RNA BIOLOGY, 2018

VOL. 15, NO. 3, 327–337

https://doi.org/10.1080/15476286.2017.1414131

REVIEW

Aptamers in HIV research diagnosis and therapy

Jyoti Bala, Srinivasan Chinnapaiyan, Rajib Kumar Dutta and Hoshang Unwalla
Department of Immunology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, USA

ABSTRACT ARTICLE HISTORY

Aptamers are high affinity single-stranded nucleic acid or protein ligands which exhibit specificity and Received 8 August 2017
avidity comparable to, or exceeding that of antibodies and can be generated against most targets. The Revised 7 November 2017
functionality of aptamers is based on their unique tertiary structure, complexity and their ability to attain Accepted 3 December 2017
unique binding pockets by folding. Aptamers are selected in vitro by a process called Systematic Evolution KEYWORDS
of Ligands by Exponential enrichment (SELEX). The Kd values for the selected aptamer are often in the Aptamers; SELEX; Acquired
picomolar to low nanomolar range. Stable and nontoxic aptamers could be selected for a wide range of Immune Deficiency
ligands including small molecules to large proteins. Aptamers have shown tremendous potential and have Syndrome; HIV; retrovirus;
found multipurpose application in the field of therapeutic, diagnostic, biosensor and bio-imaging. While Anti-viral Therapy
their mechanism of action can be similar to that of monoclonal antibodies, aptamers provide additional
advantages in terms of production cost, simpler regulatory approval and lower immunogenicity as they
are synthesized chemically. Human immunodeficiency virus (HIV) is the primary cause of acquired
immune deficiency syndrome (AIDS), which causes significant morbidity and mortality with a significant
consequent decrease in the quality of patient’s lives. While cART has led to good viral control, people
living with HIV now suffer from non-HIV comorbidities due to viral protein expression that cannot be
controlled by cART. Hence pathophysiological mechanisms that govern these comorbidities with a focus
on therapies that neutralize these HIV effects gained increased attention. Recent advances in HIV/AIDS
research have identified several molecular targets and for the development of therapeutic and diagnostic
using aptamers against HIV/AIDS. This review presents recent advances in aptamers technology for
potential application in HIV diagnostics and therapeutics towards improving the quality of life of people
living with HIV.

Introduction
in terms of production cost, simpler regulatory approval and
Oligonucleotide aptamers are single stranded, short RNA or lower immunogenicity when administered in preclinical doses
DNA molecules of 20–80 nucleotide length, capable of attain- 1000-fold greater than those used for animal and human thera-
ing tertiary complex structures to bind specifically to targets peutic application [9,13]. Aptamers are highly specific and can
against which they are selected. These aptamers are selected by discriminate between related proteins that share common sets
an iterative process called SELEX [1,2]. With increased rounds of structural domains [16,17]. Aptamers are already in clinical
of selection, possible complex tertiary structures present in the use with MacugenTM approved by the FDA in 2004 for macular
random library are enriched to obtain increased binding effi- degeneration [18-20]. Clinicaltrials.gov currently lists 19 trials
cacy against varied target molecules ranging from small mole- with aptamers (completed, in progress or expected to begin).
cules, peptides or protein targets. Aptamers can be considered Combination antiretroviral therapy (cART) has made HIV a
functional analogs of antibodies. Binding of aptamers to its spe- treatable but a chronic disease. However, HIV patients con-
cific ligand is governed by specific structures, shape comple- tinue to die of non-AIDS comorbidities almost a decade earlier
mentarity and conformation, involving binding pockets and that their non-HIV counterparts [21]. Despite this progress,
bond formation such as hydrogen bond, electrostatic interac- some comorbidities continue to remain highly prevalent among
tion and van der waals forces. Aptamers can be used as antago- HIV-infected individuals. Most of these comorbidities are asso-
nists or agonists to modulate cell signaling [3,4] or as targeting ciated with underlying chronic inflammation or the actions of
ligands for delivery of drugs and other cargo in a tissue specific HIV proteins expressed by infected cells [21,22]. For instance,
or cell type specific manner [5,6]. Recent advances in aptamer Tat is an immediate early gene of HIV and its expression can-
technology and automation of SELEX has increased the poten- not be suppressed by antiretrovirals [23,24]. Its protein trans-
tial for aptamer use in therapeutics and diagnostics [7–10]. duction domain allows its secretion by infected cells and
Aptamers can be used in an extensive array of applications in uptake by bystander cells where it mediates pleotropic effects
diverse fields of diagnostics, delivery, bio-imaging, bio sensing [25–28]. While aptamers hold great promise for HIV research,
and therapeutics. [11–15] Unlike antibodies, aptamers, can be diagnostics and therapy, their application remains limited. Sev-
synthesized chemically and hence offer significant advantages eral aptamers for HIV-1 proteins as well as host proteins that

