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A REVIEW ON APTAMERS TECHNOLOGY AND THEIR THERAPEUTIC APPLICATION IN VIRAL INFECTIONS BY

A REVIEW ON APTAMERS TECHNOLOGY AND THEIR THERAPEUTIC APPLICATION IN VIRAL INFECTIONS
BY:
NATO HUNDESSA
“A SEMINAR PAPER PRESENTED FOR THE COURSE: SENIOR SEMINAR ON CURRENT TOPICS OF VETERINARY EPIDEMIOLOGY”
ADDIS ABABA UNIVERSITY
COLLEGE OF VETERINARY MEDICINE AND AGRICULTURE
ADVISOR:
TESHALE SORI (DVM, MSc, Asso. Professor)
SEPTEMBER, 2018
BISHOFTU, ETHIOPIA
Table of Contents PAGE
TOC o “1-3” h z u ACKNOWLEDGEMENTS PAGEREF _Toc523240320 h iiLIST OF ABBREVIATIONS PAGEREF _Toc523240321 h iiiLIST OF FIGURES PAGEREF _Toc523240322 h vLIST OF TABLES PAGEREF _Toc523240323 h viSUMMARY PAGEREF _Toc523240324 h vii1. INTRODUCTION PAGEREF _Toc523240325 h 12. HISTORY OF EVOLUTION OF APTAMERS PAGEREF _Toc523240326 h 43. APPLICATION OF APTAMERS IN VIRAL INFECTION DIAGNOSTICS AND THERAPEUTICS PAGEREF _Toc523240327 h 53.1. Generation of aptamers PAGEREF _Toc523240328 h 53.2. Characteristic of aptamers PAGEREF _Toc523240329 h 63.3. Target applicability of aptamers to viral detection and inhibition PAGEREF _Toc523240330 h 83.3.1. Aptamers against viral infections; antiviral aptamers PAGEREF _Toc523240331 h 114. NEW THERAPEUTIC INNOVATIONS AND APPLICATIONS PAGEREF _Toc523240332 h 154.1. Aptamers to neutralize lethal viruses PAGEREF _Toc523240333 h 154.2. Aptamers or aptamer conjugates to kill drug-resistant pathogenic bacteria PAGEREF _Toc523240334 h 154.3. Aptamers to neutralize toxins and venoms PAGEREF _Toc523240335 h 164.4. Aptamers to potentially induce stem cell differentiation and trans-differentiation PAGEREF _Toc523240336 h 165. LIMITATIONS AND CHALLENGES OF BIOLOGICAL APPLICATOINS OF PAGEREF _Toc523240337 h 18APTAMERS PAGEREF _Toc523240338 h 186. CONCLUSIONS AND RECOMMENDATIONS PAGEREF _Toc523240339 h 197. REFERENCES PAGEREF _Toc523240340 h 20
ACKNOWLEDGEMENTS
First and for most, I am so grateful to praise my Heavenly Father, the almighty GOD, for guiding me throughout the paths of my life routines.
I would like to give my deepest gratitude for my advisor Dr. Teshale Sori for his intellectual advice and support in the writing up of this manuscript.

Last but not least, I owe my deepest gratitude to all my friends and those who were aspiring and generous towards me.

LIST OF ABBREVIATIONS
Ag Antigen
AIV Avian influenza virus
ATP Adenosine triphosphate
CD81 Cluster of Differentiation-81
CRE Carbapenem-resistant EnterobacteriaceaeDENV-2 Dengue virus-2
DNA Deoxyribonucleic acid
dsRNA Double stranded ribonucleic acid
EBOV Ebola virus
eVP35 Ebola viral protein 35
gD Protein Glycoprotein D
HA HemagglutininHBV Hepatitis B virus
HCV Hepatitis C virus
HIV-1 Human immunodeficiency virus
HPV16 Human papillomavirus-16
HSV Herpes simplex virus
HVEM Herpesvirus entry mediator
IRES Internal ribosome entry site
KPC Klebsiella pneumoniae carbapenemaseMDR Multiple drug resistance
MERS Middle east respiratory syndrome
mRNA Messenger ribonucleic acid
MRSA Methicillin-resistant Staphylococcus aureusNP Nucleocapsid protein
NS5B Nonstructural protein 5B
PCR Polymerase chain reaction
PEG Polyethylene glycol
RABV Rabies virus
RNA Ribonucleic acid
RVF Rift valley fever virus
SARS Severe acute respiratory syndrome
SELEX Systematic evolution of ligands by exponential enrichment
siRNA Small interfering ribonucleic acid
ssDNA Single-stranded deoxyribonucleic acid
VRE Vancomycin-resistant Enterococcus
WNV West nile virus

