecular mimics as well Thoseaptamers can be selected employing simple chemistry ofselection Pathogen induced immune defense involves initi-ation of recruitment of inflammatory cells (leukocytes andplatelets) to target sites (endothelial cells) [96] In suchprocesses cell-adhesion molecules are involved in threetypes of cell-cell interactions (circulatory inflammatory cellsto endothelium circulatory inflammatory cells to arrestedinflammatory cells and among circulatory inflammatorycells) [96] P- or L-selectin binding nucleic acid aptamerson mesenchymal stem cells (MSCs) are selected and bindto protein receptors on adjacent cells [97] Such aptamersresemble artificial ldquoadhesion ligandsrdquo [97] Further chemi-cally engineered MSCs modulate cellular functions via cell-cell interaction To expand the utility of aptamers compositeRNA aptamers (aptabody) were also designed and selected tofunction as antibodies in immunological assays [98] Anotherexample of aptamers as molecular mimics is a Spinachaptamer based on RNA mimicking green fluorescent protein[99] Certain RNA sequences complexed with flurophoremimic GFP in living cells [99] Generally proteins are fusedwith reporter molecule such as green fluorescent protein orits derivatives to measures the expression in vivo or inside theliving cells Similarly Spinach aptamer fluoresces inside livingcells in presence of provided fluorophore molecule that is3 5-difluoro-4-hydroxybenzylidene imidazolinone (DIHBI)[99] A report of mRFP1-Spinach construct showed in vivotranscript measurement and protein synthesis from the samemRNA simultaneously Spinach aptamer sequences can serveas good tools to characterize transcription and translationinside cells Such aptamer requires short RNA sequences withprovided fluorophore [99]

5 Conclusion and Future Prospects

The importance of the applicability of aptamers has signif-icantly increased with recent advancement in nanotechnol-ogy microfluidics microarray and other technologies forapplication in the clinical field Aptamers have been proven asmultifunctional molecules having tremendous paramedicaland medical applications A number of such aptamers arein various stages of clinical trials Recent advancement inaptamers selection and development can potentially dealwith targeted drug delivery targeted inhibition of proteinfunction and regulation of gene expression Aptamers canalso help in early cancer detection and alleviate detectioncomplications Aptamers compete with antibodies and offerpromising tools in different applications Targeted deliverytarget specificity and high affinity sum up into a higher appli-cation index in favor of aptamers although some limitationsalso exist with aptamers tools such as the stability of aptamerswith modified nucleotides intake and its retention timeinside the living system Despite the enormous possibilitiesnucleic acid aptamers suffer from certain limitations Theseinclude a typical molecular mass being in excess of 10 kD rel-ative unstability of unmodified RNA aptamers and possibleoff-target effects However further studies can confirm andvalidate outcomes of this selective approach by profiling thetargeting specificity of the chosen ligand Aptamers bindingwith transition state analogues of biochemical reaction maybe expected to catalyze the respective biochemical reactionsAn aptamer called ldquoSpinachrdquo mimics green fluorescent pro-tein and promises use as tool for characterization of geneexpression This could be used in other applications such asRNA imaging Variants of such aptamers offer great potentialapplications in synthetic biology

Abbreviations

SELEX Systematic evolution of ligand byexponential enrichment

IVT In vitro transcriptionSEB Staphylococcal enterotoxin BKd Dissociation constantrRNA Ribosomal ribonucleic acidV3 Third variable loop of HIV gp120RT Reverse transcriptaseHIV-1 Human immunodeficiency virus type 1PDGF Platelet derived growth factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Baby Santosh is an INSPIRE Fellow of the Department ofScience and Technology Financial support received fromUGC DST and DBT is gratefully acknowledged The cost ofpublication of this paper was defrayed out of grants receivedfrom DRDO and ICMR Government of India


References

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[2] C Tuerk and L Gold ldquoSystemic evolution of ligands byexponential enrichment RNA ligands to bacteriophage T4DNApolymeraserdquo Science vol 249 no 4968 pp 505ndash510 1990

[3] D H J Bunka and P G Stockley ldquoAptamers come of agemdashatlastrdquo Nature Reviews Microbiology vol 4 no 8 pp 588ndash5962006

