Aptamer-Based Therapeutics: New Approaches to Combat Human Viral Diseases

1. Introduction

2. Aptamers as Antiviral Therapeutics

2.1. Inhibition of Human Immunodeficiency Virus-1

HIV-1/AIDS is a chronic disease of the human immune system. Although combination antiretroviral therapy (cART) can slow the progress of the disease and reduce the risk of death and disease complication, it is not curative. Moreover, many patients do not tolerate cART because of its severe side effects, and it is unaffordable for patients in developing countries [56]. In this regard, aptamers have been considered an alternative or adjuvant to the chemical antiviral agents in cART to overcome these limitations [30,57,58]. To date, highly specific, nucleic acid-based aptamers that target various parts of HIV-1 genomes and HIV-1 proteins, including HIV-1 reverse transcriptase (RT), integrase, nucleocapsid, Gag, gp120, TAR, Rev and Tat, have been isolated and shown to effectively suppress viral replication (Figure 1) [57,59]. These aptamers are discussed further below.

2.1.1. HIV-1 Reverse Transcriptase

HIV-1 RT is a key, multifunctional enzyme during the retroviral life cycle. It has a DNA polymerase activity on both RNA and DNA templates and a ribonuclease H activity on RNA-DNA hybrid template [60]. HIV-1 RT catalyzes conversion of the single stranded genomic viral RNA into double stranded proviral DNA, which in turn is integrated into the host genome.

Currently, RT is one of the main drug targets for HIV/AIDS, and many nucleoside inhibitors (e.g., AZT, 3TC, ddI, ddC, d4T) and non-nucleoside inhibitors (e.g., snevirapine, delavirdine, efavirenz) are currently used in patients [61,62].

Table 1. List of representative antiviral aptamers and their properties.

Table 1. List of representative antiviral aptamers and their properties.

Virus	Aptamer Selection Target	Aptamers	Binding Affinity, KD	Inhibitory capabilities	References
HIV	Reverse transcriptase	Pseudoknot; RNA	25 pM	Inhibition of polymerase activity; 95% reduction of HIV particle formation by transient expression of aptamers	[42,63,64]
HIV	Drug resistant Reverse transcriptase mutant 3	M302; N-methyl-isatoicanhydride modified RNA	30 nM	No inhibition of polymerase and RNase H activities	[65]
HIV	Reverse transcriptase	ODN112; G-quadruplex DNA	N.A.	Inhibition of RNase H and polymerase activities with IC50 = 500 nM; Reduction of viral infectivity in cell culture with IC50 < 100 nM	[66]
HIV	RNase H domain of Reverse transcriptase	R12-2; Thiolated DNA aptamer	70 nM	Inhibition of RNase H activity; Reduction of viral infectivity in cell culture with IC50 < 100 nM	[67]
HIV	Reverse transcriptase	37NT; DNA	660 pM	Inhibition of primer-template binding and polymerase activity	[68]
HIV	Reverse transcriptase	RT1t49; DNA	~1 nM	Inhibition of polymerase activity with IC50 = 0.3 nM	[69,70]
HIV	Reverse transcriptase	PF1; DNA	82 nM	Inhibition of polymerase activity with IC50 = 60 nM	[71]
HIV	Reverse transcriptase	RU25-80;	N.A.	Inhibition of primer extension with IC50 = 60 nM and viral replication. Dependent on the nonpseudoknot UCAA motif	[72]
		RNA
HIV	Reverse transcriptase	6/5 asymmetric loop;	N.A.	Inhibition of primer extension in vitro.	[73]
		RNA		Inhibition of viral replication in cell culture
HIV	Integrase	93del; G-quadruplex DNA	N.A.	Inhibition of viral entry, reverse transcriptase and integration activities; Inhibition of cell fusion in cell at 1 µM	[74,75]
HIV	Integrase	T30695; G-quadruplex DNA	N.A.	Inhibition of integrase activities with IC50 < 100 nM	[76]
HIV	DP6 truncated Gag lacking p6 and the	DP6-22; RNA	100 nM	Inhibition of Gag-genomic interactions that negatively affects RNA transcription, processing or stability	[77]
	N-terminal myristate
HIV	Nucleocapsid	N70-13; RNA	0.6 nM	Inhibition of nucleocapsid and HIV psi-RNA interaction	[78]
HIV	Nucleocapsid	N50-20; RNA	0.5 nM	Not Determined	[79]
HIV	Gp120 of HIV strain HXB2	J58; 2' Fluoro modified RNA	210 nM	No neutralization of infection	[80]
HIV	Gp120 of HIV-1BAL	B40; 2' Fluoro modified RNA	21 nM	Inhibition of gp120-CCR5 interactions
HIV	Gp120 of HIV-1BAL	UCLA1 (truncated B40); modified RNA with an inverted thymine at 3' end and a dimethoxyltrityloxy-(CH2)6-SS-(CH2)6-phospholinker at 5' end	150 pM	Neutralization of isolated of R5 strains with IC50 < 100 nM;	[81]
				Synergistic effect with a gp41 fusion inhibitor
				and an anti-CD4 antibody
HIV	Gp120 of HIV-1BAL	A-1; 2' Fluoro modified RNA	52 nM	Conjugation with siRNAs targeting HIV tat/rev and TNPO3 to inhibit HIV-1 infection in primary human peripheral blood mononuclear cells and humanized mice	[82,83]
HIV	Tat-1- derived peptide	RNA Tat; RNA	120 pM	Competition with TAR for sequestering Tat-1 in cell culture	[84]
HIV	Rev	RBE(apt); RNA	N.A.	Conjugation with ribozyme targeting HIV Env for gene therapy	[85]
HIV	TAR	IV04; 2'-O-Methyl modified DNA	20 nM	Disruption of TAR secondary structure by formation of RNA-DNA kissing complexes	[86]
HIV	TAR	R-0624; RNA	32 nM	Formation of TAR RNA-aptamer complexes by 8-nt complementary base-pairing	[87]
HIV	TAR	a1.16; RNA	17 nM	Formation of TAR RNA-aptamer complexes by 5-nt complementary base-pairing	[88]
HIV	TAR	B22; DNA	50 nM	No inhibition of TAR function	[89]
HCV	NS3	G6-16; RNA	238 nM	Inhibition of protease activity with IC50 = 3 µM	[90]
HCV	Truncated protease domain of NS3	G9-I; RNA	10 nM	Inhibition of protease activity with IC50 = 100 nM	[91]
HCV	Helicase domain of NS3	G5; RNA	25 nM	Inhibition of helicase activity with IC50 = 50 nM	[92]
HCV	NS5B∆C55	B.2; RNA	1.5 nM	Inhibition of HCV RNA polymerase in a non-competitive manner; IC50 = 10 nM	[93]
HCV	NS5B	27v; DNA	132 nM	Competition with the RNA template for binding to the RNA polymerase and blocked both the initiation and the elongation of RNA synthesis; IC50 = 190 nM	[94]
HCV	NS5B	R-F t1; 2' Fluoro modified RNA	8 nM	Competition with the RNA template for binding to the RNA polymerase and blocked both the initiation and the elongation of RNA synthesis	[95]
HCV	NS5B	P-58; RNA	570 nM	Interference with HCV replication by targeting the essential 5BSL3.2 domain within the cis-acting replication element; IC50 =185 nM	[96]
HCV	Domain II of IRES	2-02; RNA	11 nM	Inhibition of IRES-dependent translation	[97]
HCV	Domain III-IV of IRES	3-07; RNA	9 nM	Inhibition of IRES-dependent translation	[98]
HCV	Entire IRES	AP50; RNA	5 nM	Inhibition of IRES-dependent translation	[99,100]
HBV	Surface antigen	HBs-A22;RNA	N.A.	Inhibition of receptor binding	[101]
HBV	Truncated polymerase protein	S9; RNA	N.A.	Competition with RNA to inhibit P protein binding to	[102]
				ε signal on pgRNA
SCoV	Helicase	NG8; Modified DNA	5 nM	Inhibition of nucleic acid unwinding activity; IC50 = 91.0 nM	[103]
SCoV	Helicase	ES15; RNA	N.A.	Inhibition of nucleic acid unwinding activity; IC50 = 1.2 nM	[104]
Influenza A virus	Hemagglutinin [HA-(91-261) from H3N2]	A22; DNA	N.A.	Inhibition of receptor binding; 95% reduction of viruses in mice	[105]
Influenza A virus	Hemagglutinin	A10; DNA	Strong binding	Inhibition of receptor binding, dose-dependent inhibition demonstrated	[106]
	(HA from H5N1)		measured by ELISA
Influenza A virus	Whole H3N2 virus	P30-10-16;	0.2 nM	Inhibition of receptor binding; 95% inhibition of viral fusion efficiencies in the presence of 5 µM aptamers	[107,108]
	[A/Panama(H3N2) strain]	RNA
Influenza A virus	Hemagglutinin	HA68; DNA	7 nM	Inhibition of receptor binding; complete inhibition of the agglutination of RBC in the presence of 2.5 µM aptamers	[109]
	(HA from H3N2)
Influenza A virus	Hemagglutinin	C7; DNA	Strong binding	Inhibition of receptor binding; 55% inhibition of the viral infection	[110]
	[HA-(101-257) from H9N2]		measured by ELISA
				at 1nmol in the cell viability assay
Influenza A virus	Hemagglutinin	HAS15-5; RNA	Strong binding	Inhibition of receptor binding	[111]
	(HA from H5N1)		using RT-PCR
Influenza A virus	Hemagglutinin	D-26, 2' Fluoro modified RNA	67 fM	Inhibition of receptor binding, Complete inhibition of the agglutination of RBC in the presence of 200 nM aptamer
	(HA from H1N1)
Influenza B virus	Hemagglutinin (HA from B/Johannesburg strain)	A20; RNA	0.7 nM	Inhibition of receptor binding; 93% inhibition of viral fusion efficiencies in the presence of 25 µM aptamers	[112]
Rabies virus	Rabies virus (CVS-11) infected BHK-21 cells	F34; DNA	28 nM	Inhibition of CVS-11 infectivity with a dose-dependent manner	[113]
Rabies virus	Rabies virus (CVS-11) infected BHK-21 cells	PEG-FO24; PEGylated DNA	N.A.	87.5% survival rate of mice inoculated with aptamers for 24 h prior to challenge with rabies virus	[114]
HPV	HPV16 E7	G5α3N.4; RNA	1.9 µM	N.A.	[115]
HPV	HPV transformed HeLa cells	Aptamer 20, DNA	1 nM	N.A.	[116]
HPV	HPV16 E7	A2; RNA	107 nM	Induction of apoptosis in HPV infected cancer cells	[117,118]
HSV-1	Glycoprotein D protein	Aptamer-1; 2' Fluoro modified RNA	170 nM	Inhibition of gD protein and HSV-1	[119]
				target cell receptor (HVEM) interactions, EC50 = 60 nM
HSV-2	Glycoprotein D protein	G7a; 2' Fluoro modified RNA	N.A.	Inhibition of receptor binding, IC50 = 20 nM	[120]