CONTACT Hoshang Unwalla hunwalla@fiu.edu Department of Immunology, Herbert Wertheim College of Medicine, Florida International University, 11200 SW
8th Street, AHC-1 #421, Miami, FL 33199, USA.
© 2018 Informa UK Limited, trading as Taylor & Francis Group
328 J. BALA ET AL.

interact with HIV-1 have been generated and characterized biological application. The final selected and characterized
with functional role like neutralization of virions or its proteins aptamers could be used for several applications [13,37–42].
[29–32]. This review focuses on the prospects of aptamers as From the discovery of the first SELEX protocol till now, differ-
therapeutic leads and diagnostic tools for HIV/AIDS. ent types of SELEX have been established and merged with
other advance technology based on their application in diverge
fields [10,15,43,44].
Systematic evolution of ligands by exponential
enrichment (SELEX)
Functional aptamers and its applications
Aptamers are isolated and characterized for their binding affin-
ity and specificity against the chosen ligands through an itera- Aptamers have found diverse application in the field of bio-
tive in vitro selection process called SELEX [(Fig. 1), 1,2]. A medical research. Aptamers have been used to target small mol-
randomized oligonucleotide library folds into complex struc- ecules, large complex molecules, agonists and antagonists and
tural pool. Considering the susceptibility of oligonucleotides to function as inhibitors or activators of cell signaling in specific
serum nucleases, modified oligonucleotide are widely used for diseases [41,45–48]. Naturally occurring nucleic acid aptamers
generating nuclease resistant aptamers [33–35]. A starting are present in the genome and also as a constituent of ribos-
aptamer library mostly consists of a central random region witches which can be used to elucidate the role of nucleic acids
ranging from 20–60 nucleotide long, flanked by known sequen- in signal transduction [49–52]. Aptamers are functionally com-
ces to allow primer binding for amplification, enrichment, parable to antibodies and possess a wide repertoire of charge
cloning and characterization. Briefly, a usual SELEX protocol and structure combinations for extensive use in various bio-
requires preclearing to remove library members that bind to medical, diagnostic, in vitro or in vivo bioimaging, and thera-
the support, matrix or a ligand attached to your target of inter- peutic applications. These are also broadly used as research
est to facilitate separation. This is followed by incubation of the tools in diverse areas like bioassays, imaging, pathogen detec-
precleared library with the chosen ligand for their binding, tion, tissue staining, targeted delivery vehicles, aptazyme, bio-
nonspecific binders are removed by employing washes with sensor and nanomedicine [43,44,53–55]. Aptamers also have
increasing stringency and specific binding variants are ampli- ability to recognize toxin molecules and hazardous chemicals
fied. In order to effectively obtain bound species with higher which makes them suitable candidates for use in food safety
affinity, the selection stringency is cautiously controlled by and environment monitoring [56–58]. Aptamers can be mini-
adjusting conditions and adding negative or counter completive mally truncated so as to retain only the biologically active por-
selections. The minimization of selection round is possible tion from the initial SELEX without losing the functionality. In
using advanced or automated SELEX protocols such as RAPID vitro modification of aptamers without loss of functionality (for
SELEX [7,8,36]. Specificity check and counter negative selection eg; enhancing stability)[34,35,59] combined with cost effective
are usually perform to get rid of weak binders and nonspecific bulk synthesis makes them ideal candidates for therapeutic
variants. Aptamers selected from SELEX further need structural leads or for targeted drug delivery. For instance a number of
and binding characterization and based on such analysis best reports have demonstrated the use of aptamers for cell type
aptamer candidates are selected for biochemical, functional and specific delivery of siRNAs [60–62]. Aptamers have also found

Figure 1. (1A) Representation of aptamer selection. Briefly random oligonucleotide libraries with diverse structural complexities are generated and incubated for ligand
binding. Non-specific binders are removed and high affinity binders are amplified. The selection cycle are usually repeated around 5-20 cycles. The sequences and struc-
tural characterization from chosen binders are done to achieve the best binding aptamer. (1B) Basic strategy of SELEX protocol showing incubation of random library
with chosen ligands, their incubation for binding, removal of non-specific binder and finally the enrichment of the best binders for chosen targets.
 RNA BIOLOGY 329