[4] R Stoltenburg C Reinemann and B Strehlitz ldquoSELEXmdasha(r)evolutionary method to generate high-affinity nucleic acidligandsrdquo Biomolecular Engineering vol 24 no 4 pp 381ndash4032007

[5] T Hermann and D J Patel ldquoAdaptive recognition by nucleicacid aptamersrdquo Science vol 287 no 5454 pp 820ndash825 2000

[6] L Gold ldquoOligonucleotides as research diagnostic and thera-peutic agentsrdquoThe Journal of Biological Chemistry vol 270 no23 pp 13581ndash13584 1995

[7] J Pollard S D Bell and A D Ellington ldquoDesign synthesis andamplification of DNA pools for in vitro selectionrdquo in CurrentProtocols inNucleic Acid Chemistry JohnWileyamp Sons Chapter9 Unit 92 edition 2001

[8] A V Kulbachinskiy ldquoMethods for selection of aptamers toprotein targetsrdquoBiochemistry vol 72 no 13 pp 1505ndash1518 2007

[9] Y Yang D Yang H J Schluesener and Z Zhang ldquoAdvancesin SELEX and application of aptamers in the central nervoussystemrdquo Biomolecular Engineering vol 24 no 6 pp 583ndash5922007

[10] S-Y Hwang H-Y Sun K-H Lee et al ldquo51015840-Triphosphate-RNA-independent activation of RIG-I via RNA aptamer withenhanced antiviral activityrdquo Nucleic Acids Research vol 40 no6 pp 2724ndash2733 2012

[11] Y J Lee and S-W Lee ldquoRegression of hepatocarcinoma cellsusing RNA aptamer specific to alpha-fetoproteinrdquo Biochemicaland Biophysical Research Communications vol 417 no 1 pp521ndash527 2012

[12] J Bala A Bhaskar A Varshney A K Singh S Dey and PYadava ldquoIn vitro selectedRNAaptamer recognizing glutathioneinduces ROS-mediated apoptosis in the human breast cancercell line MCF 7rdquo RNA Biology vol 8 no 1 pp 101ndash111 2011

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[14] W Xu and A D Ellington ldquoAnti-peptide aptamers recognizeamino acid sequence and bind a protein epitoperdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 93 no 15 pp 7475ndash7480 1996

[15] T M A Gronewold A Baumgartner J Hierer et al ldquoKineticbinding analysis of aptamers targeting HIV-1 proteins by acombination of a Microbalance Array and Mass Spectrometry(MAMS)rdquo Journal of Proteome Research vol 8 no 7 pp 3568ndash3577 2009

[16] J Zhou H Li S Li J Zaia and J J Rossi ldquoNovel dual inhibitoryfunction aptamer-siRNA delivery system for HIV-1 therapyrdquoMolecular Therapy vol 16 no 8 pp 1481ndash1489 2008

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[19] N Li H HNguyenM Byrom andAD Ellington ldquoInhibitionof cell proliferation by an anti-EGFR aptamerrdquo PLoS ONE vol6 no 6 Article ID e20299 2011

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2

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[53] B J Tucker and R R Breaker ldquoRiboswitches as versatile genecontrol elementsrdquoCurrent Opinion in Structural Biology vol 15no 3 pp 342ndash348 2005

[54] K Matylla-Kulinska J L Boots B Zimmermann and RSchroeder ldquoFinding aptamers and small ribozymes in unex-pected placesrdquoWiley Interdisciplinary Reviews RNA vol 3 no1 pp 73ndash91 2012

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of ricin A-chainrdquo Journal of Biological Chemistry vol 275 no 7pp 4937ndash4942 2000

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[75] S Jeong H K Lee and M Y Kim ldquoUse of RNA aptamers forthe modulation of cancer cell signalingrdquo Methods in MolecularBiology vol 542 pp 363ndash377 2009

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[78] S D Jayasena ldquoAptamers an emerging class of molecules thatrival antibodies in diagnosticsrdquo Clinical Chemistry vol 45 no9 pp 1628ndash1650 1999