Figure 1. (A) HIV-1 genome and (B) HIV-1 virion and potential antiviral targets.

In 1992, Tuerk and Gold identified RNA aptamers against HIV-1 RT from an RNA pool that spanned a 32-nt random region by using in vitro SELEX [42]. The selected anti-RT aptamer contained a consensus sequence that resulted in the formation of an RNA pseudoknot. This RNA pseudoknot was reported to bind to HIV-1 RT in the picomolar range [63] and had an inhibitory effect on HIV-1 replication [64,121,122]. Since this discovery, numerous RNA or DNA aptamers of various lengths have been raised against HIV-1 RT and their uses for inhibiting the virus replication have been explored. For example, single stranded DNA aptamers (ODN112) with high affinity for the RNase H domain of HIV-1 RT were isolated by using recombinant RTs [66]. The selected DNA ligands could greatly diminish the infectivity of HIV-1 in human cells [66]. Similarly, double-stranded DNA thioaptamers (R12-2) that contained thiophosphate backbones were selected against the RNase H domain of HIV-1 RT by using in vitro combinatorial selection methods [67]. One lead thioaptamer could specifically bind to HIV-1 RT (dissociation constant (K_D) of 70 nM) and significantly inhibited HIV-1 infection in a dose-dependent manner [67]. In addition to aptamers against wild-type (WT) HIV-1 RT, several RNA aptamers targeting a drug-resistant HIV-1 RT, mutant 3 (M3) have been isolated [65]. One of these aptamers, M302, bound M3, but did not have significant affinity for WT HIV-1 RT, which would likely allow specific detection of HIV-1 RT variants [65].

Anti-RT aptamers have been intracellularly expressed together with flanking, self-cleaving ribozymes to generate aptamer RNA transcripts that have minimal flanking sequences [122]. The expression of aptamers in the HIV-1 infected cell led to encapsidation of the aptamer in the virion particles, which subsequently blocked the HIV-1 replication [122]. These aptamers also effectively suppressed drug-resistant variants and other HIV subtypes (e.g., subtypes A, B, D, E, and F) [122]. More recently, Lange et al. further optimized this expression cassette, in which an extended, tertiary-stabilized hammerhead ribozyme was replaced to enhance its self-cleavage activity [123]. Stable clonal cell lines that expressed aptamers from these optimized constructs strongly suppressed infectious virus when used at a high multiplicity of infection (MOI) [123].