application in biomarker discovery during HIV infection and and tested in preclinical and clinical studies as a potential new
host interaction [63–65]. Few aptamers have also been reported entry inhibitor drug against HIV/AIDS.
with ability to differentiate between cancerous and non-cancer-
ous receptor that could be used as specific drug delivery agent
Potential HIV aptamer for diagnostic application
and for biomarkers identification [66–69].
While most of the work with anti-HIV aptamers has been done
with the intention of generating therapeutic aptamers, some
Stages of the HIV life cycle studies have also identified aptamers that can be used for diag-
nostics. In the subsequent sections we will discuss the aptamers
HIV infection promotes immunodeficiency by selectively tar-
that have found application in HIV either as diagnostics or
geting CD4 T-cells. CD4 cells are involved in crucial immune
therapeutics. Initial diagnosis of HIV in human are mostly
functioning. The HIV infection cycle can broadly be divided
done by ELISA and real time based an antigen/antibody combi-
into seven steps or stages (binding, fusion, reverse transcrip-
nation immunoassay that generally identifies HIV-1 and HIV-2
tion, integration, replication, assembly and budding) [(Fig. 2),
antibodies and HIV-1 p24 antigen HIV-1 infection. Aptamers
70]. An insight into the mechanism of HIV infection cycle is
that diagnose HIV proteins such as Tat, gp120 and Rev, have
important to develop efficient anti HIV therapeutics and diag-
been primarily used in several biosensor systems and hold great
nostics. Therapeutic agents against HIV are designed to target
significance as diagnostic tool development for HIV. Tat is an
one or more of the several stages of the HIV infection cycle.
immediate early gene of HIV and its expression is suppressed
Currently six classes of anti-HIV drugs target four steps of
by antiretrovirals [73–76]. Its protein transduction domain
HIV’s lifecycle and are often used in combination called combi-
allows secretion by infected cells and uptake by bystander cells
nation antiretroviral therapy. However, a number of these
where it mediates pleotropic effects [24–26] leading to several
drugs can have side effects and in some cases also contribute to
HIV associated comorbidities in the cART era. Hence deter-
HIV related comorbidities. Aptamer-based targeting of HIV at
mining amounts of Tat in the serum can be a useful biomarker
multiple stages can be more specific, cheaper to produce syn-
for subclinical HIV associated comorbidities before manisfesta-
thetically and can be similarly designed to target multiple stages
tion of clinical disease. Tombelli et al., have designed and devel-
of the HIV infection cycle. Fig. 2 shows the different stages in
oped aptamer-based biosensors for the detection of HIV-1 Tat
the HIV infection cycle that can be targeted by aptamers.
protein [77]. High affinity aptamers selected to specifically bind
HIV-1 Tat protein were immobilized on gold surface of piezo-
electric quartz crystals SPR chips. A quartz crystal microbal-
Aptamers for human immunodeficiency virus (HIV)
ance (QCM)-based and SPR-based biosensor were constructed
Human immunodeficiency virus is the primary cause of AIDS using biotinylated aptamer. Binding characterization and speci-
and AIDS related comorbidities . The primary targets of HIV ficity checks were performed. Rev proteins was used as negative
are CD4+ T cells, macrophages, and dendritic cells. Depletion control during for the specificity assays. They have shown that
of immune cells and immune dysregulation result in the AIDS. two biosensors were reproducible in terms of immobilization
Since the advent of antiretroviral therapy, HIV infected patients and binding [77]. In another study by Minunni et al., [78] RNA
have lived longer than ever before. However, viral replication aptamer against HIV-1 Tat was used for the development of
persists even in presence of cART and leading to expression aptasensor. Development of aptamer was based on aptamer
and secretion of viral proteins [21,23,24] that can have pleo- region with bio-recognition element. This designed biosensor
tropic effects on bystander cells [25–27]. Several aptamers have was standardized using piezoelectric quartz-crystals. Concisely,
been selected based on their ability to neutralize HIV and/or its biotinylated aptamer was immobilized on the gold electrode of
proteins with a goal towards developing them for therapeutic the crystal using biotin-streptavidin interaction. The authors
and diagnostic purposes. Aptamers could be further established also investigated aptasensor specificity, sensistivity and

Figure 2. General layout of potential HIV aptamers: The whole cycle of HIV infection and replication consist of seven steps or stages (binding, fusion, reverse transcription,
integration, replication, assembly and budding) called as “the HIV life cycle”. Aptamer targeted for different stages of HIV and its potential use for development of anti HIV
medicines to treat HIV infection hold great significance.
330 J. BALA ET AL.