LIST OF FIGURES PAGE
TOC h z c “Figure” Figure 1. Schematic presentation of SELEX procedure for aptamer selection PAGEREF _Toc522057075 h 6Figure 2. Diagramatic presentation of structural anatomy of a virus PAGEREF _Toc522057076 h 9Figure 3. Strategies of antiviral aptamer therapy PAGEREF _Toc522057077 h 10
LIST OF TABLESPAGE
TOC h z c “Table” Table 1. The relative advantages of aptamers over antibodies…………………………7Table 2. Current promising aptamers evaluated in preclinical models for treatment and diagnosis of various viral infections…………………………………………………… 14
SUMMARYAptamers, simply described as chemical antibodies, are synthetic oligonucleotide ligands or peptides that are composed of single-stranded DNA (ssDNA) or RNA that can be isolated in-vitro against diverse targets including viral proteins, virus-infected cells and whole pathogenic microorganisms. They possess a defined tertiary conformation and generally bind functional sites on their respective targets. They mimic the molecular recognition properties of monoclonal antibodies in terms of their high affinity and specificity. Their additional advantages; structural flexibility, stability, lower immunogenicity, and comparably smaller size than antibodies, enable them for precise recognition of cellular elements as diagnostic and therapeutic tools. The protective effects of the aptamers have been weighed up in their ability to prevent infection and also to control infection. Aptamers can be generated against most target proteins since in most cases they are able to inhibit the activity of the target protein and could also block virus replication. It can be used against various viral diseases that have been given most attention due to their severe complications and therapeutic problems, such as, hepatitis, herpes simplex virus, influenza virus, rift valley fever virus, rabies virus and others. Challenges and limitations of aptamers hinge on issues of therapeutic formulations and their bioavailability. Nevertheless, the advantages and future prospects of aptamers outweigh their limitations. With remarkable target specificity and sensitivity, versatile biophysical and pharmacokinetic properties, targeting and regulating the function of various biomedical relevant proteins, aptamers have found themselves a substantial niche and are becoming established as a promising new class of medicines which augurs well for future aptamer-based drugs and theranostic development. Therefore, this manuscript confers the biochemical properties of aptamers and their applications in the therapeutics of viral infections.

Key words: Aptamers, Antibodies, Diagnostics, Oligonucleotide ligands, Theranostic, Therapeutic, Virus, Viral protein.

1. INTRODUCTION
Infectious diseases in both animals and humans are caused by a wide range of disease agents including viruses, bacteria, fungi and protozoa (WHO, 2007). Infectious diseases that result from the invasion of viral agents into a living organism are the major cause of pathogenesis and mortality, in both industrialized and developing countries. Viruses are infective agents that enter, reproduce or multiply only within living cells of other organisms. After the virus replicates inside the cell, it may remain inactive for lengthy period or released immediately and fused to other healthy cells to commence the infection process (Shinde et al., 2012).
As part of viral replication, assembly, and release, the cell surface is modified by the insertion of viral proteins (Bayry and Kaveri, 2006). The cell surface markers confer virus-specific targets to designing specific molecular probes which recognizes and provides molecular marker for virus-infected cell unit; since few biomarkers are known and accessible for successful diagnosis of viral disease, investigators ability to target and study such viral proteins and infected cells has thus far been limited. Chemical antibodies called aptamers targeting microorganisms and viruses have attracted attention offering a promising technology for addressing this deficiency during the last decade (Barrett et al., 2014; Llor and Bjerrum, 2014).