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[80] E W M Ng D T Shima P Calias E T Cunningham JrD R Guyer and A P Adamis ldquoPegaptanib a targeted anti-VEGF aptamer for ocular vascular diseaserdquo Nature ReviewsDrug Discovery vol 5 no 2 pp 123ndash132 2006

[81] FMongelard and P Bouvet ldquoAS-1411 a guanosine-rich oligonu-cleotide aptamer targeting nucleolin for the potential treatmentof cancer including acute myeloid leukemiardquo Current Opinionin Molecular Therapeutics vol 12 no 1 pp 107ndash114 2010

[82] G Sinha ldquoRegados aptamer lines up against anticoagulantsrdquoNature Biotechnology vol 31 no 12 p 1060 2013

[83] P Jilma-Stohlawetz J C Gilbert M E Gorczyca P Knobl andB Jilma ldquoA dose ranging phase III trial of the von Willebrandfactor inhibiting aptamer ARC1779 in patients with congen-ital thrombotic thrombo-cytopenic purpurardquo Thrombosis andHaemostasis vol 106 no 3 pp 539ndash547 2011

[84] A D Keefe S Pai and A Ellington ldquoAptamers as therapeuticsrdquoNature Reviews Drug Discovery vol 9 no 7 pp 537ndash550 2010

[85] Y Nakamura A Ishiguro and S Miyakawa ldquoRNA plasticityand selectivity applicable to therapeutics and novel biosensordevelopmentrdquo Genes to Cells vol 17 no 5 pp 344ndash364 2012

[86] H Akiyama S Kachi R L E Silva et al ldquoIntraocular injectionof an aptamer that binds PDGF-B a potential treatment forproliferative retinopathiesrdquo Journal of Cellular Physiology vol207 no 2 pp 407ndash412 2006

[87] M N Darisipudi O P Kulkarni S G Sayyed et al ldquoDualblockade of the homeostatic chemokine CXCL12 and the proin-flammatory chemokine CCL2 has additive protective effects ondiabetic kidney diseaserdquoTheAmerican Journal of Pathology vol179 no 1 pp 116ndash124 2011

[88] F Schwoebel L T van Eijk D Zboralski et al ldquoThe effectsof the anti-hepcidin Spiegelmer NOX-H94 on inflammation-induced anemia in cynomolgus monkeysrdquo Blood vol 121 no12 pp 2311ndash2315 2013

[89] L A Parunov O A Fadeeva A N Balandina et al ldquoImprove-ment of spatial fibrin formation by the anti-TFPI aptamerBAX499 changing clot size by targeting extrinsic pathwayinitiationrdquo Journal of Thrombosis and Haemostasis vol 9 no 9pp 1825ndash1834 2011

[90] C-H B Chen G A Chernis V Q Hoang and R LandgrafldquoInhibition of heregulin signaling by an aptamer that pref-erentially binds to the oligomeric form of human epidermalgrowth factor receptor-3rdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 16 pp9226ndash9231 2003

[91] S E Lupold B J Hicke Y Lin and D S Coffey ldquoIdentificationand characterization of nuclease-stabilized RNA moleculesthat bind human prostate cancer cells via the prostate-specificmembrane antigenrdquo Cancer Research vol 62 no 14 pp 4029ndash4033 2002

[92] T C Chu K Y Twu A D Ellington and M Levy ldquoAptamermediated siRNA deliveryrdquo Nucleic Acids Research vol 34 no10 article e73 2006

[93] X Ni Y Zhang J Ribas et al ldquoProstate-targeted radiosensitiza-tion via aptamer-shRNA chimeras in human tumor xenograftsrdquoThe Journal of Clinical Investigation vol 121 no 6 pp 2383ndash2390 2011

[94] J Zhou Y Shu P Guo D D Smith and J J Rossi ldquoDualfunctional RNA nanoparticles containing phi29 motor pRNAand anti-gp120 aptamer for cell-type specific delivery and HIV-1 inhibitionrdquoMethods vol 54 no 2 pp 284ndash294 2011

[95] S D Seiwert T S Nahreini S Aigner N G Ahn and O CUhlenbeck ldquoRNA aptamers as pathway-specific MAP kinaseinhibitorsrdquoChemistryampBiology vol 7 no 11 pp 833ndash843 2000