reproducibility in the detail. The developed aptasensor was blocking vital entry. Mufhandu et al[30] have demonstrated a
compared with an immunosenor where Tat specific monoclo- family of gp120 binding RNA aptamers that can block viral
nal anti-Tat antibody was immobilized on carboxylated dex- entry by binding to and neutralizing HIV gp120. Briefly, a syn-
tran. The sensitivity of the Tat aptasensor was found to be thetic derivative of aptamer, called UCLA1 was used against a
comparable to that of the immunosensor as an effective diag- large panel of HIV-1 subtype C viruses. The authors have pre-
nostic tool. Similar to Tat, anti-Rev aptamers have also been viously isolated 2 0 -F-pyrimidine substituted RNA aptamers
used in several biosensor systems using methods such as based against HIV-1BaLgp120 and shown that they neutralized infec-
in SPR, QCM and the diamond field-effect transistor (FET) tivity in cell-based assays [94]. The binding of UCLA1 to HIV-
technique [79]. Yamamoto et al 2000 have demonstrated 1 subtype C gp120 resulted in neutralization of the same sub-
molecular beacon aptamer fluorescence in the presence of Tat type. The chosen aptamers had been tested both in cell lines
protein of HIV-1 [80]. They have described Tat specific RNA and primary cells for their toxicity and their binding sites on
aptamers with higher binding affinity than its canonical TAR gp120 were characterized. Furthermore UCLA1 demonstrated
RNA binding partner on HIV-1 RNA. They also demonstrated high efficacy at low doses, no toxicity, while also demonstrating
inhibition of Tat dependent transactivation in vitro and in vivo a synergistic effect with entry inhibitors [30,94]. Overall, these
[81]. They have suggested the use of aptamer derived oligomers studies suggest that UCLA1 has potential for further develop-
to evaluate the HIV Tat and the potential applications of such ment as a HIV-1 entry inhibitor. The fluoro-modification of
tools as biosensors. the aptamer backbone also resulted in the UCLA1 aptamer
CD4 is the primary HIV receptor on target cells. Presence of being more stable in biological fluids. An additional derivative,
CD4 receptor on target cells is critical for productive HIV named UCLA005 with a further chemical modification
infection [82–84]. Blocking CD4 using monoclonal antibodies (UCLA005v11) lead to a 4-fold increase in aptamer stability,
results in inhibition of viral entry [85–87]. Kenneth et al., used consequently suggesting that modified aptamers are highly sta-
recombinant human CD4 on bead based affinity matrix to ble. [95] Zhou et al 2011 used a nitrocellulaose based SELEX
screen 2-F modified random RNA library to select high affinity approach to isolate 2'-F modified aptamers against gp120 with
CD4 aptamers [88]. Aptamers were conjugated with different nanomolar (nM) affinity . The selected aptamers could be rap-
fluorophores like phycoerytherin and fluorescein. Fluorophore idly internalized into cells expressing HIV-1 envelope protein
conjugated aptamers were used for cell surface CD4 staining and neutralize HIV-1 infectivity. With aptamer A-1, the
followed by flow cytometry analysis demonstrating that authors generated an exclusive dual inhibitory anti-gp120
aptamers could be developed for diagnostic applications. aptamer-siRNA chimera with effective anti-HIV activities.
Aptamers can be easily modified for diverse applications for They have also used this chimeras for cell-type specific delivery