Aptamers are single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) oligonucleotides that can be selected to bind to a variety of target molecules, including whole cells, large purified molecules such as proteins, and small molecules such as adenosine tryphosphate (ATP). As a result of their well-defined spatial conformation, aptamers are able to specifically recognize their target molecules with high affinities. Specific aptamers are selected by an in vitro selection process, termed SELEX (systematic evolution of ligands via exponential enrichment) (Zhou et al., 2012). 
The systematic evolution of ligands via exponential enrichment (SELEX) consists of a series of repeated enrichment cycles and counter-selections based on competitive binding that ultimately selects for a group of aptamers that will bind specifically to a given specific target (Breaker, 2004).
In comparison with the use of antibodies in traditional probes, aptamers have advantages for targeting virally infected cells, including stable performance, reproducible properties, low immunogenicity, high selectivity, strong affinity, and capabilities of facile modification for further optimization (Guo et al., 2005; Keefe et al., 2010). Most of all, aptamers can be used as a discovery tool to explore the molecular basis of a specific disease or infection process. Moreover, aptamers can be selected to recognize infected cells without prior knowledge of the new potential biomarkers after cells are infected (Navani and Li, 2006). Such advantages have attracted much scientific attention, leading to the conclusion that aptamers also maintained for the detection of virus-infected cells and for related bioassays required for early detection of infection and, inhibitory aptamers, for the treatment of diseases (Smith et al., 2007).
Aptamers have also been used extensively in various biomedical applications that were once the sole realm of monoclonal antibodies, including as research tools, in bioassays, for cell detection and biomarker discovery, tissue staining, in vitro and in vivo imaging, targeted therapy and nanomedicine, and food safety and environment monitoring. They can be easily combined or conjugated with therapeutic substances such as drugs, carriers containing drug, toxins, or photosensitizers. Moreover, high affinity and selectivity allow aptamers to discriminate very close-related targets, for example, different protein isoforms (Conrad et al., 1996). In some cases, aptamers exploit as antiviral agents, can also block the functions of target proteins and thereby, could serve as a basis for the development of novel therapeutics for the treatment of viral infections (Gedi and Kim, 2014; Ozalp et al., 2013).

As a matter of fact, viruses use vital metabolic pathways within host cells to replicate, they are difficult to eliminate without using drugs that cause toxic effects to host cells in general. The most effective medical systems to viral diseases are vaccinations to offer immunity to infection, and antiviral drugs that selectively impede with viral replication. However, it can hardly be cured using standard therapeutics; this fact dictates a necessity to search for new approaches of fast and reliable diagnostics and treatment (Kaittanis et al., 2010; Shinde et al., 2012). Aptamers are the best alternatives that can be used for rapid and accurate diagnosis and treatment of infections caused by viral agents.
Therefore, the aim of this seminar paper is:
To highlight the current advances of aptamer technology and their applications against viral infection diagnosis and treatment.

2. HISTORY OF EVOLUTION OF APTAMERS
The idea that nucleic acids, whether DNA or RNA, could function as ligands and modulate the activity of target proteins was the result of research performed on viruses. Specifically, studies into the molecular biology of human immunodeficiency virus (HIV) and adenovirus demonstrated that these viruses encoded small, structured RNA that bind to endogenous proteins. These RNA ligands either facilitated viral replication or mitigated antiviral activity by the host. These findings suggested to virologists that nucleic acid molecules that bind proteins have the potential to become therapeutic drug agents (Cullen and Greene, 1989; Marciniak et al., 1990).
In 1990, a groundbreaking work was performed by Sullenger et al. (1990) demonstrating that an RNA aptamer designed to bind a viral protein prevented viral RNA-protein binding, thereby preventing viral replication and this signifies that RNA ligands serve potentially as therapeutic agents (Sullenger et al., 1990; Sullenger et al., 1991). Two pioneering studies performed simultaneously and independently verified that nucleic acid ligands or aptamerrs could be isolated against virtually any protein. Concurrently, Tuerk and Gold demonstrated that a large library of RNA molecules could be screened to find ligands that bound to T4 DNA polymerase with high affinity, where as Ellington and Szostak isolated RNA molecules that bound to organic dyes. Moreover, they stated that these RNA molecules folded into specific confirmations to bind to these dyes. They subsequently used this technique, known as SELEX, to demonstrate that this could also be performed using DNA ligands. The subsequent work on manipulating the structure and formulation of aptamers has expanded their pharmacological properties to make them a versatile class of compounds that can be tailored to specific clinical needs (Ellington and Szostak, 1992).