2.1.2. HIV-1 Integrase

HIV-1 integrase (IN), a retrovirus encoded enzyme, catalyzes the insertion of proviral DNA into the host-cell genome [124]. Because HIV-1 IN is essential for retrovirus replication, it is a promising target for the development of antiretroviral drugs.

Soultrait et al. isolated several DNA aptamers (ODN93 and ODN112) that could strongly inhibit the RNase H activity associated with HIV-1 RT [74]. Because X-ray structure analysis indicated strong similarities between the RNase H domain of HIV-1 RT and the catalytic core of HIV-1 IN, the authors investigated if the truncated version of DNA aptamers against HIV-1 RT RNase H (ODN93del and ODN112del) could also bind to HIV-1 IN [125] and found that that was the case. The DNA anti-RT aptamers efficiently blocked the processing and strand transfer activity of HIV-1 IN when used at nanomolar concentrations in vitro and abolished HIV-1 replication in infected human cells [75]. Structural studies revealed the DNA aptamers contained G-rich sequences that could form stable and non-canonical G-quadruplex structure. A docking-based model of the OND93del-IN complex suggested that the DNA aptamer was located within a channel of the tetrameric integrase [126]. Because this mutual fitting could block several catalytic amino acid residues that are essential for integrase function, the binding site of the OND93del aptamer may contribute to its anti-HIV-1 activity. Additionally, various G-quadruplex aptamers (e.g., T30695 and AID-1) have proven effective for inhibiting HIV-1 IN in vitro, and are therefore regarded as potential anti-HIV therapeutic agents [127,128,129].

2.1.3. HIV-1 Gag Protein

Gag polyprotein is a major HIV-1 structural protein that is synthesized in the cytoplasm of infected cells, orchestrates the assembly and release of HIV-1 particles, and is necessary and sufficient for the formation of noninfectious virus-like particles [130,131]. It is involved in both assembly and virion maturation after particle release, as well as early post-entry steps in virus replication. During virion maturation, Gag is cleaved into several component proteins: matrix protein (MA), capsid protein (CA), nucleocapsid protein (NC), the late domain (p6), and two small, spacer peptides, SP1 and SP2 [132]. These proteins play specific roles in the life cycle of HIV-1, and mutations in any domain of Gag lead to defects in particle assembly and loss of infectivity.

The abilities of RNA aptamers specific to the Gag polyprotein to inhibit HIV-1 replication have been explored [77]. Ramalingam et al. selected RNA aptamers against an HIV-1 Gag protein, lacking the N-terminal myristate and the C-terminal p6 (DP-6 Gag). The selected aptamers (DP6-22) showed strong binding affinity to the MA, NC or entire Gag protein. Binding studies revealed that the “NC-binder” could specifically compete with the packaging signal (ψ) of HIV-1 for binding to DP6-Gag [77]. Moreover, NC-binding aptamers disrupted Gag-genomic RNA interaction and negatively affected genomic RNA transcription, processing, or stability. The NC protein of HIV-1 plays an important role in encapsidating of viral RNA and assembling viral particles [133]. The NC protein specifically recognizes the viral genomic RNA through specific binding to the viral psi (ψ) sequences, and thus selectively encapsidates the viral RNA among different cellular RNA pools [134]. The NC is highly conserved and therefore is an excellent target for anti-viral therapy.

Kim et al. described the selection and stabilization of RNA aptamers against HIV-1 NC [78]. Surface plasmon resonance (SPR) analysis and gel shift assays showed that their selected RNA aptamer specifically bound to target NC protein with high affinity and competed with psi RNA binding to the NC protein [78]. Furthermore, the linear aptamer was tailored to form circular RNA, after which it showed higher stability and the same binding affinity to NC protein, thus providing a valuable inhibitor for viral packaging. In a similar study, an anti-NC RNA aptamer (N70-13) was shown to completely abolish NC binding to the stable trans-activation response hairpin and psi RNA stem-loops of HIV-1 RNA. When this aptamer was expressed in cells, it suppressed the packaging of viral genomic RNA [79,135].

2.1.4. HIV-1 gp120 Protein

The envelope glycoprotein of HIV consists of an exterior glycoprotein (gp120) and a transmembrane domain (gp41) and plays an important role in viral entry into cells [136]. HIV-1 infection is initiated by interactions between HIV-1 gp120 and the human cell surface receptor CD4, subsequently leading to fusion of the viral membrane with the target cell membrane [137,138]. This interaction suggests HIV-1 gp120 as another potential therapeutic target to block HIV-1 infection.

RNA aptamers that bind the HIV-1 gp120 protein have been shown to neutralize HIV-1. For example, using SPR, James and colleagues developed several 2'-F-modified anti-HIV gp120 RNA aptamers that block the interaction of gp120 and CD4 [80,139]. Moreover, mutation analysis and functional studies of these aptamers demonstrated that they specifically interact with the conserved coreceptor region of an R5 stain derived gp120 and neutralize HIV-1 infectivity in human peripheral blood mononuclear cells [140,141]. The selected aptamers suppressed the infectivity of R5 clinical isolates of HIV-1 derived from group M (subtypes A, C, D, E and F) and group O. In addition, the researchers conducted a detailed characterization of one neutralizing aptamer, B40 (117-nt). They found the minimal region of the aptamer essential for binding gp120 comprises a three-way junction linking two helix-loops and a closing helix [141]. Accordingly, a 77-nt truncated version of the aptamer (B40t77) was designed, which efficiently prevented gp120 from interacting with host cells [142]. Recently, several shortened synthetic derivatives of the B40 aptamer were designed and assessed for their anti-viral activity. One such aptamer, UCLA1, tightly bound to a consensus HIV-1 subtype C gp120 and neutralized isolates of the same subtype with 50% inhibitory concentrations (IC₅₀) in the nanomolar range and without causing toxicity in tested primary cells [81]. Furthermore, when UCLA1 was combined with T20, a gp41 fusion inhibitor, and IgG1b12, an anti-CD4 binding site monoclonal antibody, a synergistic effect was observed, suggesting UCLA1 may be a potential adjuvant anti-HIV agent [81].