instance Zhou et al., generated aptamer coated surface to cap- of the siRNA into HIV-1 infected cells and proposed the com-
ture the cells expressing CD4 antigen [89]. Briefly, glass or sili- binatorial of nucleic-acid based therapeutic agents such as
con surfaces were modified with amine-terminated silanes aptamer, miRNA and siRNA for effective suppression of HIV.
followed by coating with thiolated anti-CD4 RNA aptamers. The aptamer-siRNA chimeras demonstrated by them can be
Data showed aptamer-functionalized surfaces have comparable developed as therapeutic leads for patients failing other current
capture efficiency to substrates containing anti-CD4 antibody. antiretroviral therapy . Likewise, Khati et al. in 2003 and Dey et
These aptamers have potential scope in diagnosis and in the al 2005 showed the isolation of 2-fluoro-modified RNA
generation of aptamer based monitoring and diagnostic kits for aptamers that bind specifically to the surface gp 120 [94,97].
HIV/AIDS. Jing et al., developed two families of G-quartet oli- Later, Dey et al 2005 has shown an anti-gp120 RNA aptamer
gonucleotides named as T40217-T40222, with possible forma- neutralizes R5 strains of HIV-1 [32]. They have also reported a
tion of a tail to tail G-quartet dimer and T40224-T40227, with comprehensive structural characterization of the selected
phosphorothioate (PT) linkages in the guanine loops [90]. The aptamer. Binding and structural characterization were done by
authors suggested that G-quartet motif is the crucial prerequi- enzymatic, mutagenesis, secondary-structure analysis, surface
site for the nuclease resistance. Such stable and resistant plasmon resonance (SPR) and chemical probing methods. The
aptamer hold great promise in development as effective and study provide information to understand the molecular interac-
diagnostic tool. tions between the virus and its host cell, besides this the selected
aptamer possess properties to be developed as clinically rele-
vant anti-viral HIV therapeutic. Cohen et al., have developed
Potential therapeutic aptamers against proteins an aptamer that neutralizes R5 strains of HIV-1 by binding to
involved in HIV infection the core residues of gp120 in the CCR5 binding site. They have
earlier reported aptamers that bind the surface envelope gp120,
Aptamers against Gp120
of HIV-1, which have shown to neutralize infection of diverse
Entry of HIV-1 into host cells requires the virion surface enve- sub-types of virus. Later truncated and chemically modified
lope (Env) glycoproteins to interact with specific cell surface derivatives were generated and studied by the solid-phase syn-
receptors on target cells [91]. Hence targeting Env proteins for thesis. The structural understanding from their data may
inhibition of viral entry has great therapeutic potential. HIV deliver a scope for development of possible anti-viral agents
Gp120 is plays a critical role by interacting with its primary [98]. Dey et al 2005 have reported an aptamer that neutralizes
receptor CD4 in combination of either CCR5 or CXCR4 to R5 strains of HIV-1 blocks via disturbance of gp120-CCR5
effect viral entry [92,93]. A number of aptamers selected against interaction. They isolated and characterized 2 0 -F-pyrimidine
gp120 have demonstrated neutralization of HIV infectivity by -substituted RNA aptamers that bind to gp120 of R5 strains of
 RNA BIOLOGY 331