3. APPLICATION OF APTAMERS IN VIRAL INFECTION DIAGNOSTICS AND THERAPEUTICS
3.1. Generation of aptamers
Aptamer, a term derived from the Latin word “aptus”, meaning “to fit” and the Greek word meros, meaning “part”, are short single-stranded DNA (ssDNA) or RNA oligonucleotides (Ellington and Szostak, 1990). Aptamers possess the ability to fold into a variety of secondary structural elements providing multiple recognition surfaces for target binding. Due to their well-defined spatial conformation, they are able to specifically recognize their target molecules with high affinities (Breaker, 2004). As a molecular probe, they have been integrated and utilized in bioanalysis and biomedicine (Ku, 2015). Specific aptamers are selected from a nucleic acid library and generated through an in vitro iterative selection and screening process known as SELEX. The whole process starts from generating a randomized nucleic acid (DNA or RNA) sequence library, which is normally composed of approximately 1015 different aptamer sequences that theoretically can distinguish any several targeted molecules (Ellington, and Szostak, 1990; Tuerk and Gold, 1990).
Systematic evolution of ligands by exponential enrichment, an interactive in vitro selection procedure, is the basic method used to engineer aptamers (Sorlie et al., 2001; Siegel et al., 2013). As a standard, in principle, aptamers are selected initially from a randomly synthesized ssDNA pool based upon binding of the oligonucleotides to target molecules under favorable conditions. The pool contains 1014–1015 random sequences of synthetic ssDNA/RNA (the typical length is 15–70 nucleotides, flanked by two constant segments with primer sites for polymerase chain reaction (PCR) amplification). The unbound sequences are discarded from the bound molecules, and the target-bound sequences are then amplified by PCR (Siegel et al., 2016).
The amplified products (double stranded DNA) are converted to ssDNA through various ways and then used as a new aptamer pool for the next selection round. The amplified molecules, enriched aptamer sequences are finally cloned and identified by sequencing. Usually, after 10–20 rounds of selection, the specific aptamers with the strongest affinity for the target molecules are obtained (Siegel et al., 2016). The conventional SELEX procedures are illustrated in Figure 1.

Figure SEQ Figure * ARABIC 1. Schematic presentation of SELEX procedure for aptamer selection
Source: (Ray and White, 2010).

3.2. Characteristic of aptamers
Antibodies have, for many decades, been at the forefront of biological therapies, but their swift development has been countered by a variety of barriers that are being slowly overcome (Cosma et al., 1993). Although aptamers are similar to antibodies in terms of their affinity and specificity to targets, they offer several advantages over their antibody counterparts. Their chief advantage lies on their technical versatility and applicability to a wide array of target molecules (Sun and Zu, 2015). Fundamentally, aptamers are selected through an in vitro process and do not depend on animals or cells. Along with that, they are stable at room temperature, whereas antibodies require refrigeration to avoid denaturation. Moreover, aptamers have been shown to have low immunogenicity and toxicity so far (Ireson and Kelland, 2006), whereas, antibody generation requires the use of a live animal to stimulate an immune response, and the target molecule needs to be immunogenic as well as nontoxic, thus, evoke a negative immune response. Finally, aptamers are small molecules and may effectively penetrate into tissue barriers and have effects on cells (Ireson and Kelland, 2006; Sun and Zu, 2015). The relative advantages of aptamers over antibodies are given in Table 1.

Table SEQ Table * ARABIC 1. The relative advantages of aptamers over antibodies
Aptamers Antibodies
Binding affinity in low nano to pico molar range Binding affinity in low nano to pico molar range
Selection is entirely by a chemical process carried out in vitro and can therefore target every protein
Selection requires a biological system, therefore difficult to raise antibodies to toxins (not tolerated by animal) or nonimmunogenic targets
Can select for ligands under a variety of conditions for in vitro diagnostics Limited to physiological conditions for optimizing antibodies for  diagnostics
Iterative rounds against known target limit screening processes Screening monoclonal antibodies is time-consuming and expensive
Uniform activity regardless of batch synthesis Activity of antibodies varies from batch to batch
Pharmacokinetic parameters can be altered on demand Difficult to modify pharmacokinetic parameters
Investigator verify the target site of protein Immune system determines target site of protein
Wide variety of chemical modifications to molecule for diverse functions Limited modifications to molecule
Return to its original conformation and regain its activities after temperature insult Temperature sensitive and undergo irreversible denaturationUnlimited shelf life Limited shelf life
No evidence of immunogenicity Significant immunogenicity
Cross reactive compounds can be isolated using toggle strategy to facilitate preclinical studies No method for isolating cross-reactive compound
The inhibitory activity of the drug can be reversed by aptamer specific antidote No rational method to reverse molecules
Source: (Shahid et al., 2017).