Recently, using a nitrocellulose membrane based in vitro SELEX procedure on an RNA library, our group also succeeded in selecting several 2'-F substituted RNA aptamers that bind to HIV-1_Bal gp120 protein with nanomolar affinity [83]. The evolved anti-HIV-1 gp120 aptamers efficiently inhibited the replication of CXCR4-tropic (NL4-3 and IIIB) and CCR5-tropic (BaL and JRFL) HIV-1, as well as several clinical HIV-1 isolates [50], and were specifically internalized by cells that expressed HIV-1 gp120. By taking advantage of the gp120 binding properties of an anti-gp120 aptamer, A-1, we tested the concept of using the gp120 aptamer to deliver anti-HIV-1 small interfering (si)RNAs into HIV-1 infected cells, with the goals to enhance therapeutic efficacy and reduce the off-target effect of siRNAs. siRNA is being exploited as a new class of medicine for a variety of diseases to inhibit expression of complementary RNA transcripts, but its clinical translation is dampened by specific cell delivery and internalization. In the chimera approach, we made two designs of gp120 aptamer-Dicer substrate siRNA (DsiRNA) conjugates for cell type-specific siRNA delivery [83]: A covalent aptamer-siRNA chimera and a non-covalent aptamer-stick-siRNA chimera (Figure 2). In the covalent aptamer-siRNA chimera (Figure 2A), we first identified that the anti-gp120 aptamer provided HIV-1 neutralizing activity, even with the siRNA conjugation, and served as a vehicle for selectively delivering anti-HIV-1 siRNAs into HIV-1 infected cells [50]. The activities of the anti-gp120 aptamer-siRNA chimera were further evaluated in a humanized RAG-hu mouse model of HIV-1 infection. The chimera significantly reduced the plasma viral load and maintained CD4 levels equivalent to those of uninfected control mice [143]. In the case of the aptamer-stick-siRNA chimera (Figure 2B), a single gp120 aptamer was used in combination with three different siRNAs that targeted HIV-1 tat/rev transcripts and HIV-1 host dependency factors (CD4 and TNPO3), resulting in knockdown of target mRNAs and potent inhibition of HIV-1 replication, as well as protection against viral-induced CD4+ T-cell depletion in vivo [83,144]. The advantage of the stick design facilitated the effective interchange of different siRNAs with a single aptamer, which is required to avert viral resistance to the siRNA component [24]. A follow-up treatment with the aptamer-cocktail DsiRNA conjugates after viral rebound resulted in complete, long-term suppression of HIV-1 viral loads, suggesting a facile, targeted approach for combinatorial delivery of antiviral and host siRNAs for HIV-1 therapy in vivo [50,144].

2.1.5. HIV-1 Tat Protein

The HIV-1 viral trans-activator Tat protein consists of 86 amino acids, binds to specific regulatory elements (trans activation responsive [TAR] elements) in the HIV-1 long-terminal repeats (LTRs) and regulates viral transcription [145,146]. Tat also influences the growth and metabolism of host cells, and is important for the efficient reverse transcription of HIV-1 [147]. A 37-mer RNA aptamer, RNA^Tat that bound efficiently to the Tat protein of HIV-1, was isolated through in vitro selection procedure [84]. Compared to authentic TAR RNA, the dissociation constant of RNA^Tat to Tat was 133-times higher [84]. Because the RNA^Tat aptamers showed high affinity to Tat and did not interact with cellular proteins, it is likely to be a more efficient and suitable decoy than cellular TAR RNA, which interacts with several cellular factors within cells and inhibits the transcription of various genes [84]. The results demonstrated that the RNA^Tat aptamers could specifically prevent Tat-dependent trans-activation both in vitro and in vivo.

Figure 2. (A) Aptamer-siRNA conjugates. The 2'-F-modified gp120 aptamer was covalently appended to the sense strand of a tat/rev siRNA portion, which in turn was hybridized to the antisense strand. A 4-nt linker (CUCU) was inserted between the aptamer and siRNA portions to minimize steric interference of the gp120 aptamer with Dicer processing [143]. (B) Aptamer-stick-siRNA chimeras. The 2'-F-modified gp120 aptamer and the siRNAs are shown. The antisense of the siRNA is linked to the aptamer portion by the stick sequence, which consists of 16 nt appended to the 3' end of the gp120 aptamer, allowing complementary base-pairing of one of the two siRNA strands with the aptamers [83].

2.1.6. HIV-1 Rev Protein

HIV-1 Rev, a virally encoded sequence-specific RNA-binding protein, plays a critical role in the nuclear export of intron-containing HIV-1 RNA. Rev shuttles between the nucleus and the cytoplasm, and harbors both a nuclear localization signal and a nuclear export signal [148,149]. Rev directly binds to unspliced and incompletely spliced viral RNA through the cis-acting Rev response element sequence [150].

RNA aptamers have been raised against the HIV-1 Rev protein through in vitro evolution [18]. Structural analysis and chemical modifications of these aptamers revealed that two major regions of the RNA aptamers were responsible for interacting with the Rev protein. These regions might provide a starting point for developing therapeutic nucleic acids for HIV-1 therapy [85]. Furthermore, the Rev-binding aptamer and the anti-Env ribozyme were constructed with the RSV and CMV promoters, which have a high transcription rate, produce a stable transcript and localize the transcript within the cytoplasmic compartment [151]. It is expected that the resulting cassette could effectively suppress HIV-1 production from HIV proviral clones.

2.1.7. HIV-1 TAR Element

The HIV-1 TAR element, a 59-nt-long stem loop present at the 5' end of HIV-1 RNA, mediates the trans-activation of transcription through binding of the viral protein Tat [152]. The interaction between TAR RNA and Tat protein increases the processivity of the RNA polymerase, allowing high yield synthesis of the full-length retroviral genome [149,153]. Therefore, the binding of an oligonucleotide ligand (such as an aptamer) could be able to compete with the interaction between TAR RNA and Tat, which could prevent transcription.

Boiziau et al. performed in vitro selection to identify ligands specific to HIV-1 TAR [86]. After 15 rounds of selection-amplification, several DNA aptamers were isolated. Structural analysis revealed that the selected DNA aptamers (IV04) could fold into imperfect stem-loop structures and presented a consensus motif (5'-ACTCCCAT-3') [86]. The six central bases of the consensus were complementary to the TAR apical region, allowing the formation of RNA-DNA kissing complexes in the presence of 10 mM magnesium [86]. These complexes did not interfere with TAR function and were not observed when the magnesium concentration was lowered to a more physiological concentration (3 mM). It was supposed that the aptamer that had a lower binding affinity could not efficiently compete with the viral or cellular proteins that induce transcription. Therefore, the researchers added a counter-selection step to eliminate those binders that had lower affinity [89]. One of the aptamers could be further shortened to a 19-mer DNA and had a dissociation constant of 50 nM, but their inhibitory capabilities were not significantly altered [89].