HIV-1. This modified aptamers were able to neutralize the further improved the antiviral effect of the selected aptamers
infectivity of phylogenetically diverse R5 strains. They also elu- using a ribozyme directed, with receptor ligand-facilitated cat-
cidated the mechanism involved in such neutralization by dem- ionic liposome delivery. Their data concludes strong favor for
onstrating that the aptamer binds to the CCR5-binding site on the use of in vitro isolated and enriched ligands as possible
gp120 in a relatively CD4-independent manner [97]. anti-HIV agent.

Aptamers against Gag protein Aptamers for nucleocapsid protein

Gag polyprotein can serve as another therapeutic target against Nucleocapsid (NC) protein of HIV-1 is involved in the encapsi-
HIV infection. A few studies have generated and characterized dation of viral RNA and assembly of viral particle [107–109].
aptamers against Gag protein [99,100]. Gag polyprotein is a Inhibition of nucleocapsid synthesis inhibits viral replication,
structural protein of HIV-1. Ramalingam et al., investigated the thereby making them attractive as potential targets for anti-
prospective of anti-Gag RNA aptamers to inhibit HIV-1 repli- HIV therapy [110–113]. Kim et al 2002 isolated RNA aptamers
cation. They have generated and characterized RNA aptamers that recognize the mature form of the NC protein [114]. RNA
against full-length Gag protein of HIV-1. The selected aptamers aptamers selected in vitro was able to bind NC with high affin-
were further established for their efficacy and functionality in ity, specificity and was able to compete for the packaging ele-
terms of blocking the HIV replication in cultured cells. Interest- ment (psi) RNA binding to the NC protein, suggesting that the
ingly the selected aptamers showed several fold inhibition in aptamer can be used to inhibit viral packaging [114]. Kim and
the extracellular capsid levels. Reduced cellular levels of mRNA Jeong, also screened two independent RNA libraries one of
for Gag was observed, suggesting a molecular structural control which had additional viral psi-sequences to isolate RNA
mechanism mediated via specific Gag-genomic RNA interac- aptamers that bound to the mature form of the NC protein of
tions [99]. The authors also suggested hematopoietic stem cell HIV-1. RNase foot printing was performed to characterize the
(HSC)-based gene therapy, where HSCs are engineered to RNA structures and to map the protein binding sites and data
express anti-HIV molecules such as aptamers. Lochrie et al., revealed that RNA aptamers that bind NC protein contain
isolated aptamers to HIV-1 Gag which bound to nucleocapsid pseudoknots in addition to the characteristic stem-loop struc-
(NC) region interfered with binding of psi (c) packaging signal ture to facilitate RNA and NC (HIV-1) protein interaction
[100]. Their results suggest that RNA aptamers may offer an [115]. Later Kim and Jeong, further used one of such RNA
innovative way for inhibiting HIV replication. aptamer to the NC protein, N70-13, to investigate its effect on
NC protein both in vitro and in cell culture [116]. They found
Aptamers against Rev protein complete abolishment of NC binding to the stable “transactiva-
tion response hairpin” and “psi RNA stem-loops of HIV-1
The Rev protein of HIV-1 acts post transcriptionally to mediate RNA”. The same aptamer were also expressed as an intramer
export of unspliced and partially spliced viral RNA thereby that resulted in the inhibition of packaging of viral genomic
allowing expression of late genes [101]. HIV Rev specifically RNA, underlying importance of targeting NC via aptamer tools
binds to to Rev responsive element (RRE) located on unspliced for the optional development of clinical agent.
and partially spliced viral RNA to mediate RNA export. Inhibi-
tion of rev axis using siRNA or trans-dominant negative
mutants have demonstrated significant suppression of viral Potential therapeutic aptamers against receptors
replication [102,103]. However it is not known if the binding is involved in HIV infection
“structure-specific” or sequence-specific. RNA constitutes an
Aptamers for CD4
imperative structural component of numerous ribonucleopro-
tein (RNP) complexes often functioning as a scaffold to allow Earlier, Zhang et al., have used RNA aptamers as probes spe-
oligomerization of proteins, in ribosome and spliceosome. HIV cific for human CD4 for possible use in cell phenotyping [117].
viral replication requires assembly of RNP, consisting of Rev They found the synthetic CD4 aptamer showed comparable
protein and RRE as an essential prelude to nuclear export of cell-binding specificity as standard CD4 antibody. Additionally,
unspliced viral mRNAs. The specific mechanism necessitates the aptamer can be combined with antibodies for use in multi-
protein-protein and protein-RNA interactions [104]. RNA color flow cytometry analysis with significant repercussions for
aptamers for HIV-1 Rev Protein have previously been isolated clinical application. Wheeler et al., reported aptamer-siRNA
with high affinity and specificity using in vitro selection. In vitro chimeras in which the CD4 aptamer was coupled with siRNA
studies has found that selected sequences also compete with the targeting HIV Gag and Vif or host CCR5 for targeted delivery
wild-type Rev-binding element (RBE). Symensma et al., did to HIV susceptible cells. The chimeras were specifically taken
biological functional characterization of selected aptamer to up by CD4+ cells, which subsequently resulted in vitro and in
determine whether the chosen aptamers could functionally sub- vivo inhibition of HIV infection in primary CD4+ T cells and
stitute for RRE in vivo [105]. Reporter system based hybrid macrophages. They further demonstrated that CD4 aptamer/
RREs were generated and analyzed for their ability to facilitate siRNA chimeras with hydroxyl ethylcellulose gel formulation
Rev function. Remarkably, the data suggests that aptamers resulted in complete transmission block in polarized human
were functionally comparable to the wild-type element. cervicovaginal explants and humanized mice [118,119]. Like-
Konopka et al., had shown that HIV-1 gene expression could wise, Guo et al., have also used CD4 aptamer and specific chi-
be inhibited by the anti-HIV Rev-binding aptamer [106]. They meric RNA scaffolds for targeted delivery [120]. Blocking or
332 J. BALA ET AL.