3.3. Target applicability of aptamers to viral detection and inhibition
Convenient diagnosis is the major key factor for viral diseases treatment. However, viral infections are difficult to distinguish, especially at its onset. If acute infection appears, the patient presents the set of nonspecific signs and symptoms. Time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, Ebola and Severe acute respiratory syndrome (SARS). In contrast, chronic viral diseases are asymptomatic/oligo-symptomatic. The therapeutic success, focused on organ protection from chronic destruction and failure, for example, human immunodeficiency virus-1 (HIV-1) or hepatitis C virus (HCV), depends on early detection of an infective agent (Bruno et al., 2012).

For choosing the most suitable viral target, it is vital to understand the anatomy and the replication cycle of the virus. The virus anatomy can be divided in three parts: (i) the genetic material, either RNA or DNA, inside the virus; (ii) a coat consisting of proteins on which surface epitopes essential for attachment and infection are localized; and (iii) for some viruses, a lipid bilayer or envelope to protect the protein coat (Lakshmipriya et al., 2013). In viral diagnostics, the components targeted to detect the virus are whole virus (virion), nucleic acids and/or viral coat consisting protiens (Figure 2). The envelope is not targeted, as it has hardly any particular features to enable selective recognition. During viral reproduction, virions are produced by the host cell, which expresses every component in excess and not all are integrated in a complete infectious virus. The components viral agent are therefore enormously abundant than complete virions, so they are the preferred targets. The viral proteins seem to be better targets, since nucleic acids are rapidly degraded in a clinical matrix (Park et al., 2014).

Figure SEQ Figure * ARABIC 2. Diagramatic presentation of structural anatomy of a virus
Source: (Van den Kieboom et al., 2015)
Other than to treat the infection, aptamers might be used to prevent it—viral infection can be reserved in almost any step of the disease. The most effective therapeutic strategy is to block the penetration of viruses into the cells and/or inhibition of enzymes involved in their replication (Bellecave et al., 2008; Gopinath et al., 2012). Moreover, certain aptamers are able to selectively stimulate the immune system. They are suitable subjects of structural modifications as in vivo biostability improvement and conjugation with other therapeutic molecules, such as small interfering RNA (siRNA) and ribozymes (Figure 3) (Dey et al., 2005; Romero-Lopez et al., 2012).

Figure SEQ Figure * ARABIC 3. Strategies of antiviral aptamer therapy
Source: (Tomasz et al., 2015)
Highly variable viral genome regions are the common cause of virus resistance to currently used therapies. Thus, there is a requirement to generate aptamers specific to highly conserved nucleic acid regions, where mutations appear relatively rare (Konno et al., 2008). High attention has been paid to HCV and HIV-1 infections, by the reason of their predominance, prevalence, severe complications and well-known therapeutic problems. Other viral diseases considered as aptamer targets include influenza, herpes simplex virus (HSV) and HBV/HCV infections, i.e., the diseases, with commonly occurring immune antiviral response (Biroccio et al., 2002; DeStefano and Nair, 2008).

3.3.1. Aptamers against viral infections; antiviral aptamers
Successful viral infection involves binding of the virus to the host cell surface, receptor-mediated entry, uncoating of the virus particle, production of viral proteins and replication of the viral DNA, and virus assembly followed by release of new virus particles (Bayry and Kaveri, 2006; Lewis, 2007) . As part of viral penetration, replication, assembly, and release during viral infection, the cell surface is modified by the inclusion of viral proteins. These cell surface alterations or markers provide virus-specific targets for the design of specific molecular probes that can recognize and provide molecular signatures for virus-infected cells (Wiles et al., 2006; Adams et al., 2007). Aptamers can be used to inhibit viral infectivity as therapeutic agents to counter viral infections at any stage in the viral replication cycle, including viral entry, which has the potential to prevent initial infection (Zhou et al., 2010).
The viral enzymes, RNA-dependent RNA polymerases, that catalyze the replication of RNA from an RNA template, have been a common target because viral polymerases use RNA as the template during genome replication. For example, the HCV polymerase, non-structural protein 5B (NS5B), is required for transcribing the HCV genome and has been used to generate both anti-NS5B RNA and DNA aptamers (Biroccio et al., 2002; Bellecave et al., 2008). Both sets of aptamers have been shown to bind to NS5B with high affinity and inhibit polymerase activity (Kanamori et al., 2009).