Similarly, RNA aptamers against HIV-1 TAR have also been developed [87]. The selected sequences (R06₂₄) folded into imperfect hairpins and displayed a consensus motif (5'-GUCCCAGA-3') that was complementary to the entire TAR loop, leading to the formation of TAR RNA-aptamer “kissing complexes” [87]. The lower nanomolar dissociation constant (K_D = 30 nM at 3 mM magnesium) of the selected RNA aptamers was attributed to closing of the aptamer loop by the consensus G and A residues [87]. Moreover, anti-TAR RNA aptamers were isolated from a genomic human library [88]. Theoretically, a genomic SELEX library contains all cellular RNA transcripts that can identify putative functional interactions involving RNA motifs [154]. The genomic SELEX is useful when studying for RNA-RNA interaction and searching for RNAs that might be expressed in a very low level. In the case of TAR aptamers, one of the selected aptamers, (a1), formed a complex with TAR through a kissing interaction and had ~4-fold lower dissociation constant than that of a previously identified aptamer, R06 (K_D: 4 nM vs. 17 nM) [88].

Virus-resistant transgenic T cells and macrophages that express HIV-1 TAR aptamer either alone or in combination with others nucleic acid therapeutics have been produced by lentiviral gene transduction of CD34⁺ progenitor cells. When the differentiated T lymphocytes and macrophages were challenged with HIV-1, marked resistances against HIV-1 infection was seen [58]. For example, a triple combination lentiviral construct composed of a U6-driven TAR RNA decoy, a U6-promoted HIV-1 tat/rev shRNA, and a VA1-promoted anti-CCR5 trans-cleaving hammerhead ribozyme efficiently transduced human progenitor CD34⁺ cells [155]. These transduced cells had increased suppression of HIV-1 over 42 days when compared to cells that received a single anti-tat/rev shRNA or double combinations of shRNA/ribozyme or decoy [155]. Recently, this triple construct has been used for ex vivo gene delivery to hematopoietic stem cells in a human clinical trial [156]. The lentivirally transduced cells successfully engrafted in all four infused patients by day 11 and no unexpected infusion-related toxicities were seen [156]. Long-term (up to 24 months) expression of an ectopically expressed shRNA and ribozyme in multiple peripheral blood cell lineages of two of the transplanted patients was observed [156].

2.2. Inhibition of Hepatitis C Virus

Hepatitis C infection is a serious public health problem that chronically affects 170 million people worldwide [157]. Infection with hepatitis C virus (HCV) places individuals at high risk for scarring of the liver and ultimately leads to cirrhosis and liver cancer. HCV has limited therapeutic options, including a combination therapy of PEGylated interferon-α and ribavirin, which is not effective against certain HCV genotypes [158]. Thus, there is a need for new HCV therapies. The HCV is a 9.6 kb enveloped virus with a positive linear, single stranded sense RNA genome that contains a 5' non-translated region (NTR), protein coding region and 3' NTR for viral replication [159]. The 5' coding region consists of a single polyprotein of 3,010 amino acids, which is further processed by viral and cellular proteases into structural (C, E1, E2 and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins [159]. Because some of these proteins are considered important for viral replication and proliferation, they are prime targets for the development of antiviral therapies.

2.2.1. HCV Non-Structural Protein (NS) 3

The NS3 protein of HCV is a multi-functional enzyme that has two distinct domains: an amino (N)-terminal with trypsin-like serine protease activity that comprises one-third of the protein, and a carboxyl (C)-terminal with an NTP-dependent RNA/DNA helicase unwinding activity in the remaining two thirds. Generally, the protease activity of the NS3 cleaves the junctions between the non-structural proteins [160], while the helicase activity unwinds the double-stranded RNA generated by the RNA-dependent RNA polymerase NS5B during genome replication [161]. Because NS3 is essential for the HCV life cycle, many aptamers have been evolved against NS3 as potential anti-HCV agents. Kumar et al. isolated RNA aptamers from a randomized pool comprising 120 bases [90]. The selected aptamers, G6-16, bound specifically to the protease domain of NS3 and inhibited its activity in vitro [90]. Evaluation of enzyme kinetics revealed that the G6-16 aptamers inhibited the protease activity in a mixed (competitive and non-competitive) fashion with respect to the substrate [90]. Interestingly, the aptamers moderately inhibited the helicase activity of NS3, even though the aptamer bound to the protease domain of NS3 [90]. Although these aptamers have potential to serve as a dual-function inhibitor, their inhibitory concentrations were high (K_i = 3 µM) and appear to be significantly higher than those of other aptamers that target other proteins.

Because the G6-16 aptamers had low efficacy, a new set of aptamers against the NS3 proteins was re-evolved, using a slightly modified selection strategy [91]. A truncated protease domain of NS3 was used as a selection target instead of the entire NS3 protein, and a 30-nucleotide randomized core region was used for SELEX instead of a 120-nucleotide randomized core region [91]. Consequently, three classes of conserved aptamers were identified after 9 rounds of selection. The dissociation constant of these aptamers was about 10 nM, which could inhibit 90% of the protease activity of both truncated NS3 and entire NS3 in a non-competitive manner [91]. Mutational analysis of the aptamer G9-1 revealed that the sequence required for protease inhibition was in stem I, stem III and loop III of the aptamers [91]. Use of these aptamers in vivo required that they may be expressed intracellularly. Therefore, an in vivo aptamer expression system was designed that combined the G9-1 aptamer with cis-acting genomic human hepatitis delta virus (HDV) ribozymes to construct a chimeric HDV-ribozyme-G9-aptamer [162]. The G9-1 aptamer was inserted into the non-functional stem IV region of the HDV ribozyme so that the aptamer would be protected from exonuclease degradation in the presence of the tightly packed structure of the HDV ribozyme and hence remains stable and functional in cells [162]. Furthermore, the chimera was attached with a nuclear export signal (CTE-M45) and arrayed as a tandem repeat in the expression plasmid in order to increase its cytoplasmic level in cells. Evaluation of this expression system in cultured cells showed efficient protease inhibition activities similar to the aptamer G9-1 alone [162].