modulating the interaction of viral gp120 and CD4-expressing

T cells could be a clinically significant anti-viral strategy for
suppressing HIV. Zhao et al., isolated ssDNA anti-CD4
aptamers that could interfere with HIV gp120 CD4 interaction
suggesting strong potential of such molecules in as anti HIV
therapy [29].

Aptamers for CCR5

CCR5 is a G-protein coupled seven transmembrane receptor
expressed by T cells and macrophages that act as co-receptor
for macrophage-tropic HIV-1 [121,122]. Many reports have
demonstrated selection of anti-CCR5 aptamers to inhibit HIV
entry. Zhou et al., used live cell based SELEX combined with
high-throughput sequencing technology to select cell-specific
RNA aptamers against human CCR5 [31]. These aptamers tar-
geted HIV-1 susceptible cells and inhibited HIV-1 infectivity.
Anti-CCR5 aptamer was able to inhibit R5 virus infection in
primary peripheral blood mononuclear cells. Their data also
suggest that CCR5-targeted aptamers and aptamer-siRNA con-
jugates offer a potential anti HIV/AIDS agent with targeted
delivery of several therapeutic candidates. Scheideman et al.,
has reported peptide aptamers that bind the transmembrane
region of CCR5 and inhibit CCR5 cell surface expression and Figure 3. (a) The interaction model between HIV-1 RT (reverse transcriptase) and
the aptamer. The secondary structure of aptamer is the -HIV-1 RT complex. The
HIV co-receptor function [123]. Briefly, screening of library of p66 subdomains: fingers, palm, thumb, and RNase H and p51 interactions are
randomized transmembrane domains was performed and the- shown using a surface representation (With permission of Springer) [ref 124]. (b)
ses variants were named as termed “traptamers,” for transmem- The dimeric 93del aptamer is positioned into the channel formed by the HIV1-IN
(integrase) tetramer to block the HIV1-IN catalytic site (reproduced with permission
brane aptamers. Six 44- or 45-amino-acid proteins with from Proceedings of the National Academy of Sciences USA) [ref 134].
completely different transmembrane sequences were isolated
and characterized. Traptamers were able to inhibit both, cell
surface, and total expression of the HIV co-receptor CCR5. and aptamer interaction at molecular level that will help in the
Truncation and standardization of two traptamers augmented development of efficient anti-HIV therapeutics. Immunologic
its activity and resulted in higher activity in terms of inhibition and proteolytic analysis of HIV-1 reverse transcriptase structure
of R5-tropic reporter virus. These active traptamers specifically was first demonstrated by Ferris et al in 1990 [125]. They
recognized the transmembrane domains of CCR5. The authors reported HIV-1 reverse transcriptase from HIV type 1 has two
have also constructed novel multiple proteins not found in subunits, which have respective molecular weights of 66 and
nature that interfere with CCR5 expression and inhibit HIV 51 kDa. Importance of HIV-1 reverse transcriptase in HIV repli-
infection [123]. This novel strategy provides valuable insight to cation and infection are well established, which signify its obvi-
target organization of the transmembrane domains of CCR5 ous targeting for AIDS therapeutic claim [126,127]. A number
and to target biological cellular pathways mediated by them. of antiretroviral are designed to target and inhibit HIV reverse
These molecules could be developed as an innovative diagnostic transcriptase. DeStefano and Nair have reported DNA aptamer
approach or anti-HIV therapeutic. (37NT) for the reverse transcriptase of HIV HXB2 strain [128].
They have developed novel small single-stranded loop-back
DNA aptamer inhibitors of HIV reverse transcriptase. From the
Aptamers against enzymes involved in HIV infection selected variants, a modified and characterized 37-nt aptamer
was able to act as an effective inhibitor of HIV reverse transcrip-
Aptamers for reverse transcriptase (RT)
tase. The selected aptamer also competed with the natural tem-
HIV-1 RT has been targeted for the development of anti-HIV plate for the binding site in the enzyme. Nicken et al., showed
therapeutics drug. Aeksiri et al in 2014 have reported HIV-1 simple model systems for screening RNA protein interactions
(RT) reverse transcriptase-aptamer interaction using molecular within an intracellular context. They demonstrated inhibition of
dynamics simulations [124]. Authors have examined the confor- HIV-1 reverse transcriptase by RNA aptamers in Escherichia coli
mational dynamics of aptamer bound HIV-1 RT complex with [129]. Michalowski et al., recognized three DNA aptamers
an aptamer (Fig. 3a). Briefly, they have studied apo HIV-1 RT, named RT5, RT6 and RT47 that inhibit HIV-1 reverse transcrip-
HIV-1 RT bound RNA aptamer, and also HIV-1 RT bound tase [130]. The binding motif responsible for inhibition consists
DNA substrate. Their data establish that HIV-1 RT complex of a bimodular structure comprising a 5 0 -stem-loop module
with an aptamer was more stable than DNA substrate complex. linked to a 3 0 -G-quadruplex. Moreover, they also mentioned
Aptamer and HIV-RT binding interaction showed fingers-and- that chosen DNA aptamers inhibited reverse transcriptase from
thumb-closed conformation and key residues involved in inter- diverse primate lentiviruses with low nM IC50 values. These
action were identifies. Their data gives insight about HIV-RT results provide reverse transcriptase inhibition by oligo nucleic
 RNA BIOLOGY 333