Inhibition of protein translation of the viral genome is the other key host defense mechanism against viral infections since viruses are completely dependent on the host machinery for protein synthesis. However, viruses have developed diversed means to escape host defenses and take control the host translational machinery. For example, the HCV messanger RNA (mRNA) has an internal ribosome entry site (IRES), a structured region within the mRNA that binds the ribosome and initiates cap-independent translation, allowing HCV to avoid the necessity for host initiation and elongation factors. Aptamers targeting the HCV IRES inhibit IRES-dependent translation of HCV proteins (Aldaz-Carroll et al., 2002; Kikuchi et al., 2003; Da Rocha Gomes et al., 2004).

However, recently, several aptamers target viral surface antigens or virus-infected cells, e.g., RNA aptamer HBs-A22 specifically binds to HBV surface Ag-expressing hepatoma cells (Liu et al., 2010), ssDNA aptamer S15 targets dengue virus-2 (DENV-2) envelope protein, (Chen et al., 2015) and ssDNA aptamer GE54 targets neurotropic rabies virus (RABV) glycoprotein-expressing cells and rabies virus-infected cells, which clearly demonstrated an inhibition of RABV-infected cell (Liang et al., 2014). Some aptamers bind to viral enzymes to inhibit replication, e.g., an RNA aptamer against HBV polymerase protein and an ssDNA aptamer against SARS-coronavirus helicase bind to the nucleic acid binding site of the helicase and block the unwinding activity (Shum and Tanner, 2008). Several aptamers target viral proteins thus blocking nucleocapsid formation, e.g., ssDNA aptamers against the HBV core protein or matrix-binding domain (Orabi et al., 2015). Other aptamers target viral oncoproteins, e.g., RNA aptamers binding to oncoproteins of human papillomavirus 16 (HPV16), E7 and E6 (Nicol et al., 2011; Belyaeva et al., 2014).

Wang et al. (2000) started with a ssDNA library and performed the first rounds of selection cycles using purified hemagglutinin (HA) from avian influenza virus (AIV) H5N1, and afterwards the entire H5N1 virus was used as a target. After several SELEX rounds of positive target selection and approval of selected pools of oligonucleotides binding to nontarget AIV subtypes (H5N2, H5N3, H5N9, H7N2, H2N2, and H9N2), aptamers were cloned and sequenced. Thus, an aptamer possessing a high binding capability was identified to the H5N1 virus (Wang et al., 2000).
A particular ssDNA aptamer that specifically binds to the HCV-E2 envelope glycoprotein was named ZE2. It is believed that the ZE2 aptamer competitively inhibits the HCV-E2 envelope glycoprotein by binding to CD81 (Cluster of Differentiation 81), an important HCV protein receptor, and significantly blocks HCV cell culture infection of human hepatocytes. Thereby, the ZE2 aptamer emphasizes its potency to act as a possible novel diagnostic and therapeutic candidate in HCV infections (Chen et al., 2009).
Aptamer technology has also been used by Gopinath et al. (2012) who isolated two RNA aptamers (aptamer-1 and aptamer-5) against the ectodomain of the HSV-1 glycoprotein D (gD protein), which plays an important role in viral entry to the host cells. Both aptamers explicitly bind to gD protein of HSV-1 with high affinity. Furthermore, aptamer-1 efficiently blocked the interaction between the gD protein and the HSV-1 target cell receptor, herpesvirus entry mediator (HVEM). Anti HSV1 activity of aptamer1 showed that this aptamer efficiently inhibited viral entry. A shorter variant of aptamer-1 named mini-1 aptamer (44-mer) had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions (Gopinath et al., 2012). In a similar way, Moore et al. (2011) have reported the isolation and characterization of one aptamer, G7a, that binds the gD protein of HSV-2 and neutralizes infection through the NECTIN-1, nectin cell adhesion molecule 1, and HVEM entry receptors. Interestingly, aptamers that prevent HSV-2 infection may also reduce the morbidity associated with HIV-1 as HSV-2 is a major risk factor for the acquisition of HIV-1 (Moore et al., 2011).
Aptamers against other emerging viruses: An emergent virus is a virus that has adapted and emerged as a new pathogenic strain, with attributes facilitating pathogenicity in a field not normally associated with that of virus. Most of these viruses have newly appeared in a population or have existed but are rapidly increasing in incidence or geographic range and only recently aptamers against emergent viruses such as rift valley fever (RVF), dengue, Ebola and other viruses have been developed (Bruno et al., 2012).