[96] A D Luster R Alon and U H von Andrian ldquoImmunecell migration in inflammation present and future therapeutictargetsrdquo Nature Immunology vol 6 no 12 pp 1182ndash1190 2005

[97] W Zhao W Loh I A Droujinine et al ldquoMimicking theinflammatory cell adhesion cascade by nucleic acid aptamerprogrammed cell-cell interactionsrdquo FASEB Journal vol 25 no9 pp 3045ndash3056 2011

[98] D Xu and H Shi ldquoComposite RNA aptamers as functionalmimics of proteinsrdquoNucleic Acids Research vol 37 no 9 articlee71 2009

[99] J S Paige K Y Wu and S R Jaffrey ldquoRNA mimics of greenfluorescent proteinrdquo Science vol 333 no 6042 pp 642ndash6462011

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Enzyme Research



Microbiology


random oligonucleotide combinatorial library of large se-quence complexity typically consisting of about 1013 to 1015different variants of nucleic acid sequences and involves theselection for oligonucleotides able to efficiently bind desiredtarget molecules [4] For the selection of RNA aptamersbinding chosen target the RNA library is obtained by invitro transcription of a random DNA oligonucleotide libraryusing T7 RNA polymerase before starting the first roundof RNA SELEX process Target binding function of nucleicacid aptamers is mainly dependent on their unique three-dimensional folding The secondary structures of aptamersmainly consist of short helical arms and single stranded loopsdefined by intramolecular base complementarity whereastertiary structures of aptamers result from a combination ofthese secondary structures with pseudoknotting of segmentalsequence complementarity of loops and bulges and allowaptamers to bind target by noncovalent interactions likeVan der-Waals interactions hydrogen bonding topologicalcompatibility stacking of aromatic rings and electrostaticinteractions [5]

2 Designing Aptamer Library and BasicPrinciple Underlying SELEX

SELEX is started with a population of different randomsequences flanked by defined sequences The defined se-quences are placed to ensure amplification of all different se-quences in the selected population by polymerase chain reac-tion (PCR) The primers designed should anneal specificallyto the template without forming ldquoprimer dimerrdquo or secondarystructures Typically up to 20-nucleotide long primers areused for PCR and can be synthesized with good yield Forselection of RNA aptamers T7 RNA polymerase promotersequence is required 51015840 to the PCR template sequence withinthe primer design (Figure 2) In principle aptamer librariesup to 1020 oligonuclotides are technically feasible but arerarely used in practice [6] The major considerations madewhile designing libraries are summarized below

21 Type of Randomization NA aptamer randomizationdepends upon sequence information of aptamer randomsequence regionThree types of randomization are employedin designing aptamer random sequence region that is par-tial segmental and complete Partially randomized (doped)library is used for selecting new functional molecules bytaking in consideration the motifs whose structure andfuctions are known to bind with the target molecule [7]Knowledge of structural and functional motifs suggests andsuffices for the use of partial random sequences in randomsequence region The extent of randomization and coverageof variant sequences flanking the motif are the basis ofdesigning and synthesis of the partial randomized pool Itcorresponds to mutation at constant rate for each nucleotidein sequence resulting in an array of point mutations similarto nature but at much higher frequencies [7] In segmentalrandomization a compromise between partial and completerandomization is made in longer oligonucleotide sequencesCompletely random library design allows launching aptamer

Number of nucleotides

Max

num

ber o

f va

riant

s rep

rese

nted

per

mg

and

max

num

ber o

f var

iant

s (lo

g sc

ale)

35

30

25

20

15

10

5

00 5 10 15 20 25 30 35 40 45 50

Maximum number of variants represented per mgMaximum number of variants

Figure 1Maximumnumber of variants (filled squares) and numberof variants per milligram (solid circles) of oligonucleotides as afunction of the number of nucleotides in the nucleic acid sequencein aptamer library

selection with minimal or no prior knowledge of structure ofeither the target or the aptamer [7]