The HCV NS3 helicase has been reported to preferentially bind to poly U sequence. Therefore, Fukuda et al. added a poly U tail to the 3' end of the G9 aptamer [163]. Addition of the poly U tail not only increased binding and protease inhibition, but also resulted in an additional ability to inhibit the helicase. As compared with the original G9 aptamer, the modified G9-poly U aptamer had more potent protease activities and efficiently inhibited unwinding of DNA by the NS3 helicase [163]. The poly U tail is similar to a decoy that masks the substrate binding site of the NS3 helicase. Unfortunately, the poly U tail had only a moderate IC₅₀ (1 µM) for inhibiting the NS3 helicase. Therefore, another SELEX against the HCV NS3 helicase domain was performed to identify a specific HCV NS3 helicase aptamer with and improved IC₅₀ [92]. After 8 rounds of selection, aptamer 5 most strongly inhibited helicase activity in vitro (IC₅₀ of 50 nM) [92]. Although HCV NS3 helicase uses NTPs as an energy source to unwind nucleic acids, only the helicase activity of the protein was inhibited by the aptamer 5, while the NTPase activity was not affected, suggesting that the aptamer may bind to the nucleic acid binding site of the helicase to block the unwinding activity [92]. Furthermore, given the identification of specific and effective aptamers against the protease and helicase domains of HCV NS3, a dual-function nanoparticle was created by conjugating the protease aptamer G9 and the helicase aptamer 5 through a poly U spacer [164]. The distance between these two aptamers was carefully adjusted. The resulting chimera, G925-s50, was more effective at inhibiting NS3 protease/helicase activity in vitro. In particular, the helicase activity had four-fold greater inhibition when compared with aptamer 5 alone [164]. Cell culture experiments further demonstrated that the G925-s50 chimera effectively inhibited NS3 protease activity in living cells and in an HCV genome replication system [164].

2.2.2. HCV Non-Structural Protein 5B

The NS5B viral polymerase of HCV is an RNA-dependent RNA polymerase that is essential for transcribing the full length of the HCV genome [165]. During synthesis of viral RNA, NS5B synthesizes a negative strand RNA that serves as a template for the synthesis of new positive RNA strands. This essential role of NS5B in the HCV life cycle has made it attractive for the development of anti-HCV agents. Biroccio et al. isolated an RNA aptamer against a truncated NS5B protein that lacked the C-terminal membrane localization signal (NS5B-∆C55) [93]. Deletion of this C-terminal sequence helped to enhance stability and catalytic activity of the protein to improve the SELEX procedure. The selected aptamers shared a consensus sequence that can fold into a stem-loop structure [93]. In particular, one aptamer, B.2, bound to the NS5B protein with high affinity and non-competitively inhibited the activity of NS5B polymerase with respect to template RNA [93]. This observation suggested that the B.2 aptamers and template RNA did not bind to the same region of the protein. Similarly, Bellecave et al. identified two DNA aptamers (27v and 127v) that specifically bound to NS5B and inhibited its polymerase activity [94]. However, these aptamers used two different mechanisms to inhibit the enzyme. The 27v aptamer competed with template RNA and inhibited both steps of RNA synthesis (i.e., initiation and elongation), while the 127v aptamer competed poorly with the RNA template and inhibited only initiation [94]. Further cell culture studies revealed that the 27v aptamer could inhibit viral RNA synthesis and interfered with viral particle production in HCV JFH1 infected Huh7 cells [94].

Other HCV NS5B aptamers include 2' F modified aptamers evolved by Lee et al. Transfection of this aptamer into cells significantly suppressed replication of both HCV genotype 1b and 2a, but did not generate escape mutant viruses or cause cellular toxicity [95]. This potent aptamer was further modified through conjugation of cholesterol or galactose-polyethylene glycol ligands to increase its stability and specificity for the liver.

2.2.3. HCV Internal Ribosome Entry Site

The internal ribosome entry site (IRES) is a highly conserved and complex RNA structure located at the 5' non-translated region of the HCV genome. The HCV IRES is associated with the host cell small ribosomal unit (40S) and eukaryotic initiation factor 3 (eIF3) to mediate translation initiation of the HCV genome in a 5' cap independent manner [166]. The IRES RNA has four conserved domains (I-IV) for which the structure-function relationships are well-understood (Figure 3) [167]. To identify an RNA aptamer against domain II of IRES, Kikuchi et al. used a biotinylated DNA probe that was complementary to the 5' extension of domain II to fix the IRES on streptavidin magnetic beads [97]. After 4 rounds of selection, almost all clones contained the conserved sequence 5'-UAUGGCU-3', which is complementary to the apical loop of domain II [97]. This observation suggested that the apical loop of domain II may be exposed to interact with cellular molecules such that it is accessible to contact with aptamers. One aptamer, 2-02, had a low dissociation constant to IRES domain II (K_D of 11 nM), but this aptamer only moderately inhibited the IRES-dependent translation [97]. Similarly, the research group isolated RNA aptamers capable of binding to domain III-IV [98]. These aptamers also shared the conserved sequence 5'-ACCCA-3', which is complementary to the apical loop of domain IIId [98]. This loop is known to be a critical region for IRES-dependent translation. Notably, the 3-07 aptamer showed very high affinity to domain III and strongly inhibited the IRES-dependent translation of firefly-luciferase mRNA in mammalian cells [98]. Furthermore, conjugates of domain II-specific 2-02 aptamers and domain III-specific 3-07 aptamers not only bound to the IRES more efficiently than the two parental aptamers, but also inhibited IRES-dependent translation about 10 times as efficiently as the 3-07 aptamer [168].

Figure 3. Representation of the secondary structure of HCV IRES. Structural domains are shown as I–IV. The HH363 cleavage site is indicated by an arrow.

Romero-Lopez et al. developed an innovative aptamer-ribozyme chimera that comprised an aptamer targeted to domain IV of the IRES and a hammerhead ribozyme that targeted the HCV genome [99]. To identify a chimera that contained both cleaving and binding activity, an RNA library pool was first designed on the basis of the well-characterized hammerhead ribozyme that cleaved at the position 363 of the HCV genome [99]. A 25-nt long randomized region was attached at the 3' end of the ribozyme. Identification of potent chimera then involved two steps: selection of aptamers that specifically bound to the IRES and identification of high-affinity aptamers that retained catalytic activity for cleaving HCV RNA [99]. Consequently, a chimeric molecule (HH363-50) composed of two inhibitors was identified. HH363-50 actively functioned as a chimeric inhibitor in vitro, suggesting that conjugation of the aptamer and ribozyme domains did not cause any loss of function [100]. The aptamer domain bound to domain IV of IRES and interfered with formation of a productive 80S ribosomal complex, while the ribozyme domain reduced the HCV RNA levels in a subgenomic replicon system by cleaving the viral genome [100]. These studies confirmed the success of combining different functional nucleic acids to produce dual-function inhibitors.