Ellenbecker et al. (2012) isolated RNA aptamers that bound to the nucleocapsid protein of RVF virus, an RNA binding protein involved in several stages of viral replication for the production of viable virus (Ellenbecker et al., 2012). This protein protects the viral genome from degradation and prevents the formation of double stranded RNA (dsRNA) intermediates during replication and transcription by encapsidating viral genomic and antigenomic RNA (Ruigrok et al., 2011). The viral protein 35 (VP35) is a multifunctional dsRNA binding protein that plays important roles in the viral replication, innate immune evasion and pathogenesis of Ebola virus (EBOV). Binning et al. (2013) determine regions of Ebola virus VP35 (eVP35) to target aptamer selection. Based on their interaction properties to eVP35, two distinct classes of aptamers were characterized. The results revealed that the aptamers bind to distinct regions of eVP35 with high affinity and specificity. In addition, these aptamers compete with dsRNA for binding to eVP35 and disturb the eVP35-nucleoprotein interaction inhibiting the function of the EBOV polymerase complex (Binning et al., 2013).

Table SEQ Table * ARABIC 2. Current promising aptamers evaluated in preclinical models for treatment and diagnosis of various viral infections
Virus Aptamer name Type Target Reference
HBV HBs-A22 RNA HBsAg(Liu et al., 2010)
HCV 2-02, 3-07, 0207 and 0702 RNA
IRES element (Kikuchi et al., 2009)
P6-n, HH363-n RNA IRES element (Romero-Lopez et al., 2012)
Class A, B, C and D (ODN n) DNA
NS5B
(Bellecave et al., 2008)
ZE2 DNA glycoprotien E2(Chen et al., 2009)
HPV-16 G5?3N.4 RNA Oncoprotein E7 and E6(Toscano-Garibay et al., 2011)
HSV GC-rich RNA aptamer RNA ICP27 (Corbin-Lickfett et al., 2009)
aptamer-1 and aptamer-5 RNA gD protein (Gopinath et al., 2012)
mini-1aptamer (44-mer) RNA gD protein (Gopinath et al., 2012)
G7a RNA gD protein (Moore et al., 2011)
Influenza virus (H5N1) n.d DNA HA protein (Bai et al., 2012)
Dengue virus S15 DNA DENV-2 envelop protein (Chen et al., 2015)
Rift valley fever virus n.d.  RNA Nucleocapsid protein (Ellenbecker et al., 2012)
Ebola virus 1G8–14, 2F11- 4 RNA VP35 (Binning et al., 2013)
SARS virus n.d. DNA/RNA Nucleocapsid protein (Ahn et al., 2009; Cho et al., 2011)
Rabies virus GE54 DNA Glycoprotein (Liang et al., 2014)
*n.d –not determined

4. NEW THERAPEUTIC INNOVATIONS AND APPLICATIONS
4.1. Aptamers to neutralize lethal viruses
The emergence of new viral threats such as Middle east respiratory syndrome (MERS), dengue, west nile virus (WNV), and most recently the Bourbon virus, may shift some aptamer development efforts to these newer viral threats (Wandtke et al., 2015). It appears clear that high affinity and highly specific aptamers can bind envelope, polymerase and structural viral proteins to inhibit or block fusion, penetration, or replication of viruses (Bruno et al., 2012). Thus, as new viral threats become apparent, aptamer research and development seem likely to go along with viral emergence, because in the absence of effective vaccines or antisera, aptamers may provide a last line of defense. Indeed, in the event of another lethal pandemic influenza or other virus outbreak, aptamers may provide the only line of defense and could be developed mechanically, if necessary to avoid human exposure to the virus, for passive immunity until patients can mount their own immune responses (Lee et al., 2005; Lai et al., 2012).

4.2. Aptamers or aptamer conjugates to kill drug-resistant pathogenic bacteria
The crisis of antibiotic resistance which is already upon the human race demands either new antibiotic development or novel approaches to killing multidrug-resistant (MDR) bacteria in vivo. Recently, carbapenemresistant enterobacteriaceae (CRE) and 
Klebsiella pneumoniae Carbapenemase (KPC) have garnered most of the morbid headlines and high patient mortality while Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococcus, (VRE) continue to be significant problems. Unfortunately, the new drug pipeline for resistant bacteria is scant which has drawn attention to chimeric or humanized monoclonal antibodies to combat drug-resistant bacteria (DiGiandomenico et al., 2014; Saylor et al., 2009).
Similarly, aptamers can possibly be used as bacteriostatic agents to likely suppress the growth of bacteria by simple cell surface binding to decrease membrane potential. Aptamers are being investigated to determine if they can block or degrade key drug-resistant bacterial enzymes, thereby possibly overcoming resistance to beta-lactams and a variety of other antibiotics (Schlesinger et al., 2011). Direct killing of Gram negative bacteria and cancer cells by induction of the classic complement-mediated cell lysis system or opsonization by bifunctional (bidentate linker) aptamers or aptamer-Fc conjugates to emulate antibodies has also been demonstrated (Bruno, 2013).