22 Length of RandomSequence Region (AnAptamerUnit) Itis very important to choose the length of the random regionwhile selecting for an aptamer It is a wishful assumptionthat libraries with a longer random region may facilitateselection ofmore efficient aptamers because longer sequencesare able to form a wider range of different three-dimensionalstructures [7] The longer sequences offer a large numberof short structural motifs in addition to diverse and largemotifs [8] If the length of this region is 119873 nucleotideslong it will give a maximum of 4119873 possible sequencevariants which means a theoretical maximum of 4119873 probableconformations Short randomized nucleic acid segmentsof 20 to 50 nucleotides provide a vast sequence space fornovel nucleic acid structures and functions [8] If a targethas intrinsic nonspecific and weak affinity for nucleic acidsthen a partial or segmental random sequence pool of 30 to60 nucleotides based on natural ligands will yield aptamersSince the total mass of oligonucleotides used for startingSELEX is limited it puts a natural as well as a practical limiton the total number of variants that can be taken irrespectiveof the size of the oligonucleotides and theoretical maximumnumber of variants it may potentially form Therefore it isstatistically impossible and practically nonfeasible (presentedin Figure 1) to achieve the synthesis of a library with themaximum theoretical diversity For the practical reasons invitro selections of nucleic acids are typically limited to 430(approx 1020) different sequences

23 Chemistry of Aptamer Library Chemistry of the oligonu-cleotide pool also affects the selectionmethod to be employed


dsDNA library

RNA library


PCR



Negativeselection



RT-PCR

IVT

of SELEX


cDNA library

Step 1

Step 2

Step 3

Step 4

Oligo1+ T7 promoter

8ndash15 cycles






















agent [13]































38 plusmn 9

67 plusmn 8


[38]



Table 1 Continued


Peptide





na not available





Disease diagnosis


Biosensorsbiochips

Riboswitches

Prophylaxis

Therapeutics

Research tool


Molecular mimics


applications











AC

B

5998400

3998400

(a)

AC

B G-24

C-28

G-29

A-30C-7

G-8

(b)

AC

B

G-24

C-28

C-7

G-8

(c)
























165is the dom-


165selectively












Abbreviations





Acknowledgments



References

























2

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


dsDNA library

RNA library


PCR



Negativeselection



RT-PCR

IVT

of SELEX


cDNA library

Step 1

Step 2

Step 3

Step 4

Oligo1+ T7 promoter

8ndash15 cycles






















agent [13]































38 plusmn 9

67 plusmn 8


[38]



Table 1 Continued


Peptide





na not available





Disease diagnosis


Biosensorsbiochips

Riboswitches

Prophylaxis

Therapeutics

Research tool


Molecular mimics


applications











AC

B

5998400

3998400

(a)

AC

B G-24

C-28

G-29

A-30C-7

G-8

(b)

AC

B

G-24

C-28

C-7

G-8

(c)
























165is the dom-


165selectively












Abbreviations





Acknowledgments



References

























2

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








agent [13]































38 plusmn 9

67 plusmn 8


[38]



Table 1 Continued


Peptide





na not available





Disease diagnosis


Biosensorsbiochips

Riboswitches

Prophylaxis

Therapeutics

Research tool


Molecular mimics


applications











AC

B

5998400

3998400

(a)

AC

B G-24

C-28

G-29

A-30C-7

G-8

(b)

AC

B

G-24

C-28

C-7

G-8

(c)
























165is the dom-


165selectively












Abbreviations





Acknowledgments



References

























2

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued


Peptide





na not available





Disease diagnosis


Biosensorsbiochips

Riboswitches

Prophylaxis

Therapeutics

Research tool


Molecular mimics


applications











AC

B

5998400

3998400

(a)

AC

B G-24

C-28

G-29

A-30C-7

G-8

(b)

AC

B

G-24

C-28

C-7

G-8

(c)
























165is the dom-


165selectively












Abbreviations





Acknowledgments



References

























2

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








AC

B

5998400

3998400

(a)

AC

B G-24

C-28

G-29

A-30C-7

G-8

(b)

AC

B

G-24

C-28

C-7

G-8

(c)
























165is the dom-


165selectively












Abbreviations





Acknowledgments



References

























2

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















165is the dom-


165selectively












Abbreviations





Acknowledgments



References

























2

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Abbreviations





Acknowledgments



References

























2

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References

























2

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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