3. Conclusions

References

1. Haasnoot, J.; Berkhout, B. Nucleic acids-based therapeutics in the battle against pathogenic viruses. Handb. Exp. Pharmacol. 2009, 189, 243–263. [Google Scholar] [CrossRef]
2. Mescalchin, A.; Restle, T. Oligomeric nucleic acids as antivirals. Molecules 2011, 16, 1271–1296. [Google Scholar] [CrossRef]
3. Alter, G.; Heckerman, D.; Schneidewind, A.; Fadda, L.; Kadie, C.M.; Carlson, J.M.; Oniangue-Ndza, C.; Martin, M.; Li, B.; Khakoo, S.I.; et al. HIV-1 adaptation to NK-cell-mediated immune pressure. Nature 2011, 476, 96–100. [Google Scholar] [CrossRef]
4. Ding, Q.; Cao, X.; Lu, J.; Huang, B.; Liu, Y.J.; Kato, N.; Shu, H.B.; Zhong, J. Hepatitis C virus NS4B blocks the interaction of STING and TBK1 to evade host innate immunity. J. Hepatol. 2013, 59, 52–58. [Google Scholar] [CrossRef]
5. Arts, E.J.; Hazuda, D.J. HIV-1 antiretroviral drug therapy. Cold Spring Harb. Perspect. Med. 2012, 2, a007161. [Google Scholar]
6. Gopinath, S.C. Antiviral aptamers. Arch. Virol. 2007, 152, 2137–2157. [Google Scholar] [CrossRef]
7. Jordheim, L.P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug Discov. 2013, 12, 447–464. [Google Scholar] [CrossRef]
8. Jones, M.; Nunez, M. Liver toxicity of antiretroviral drugs. Semin. Liver Dis. 2012, 32, 167–176. [Google Scholar] [CrossRef]
9. Preston, B.D.; Poiesz, B.J.; Loeb, L.A. Fidelity of HIV-1 reverse transcriptase. Science 1988, 242, 1168–1171. [Google Scholar]
10. Brown, A.J.; Cleland, A. Independent evolution of the env and pol genes of HIV-1 during zidovudine therapy. AIDS 1996, 10, 1067–1073. [Google Scholar]
11. Kwong, A.D.; Rao, B.G.; Jeang, K.T. Viral and cellular RNA helicases as antiviral targets. Nat. Rev. Drug Discov. 2005, 4, 845–853. [Google Scholar] [CrossRef]
12. Kuritzkes, D.R. Drug resistance in HIV-1. Curr. Opin. Virol. 2011, 1, 582–589. [Google Scholar] [CrossRef]
13. Garcia-Lerma, J.G.; Nidtha, S.; Blumoff, K.; Weinstock, H.; Heneine, W. Increased ability for selection of zidovudine resistance in a distinct class of wild-type HIV-1 from drug-naive persons. Proc. Natl. Acad. Sci. USA 2001, 98, 13907–13912. [Google Scholar] [CrossRef]
14. Schmitz, S.H.; Scheding, S.; Voliotis, D.; Rasokat, H.; Diehl, V.; Schrappe, M. Side effects of AZT prophylaxis after occupational exposure to HIV-infected blood. Ann. Hematol. 1994, 69, 135–138. [Google Scholar] [CrossRef]
15. Kuehl, A.K.; Noormohamed, S.E. Recombinant erythropoietin for zidovudine-induced anemia in AIDS. Ann. Pharmacother. 1995, 29, 778–779. [Google Scholar]
16. Rachlis, A.; Fanning, M.M. Zidovudine toxicity. Clinical features and management. Drug Saf. 1993, 8, 312–320. [Google Scholar] [CrossRef]
17. Ellington, A.D.; Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818–822. [Google Scholar] [CrossRef]
18. Tuerk, C.; MacDougal-Waugh, S. In vitro evolution of functional nucleic acids: High-affinity RNA ligands of HIV-1 proteins. Gene 1993, 137, 33–39. [Google Scholar] [CrossRef]
19. Hernandez, L.I.; Flenker, K.S.; Hernandez, F.J.; Klingelhutz, A.J.; McNamara, J.O.; Giangrande, P.H. Methods for evaluating cell-specific, cell-internalizing RNA aptamers. Pharmaceuticals 2013, 6, 295–319. [Google Scholar] [CrossRef]
20. Zhou, J.; Li, H.; Zhang, J.; Piotr, S.; Rossi, J. Development of cell-type specific anti-HIV gp120 aptamers for siRNA delivery. J. Vis. Exp. 2011, 52, 357–368. [Google Scholar]
21. Liu, J.; You, M.; Pu, Y.; Liu, H.; Ye, M.; Tan, W. Recent developments in protein and cell-targeted aptamer selection and applications. Curr. Med. Chem. 2011, 18, 4117–4125. [Google Scholar] [CrossRef]
22. McNamara, J.O., 2nd; Andrechek, E.R.; Wang, Y.; Viles, K.D.; Rempel, R.E.; Gilboa, E.; Sullenger, B.A.; Giangrande, P.H. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat. Biotechnol. 2006, 24, 1005–1015. [Google Scholar] [CrossRef]
23. Zhou, J.; Rossi, J.J. Aptamer-targeted RNAi for HIV-1 therapy. Methods Mol. Biol. 2011, 721, 355–371. [Google Scholar] [CrossRef]
24. Zhou, J.; Rossi, J.J. Aptamer-targeted cell-specific RNA interference. Silence 2010, 1, 4. [Google Scholar] [CrossRef]
25. Tucker, W.O.; Shum, K.T.; Tanner, J.A. G-quadruplex DNA aptamers and their ligands: Structure, function and application. Curr. Pharm. Des. 2012, 18, 2014–2026. [Google Scholar] [CrossRef]
26. Nimjee, S.M.; Rusconi, C.P.; Sullenger, B.A. Aptamers: An emerging class of therapeutics. Annu. Rev. Med. 2005, 56, 555–583. [Google Scholar] [CrossRef]
27. Ng, E.W.; Shima, D.T.; Calias, P.; Cunningham, E.T., Jr.; Guyer, D.R.; Adamis, A.P. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat. Rev. Drug Discov. 2006, 5, 123–132. [Google Scholar] [CrossRef]
28. Doggrell, S.A. Pegaptanib: The first antiangiogenic agent approved for neovascular macular degeneration. Expert Opin. Pharmacother. 2005, 6, 1421–1423. [Google Scholar] [CrossRef]
29. Sundaram, P.; Kurniawan, H.; Byrne, M.E.; Wower, J. Therapeutic RNA aptamers in clinical trials. Eur. J. Pharm. Sci. 2013, 48, 259–271. [Google Scholar] [CrossRef]