4.3. Aptamers to neutralize toxins and venoms
To date, several highly specific aptamers have been developed against a variety of different bacterial toxins and snake venoms (Ye et al., 2014). Certainly, snake and other venoms are complex materials which can include phospholipases and other degradative enzymes as well as neurotoxins and cardiotoxins in some cases. These toxic components would all require neutralization by a combination of “polyclonal” aptamers to be effective as a single antivenom product. These opinions inspire to hope that aptamers or their conjugates can be most likely developed to effectively counteract snake, spider, scorpion, insect and other venoms. Antisera for venomous bites require cold storage, but lyophilized aptamers would not require cold storage and could be kept at ambient temperatures (even in desert or harsh environments) for long-term storage as long as humidity was sealed out (Quaak et al., 2010).

4.4. Aptamers to potentially induce stem cell differentiation and trans-differentiation
One very exciting potential application of aptamers could be to induce differentiation or trans-differentiation of stem cells, especially of bone marrow origin, to avoid the controversy surrounding the use of embryonic stem cells. Aptamers have already played a part in the affinity-based capture, isolation, concentration and collection of progenitor cells from various tissues (Guo et al., 2006), proving that whole cell-selected aptamers can be developed that bind stem cells with great affinity. Therefore, they could become agents of choice to replace more expensive recombinant antibodies or growth factors, thereby accelerating the field of regenerative medicine (Haller et al., 2015).

5. LIMITATIONS AND CHALLENGES OF BIOLOGICAL APPLICATOINS OF 
   APTAMERSAlthough aptamers are promising candidates for diagnostic and therapeutic applications, some barriers affecting the therapeutic potency in vivo still remain as they need to go across physiological barriers (i.e. cell membrane) before they reach their targets. Some of the limitations aptamers may face include the rapid clearance rate from circulation due to their small size and degradation by nucleases for the unmodified aptamers. However, these limitations can be easily resolved by chemically modifying the aptamers (Sun and Zu, 2015).
Polymers such as polyethylene glycol PEG or lipids such as cholesterol can be conjugated to the aptamers to enhance aptamer circulation time and increase their in-vivo half-life and pharmacodynamics. Moreover, those PEG-conjugated aptamers confirm higher cellular uptake than the unconjugated form. Modified nucleotides containing altered base, sugar, and internucleotide linkage groups can be used in the aptamer synthesis to increase resistance against nucleases and hence improve aptamer cell uptake and nuclear distribution. The strategies for producing chemically modified aptamers are well-established and can be scaled up for commercial manufacturing (Keefe et al., 2010).

6. CONCLUSIONS AND RECOMMENDATIONSThe unique traits, attractive biological functions and pharmacokinetic attributes of nucleic acid aptamers provide vast potential for class of theranotics towards viral infections. They routinely achieve the same affinities and specificities as therapeutic antibodies, avoid the immunogenicity concerns of protein drugs, and can be generated to a range of targets more efficiently. So that, medical sciences believe that the next generation of aptamers will be particularly useful for therapeutic and diagnostic purposes, including molecular probes, biosensors. However, aptamers have not yet demonstrated their place in the therapeutic arena as their complex syntheses make them more expensive to manufacture than small molecules. Moreover, their still largely unknown pharmacokinetic properties make them harder to develop than any given therapeutic antibody. It is hoped that further efforts to reduce the cost of synthesis, to rationally improve pharmacokinetic properties, and to develop approaches to non-antagonist modes of action will improve therapeutic opportunities.
Therefore, in reference to the above conclusion the following recommendations are pointed out:
An aptamer-based medication should be integrated to the diagnostic and treatment methods of varies microbial and viral infections since this allows more precise real-time monitoring and therapy,
The delivery methods and significance of aptamer applications in clinical and laboratory medicine should be investigated and conducted to facilitate clinical translation of aptamers mediated targeted therapy.

Research studies on the aptamer based technolologies should be carried out in the country in order to allow its successful therapeutic applications against devastating infections.

Therapeutic effects and safety of aptamers should be carefully studied and applied in medical laboratories of the country.

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