acid based therapeutics. Joshi and Prasad reported an effective recognition, protease inhibition, and advantages of aptamers
aptamer based inhibitor of HIV Type 1 replication by template for pharmacological intervention with pathophysiological func-
analog reverse transcriptase [131]. The inhibitors were generated tions of proteases [141]. Several studies have reported protease-
using SELEX. The author also demonstrated that virion-encapsi- inhibiting aptamers directed against blood coagulation factors,
dated TRTIs can predispose virions for inhibition rapidly upon which are currently undergoing clinical trials as anticoagulant
entry. In another study Ferreira-Bravo et al., selected 2 0 -deoxy- drugs [141–143]. Duclair et al., isolated high-affinity RNA
2 0 -fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 Aptamers against the HIV-1 protease to inhibit protease activ-
reverse transcriptase with high affinity [132]. They showed ity [144]. The authors also suggested that the anti-protease
strong and specific inhibition of HIV-1 reverse transcriptase in RNA aptamers have the prospective to be utilized in such an
extension assays with no effect on avian myeloblastosis or molo- approach using intracellular expression to confer resistance to
ney murine leukemia reverse transcriptase. Lai and DeStefano viral replication. Anti-HIV protease aptamers have been shown
reported and characterized DNA Aptamers to HIV reverse tran- to inhibit both in vitro protease activity and late events of viral
scriptase selected by a primer-free SELEX method with other replication.
aptamers [133].
Conclusions and future perspective
Aptamers for integrase
HIV is pandemic in many areas of the world. In spite of the
Phan et al 2005 have reported NMR-based solution structure of advances in combating HIV over many decades, there is no
the 93del (GGGGTGGGAGGAGGGT) HIV-1 integrase cure or an effective vaccine against HIV and it continues to
aptamer (Fig. 3b) [134]. They have shown stable interlocked affect many patients worldwide. Limited resources for HIV pre-
dimeric parallel-stranded DNA quadruplex which act as inhibi- vention is one of the challenge and this issue has more impact
tor of HIV-1 integrase. Guanine-rich DNA structure attains on developing countries. Given the precincts of existing HIV
unusual stable dimeric quadruplex structures. The details NMR diagnostic, treatment and prevention methods and the financial
structural insight were presented, additionally integrase inhibi- load, an alternative second line potential treatment agents, new
tion assays were performed (ref). Interestingly, they have pro- formulations, better pills and simple diagnostic tools, in low
posed a model for positioning the 93del dimeric DNA and middle income countries are desirable. Aptamers hold
quadruplex and HIV-1 integrase which can help in designing enormous potential as diagnostic agents and therapeutics for
novel strategy for HIV/AIDS. Retroviral enzyme integrase is vital HIV/AIDS. Macugen an RNA aptamer for vascular endothelial
for viral replication which facilitates the integration of proviral growth factor (VEGF)-165 was the first FDA approved aptamer
DNA in the host chromosome [135,136]. Chiu and Davies first drug targeting VEGF for use against macular degeneration dis-
reported the structure and function of HIV-1 integrase in 2004. ease and many aptamers are under clinical trial [18]. There is
HIV-1 integrase is a multidomain enzyme, a potential target for currently no cure or effective HIV vaccine, it is of the highest
therapeutic targeting [137]. Ojwang et al., demonstrated an implication to discover specific molecular probes targeting HIV
aptamer, named T30177, able to inhibit HIV integrase activity for early diagnosis and therapies using aptamer and SELEX
[138]. T30177 represents a class of integrase inhibitors with technology can play a pivotal role in this. Several aptamer-
promising properties for the development as therapeutic agent based potential anti-HIV candidates have been generated using
against HIV-1 in humans [138]. T30177 is a novel integrase SELEX technology. Aptamer research over past years has found
inhibitor and is being tested for its efficacy. T30177 was further relevance as valuable diagnostic and therapeutic leads tools that
derivatized to increase its stability and retained the efficient inhi- could cure and diagnose HIV [30,31,78]. Aptamers offer dis-
bition capability for HIV-1. T30177 inhibited replication of sev- tinct advantages over ART in terms of cost and safety as alter-
eral strains of HIV-1 in human T-cell lines, peripheral blood native anti-HIV therapeutics. Moreover, aptamers specifically
lymphocytes, and macrophages. T30177 mediated inhibition was targeted to different HIV proteins can help neutralize these
also reported in multiple clinical isolates of HIV-1 [138]. Meti- proteins and mitigate the non-AIDS comorbidities observed in
fiot et al., 2005 targeted HIV-1 integrase with aptamers using people living with HIV. In the cART era the emphasis is on
purified RNase H domain of HIV-1 RT. Aptamers containing viral control. However, cART itself has its own side-effects can
G-rich sequences were isolated and investigated. Additionally, a itself contribute to co-morbidities observed in HIV patients.
second in vitro selection was performed with an isolated RNase Hence, it is of the highest implication to discover specific
H domain of HIV-1 reverse transriptase (p15) as a target using molecular probes targeting HIV for early diagnosis and thera-
a new DNA library. Their data suggested that prototype struc- pies. The development of aptamer based biosensors can have
tures can be exploited to develop inhibitors of two related profound impact in diagnostics use of aptamers for the detec-
enzymes [139]. As novel anti-HIV agents, the G-tetrad-forming tion of receptor proteins involved HIV infection. Aptamers to
oligonucleotides have been explored for their structure activity cellular proteins can also play a role in identifying unknown
in relation with inhibition of integrase [140]. cellular pathways involved in HIV replication by functional
proteomics approach [145,146]. Remarkably, targeted delivery
of other nucleic acid based therapeutics like siRNA has been
Aptamers for proteases (PR)
accomplished using aptamers to cell-surface receptors. Further
Proteases are important therapeutic targets in various patholog- advancement in the fields of SELEX technology, nanotechnol-
ical condition. Dupont et al., has presented detailed review on ogy, array-based platforms, microfluidics, and others techni-
biochemical mechanisms of aptamer selection, protein-aptamer ques can facilitate rapid selection and application of aptamers
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