30. Burnett, J.C.; Rossi, J.J. RNA-based therapeutics: Current progress and future prospects. Chem. Biol. 2012, 19, 60–71. [Google Scholar] [CrossRef]
31. Mongelard, F.; Bouvet, P. AS-1411, a guanosine-rich oligonucleotide aptamer targeting nucleolin for the potential treatment of cancer, including acute myeloid leukemia. Curr. Opin. Mol. Ther. 2010, 12, 107–114. [Google Scholar]
32. Gilbert, J.C.; DeFeo-Fraulini, T.; Hutabarat, R.M.; Horvath, C.J.; Merlino, P.G.; Marsh, H.N.; Healy, J.M.; Boufakhreddine, S.; Holohan, T.V.; Schaub, R.G. First-in-human evaluation of anti von Willebrand factor therapeutic aptamer ARC1779 in healthy volunteers. Circulation 2007, 116, 2678–2686. [Google Scholar] [CrossRef]
33. Vinores, S.A. Pegaptanib in the treatment of wet, age-related macular degeneration. Int. J. Nanomed. 2006, 1, 263–268. [Google Scholar]
34. Zhou, J.; Rossi, J.J. Therapeutic potential of aptamer-siRNA conjugates for treatment of HIV-1. BioDrugs 2012, 26, 393–400. [Google Scholar]
35. Zhou, J.; Rossi, J.J. Progress in RNAi-based antiviral therapeutics. Methods Mol. Biol. 2011, 721, 67–75. [Google Scholar] [CrossRef]
36. Zhou, J.; Rossi, J.J. Cell-specific aptamer-mediated targeted drug delivery. Oligonucleotides 2011, 21, 1–10. [Google Scholar] [CrossRef]
37. Zhou, J.; Rossi, J.J. Bivalent aptamers deliver the punch. Chem. Biol. 2008, 15, 644–645. [Google Scholar] [CrossRef]
38. Kanwar, J.R.; Roy, K.; Kanwar, R.K. Chimeric aptamers in cancer cell-targeted drug delivery. Crit. Rev. Biochem. Mol. Biol. 2011, 46, 459–477. [Google Scholar]
39. Khati, M. The future of aptamers in medicine. J. Clin. Pathol. 2010, 63, 480–487. [Google Scholar] [CrossRef]
40. Cheung, Y.W.; Kwok, J.; Law, A.W.; Watt, R.M.; Kotaka, M.; Tanner, J.A. Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer. Proc. Natl. Acad. Sci. USA 2013, 110, 15967–15972. [Google Scholar]
41. Bunka, D.H.; Stockley, P.G. Aptamers come of age—at last. Nat. Rev. Microbiol. 2006, 4, 588–596. [Google Scholar] [CrossRef]
42. Tuerk, C.; MacDougal, S.; Gold, L. RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. Proc. Natl. Acad. Sci. USA 1992, 89, 6988–6992. [Google Scholar] [CrossRef]
43. Jaeger, J.; Restle, T.; Steitz, T.A. The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor. EMBO J. 1998, 17, 4535–4542. [Google Scholar] [CrossRef]
44. Guo, P. The emerging field of RNA nanotechnology. Nat. Nanotechnol. 2010, 5, 833–842. [Google Scholar] [CrossRef]
45. Guo, P.; Haque, F.; Hallahan, B.; Reif, R.; Li, H. Uniqueness, advantages, challenges, solutions, and perspectives in therapeutics applying RNA nanotechnology. Nucleic Acid Ther. 2012, 22, 226–245. [Google Scholar]
46. Shukla, G.C.; Haque, F.; Tor, Y.; Wilhelmsson, L.M.; Toulme, J.J.; Isambert, H.; Guo, P.; Rossi, J.J.; Tenenbaum, S.A.; Shapiro, B.A. A boost for the emerging field of RNA nanotechnology. ACS Nano 2011, 5, 3405–3418. [Google Scholar] [CrossRef]
47. Tarapore, P.; Shu, Y.; Guo, P.; Ho, S.M. Application of phi29 motor pRNA for targeted therapeutic delivery of siRNA silencing metallothionein-IIA and survivin in ovarian cancers. Mol. Ther. 2011, 19, 386–394. [Google Scholar] [CrossRef]
48. Shu, Y.; Cinier, M.; Shu, D.; Guo, P. Assembly of multifunctional phi29 pRNA nanoparticles for specific delivery of siRNA and other therapeutics to targeted cells. Methods 2011, 54, 204–214. [Google Scholar] [CrossRef]
49. Shu, Y.; Haque, F.; Shu, D.; Li, W.; Zhu, Z.; Kotb, M.; Lyubchenko, Y.; Guo, P. Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs. RNA 2013, 19, 767–777. [Google Scholar] [CrossRef]
50. Neff, C.P.; Zhou, J.; Remling, L.; Kuruvilla, J.; Zhang, J.; Li, H.; Smith, D.D.; Swiderski, P.; Rossi, J.J.; Akkina, R. An aptamer-siRNA chimera suppresses HIV-1 viral loads and protects from helper CD4(+) T cell decline in humanized mice. Sci. Transl. Med. 2011, 3. [Google Scholar] [CrossRef]
51. Bai, J.; Banda, N.; Lee, N.S.; Rossi, J.; Akkina, R. RNA-based anti-HIV-1 gene therapeutic constructs in SCID-hu mouse model. Mol. Ther. 2002, 6, 770–782. [Google Scholar] [CrossRef]
52. Ahmad, K.M.; Xiao, Y.; Soh, H.T. Selection is more intelligent than design: Improving the affinity of a bivalent ligand through directed evolution. Nucleic Acids Res. 2012, 40, 11777–11783. [Google Scholar] [CrossRef]
53. Ye, M.; Hu, J.; Peng, M.; Liu, J.; Liu, H.; Zhao, X.; Tan, W. Generating aptamers by cell-SELEX for applications in molecular medicine. Int. J. Mol. Sci. 2012, 13, 3341–3353. [Google Scholar] [CrossRef]
54. Dua, P.; Kim, S.; Lee, D.K. Nucleic acid aptamers targeting cell-surface proteins. Methods 2011, 54, 215–225. [Google Scholar] [CrossRef]
55. Tang, Z.; Parekh, P.; Turner, P.; Moyer, R.W.; Tan, W. Generating aptamers for recognition of virus-infected cells. Clin. Chem. 2009, 55, 813–822. [Google Scholar] [CrossRef]
56. Richman, D.D.; Margolis, D.M.; Delaney, M.; Greene, W.C.; Hazuda, D.; Pomerantz, R.J. The challenge of finding a cure for HIV infection. Science 2009, 323, 1304–1307. [Google Scholar] [CrossRef]
57. Joshi, P.J.; Fisher, T.S.; Prasad, V.R. Anti-HIV inhibitors based on nucleic acids: Emergence of aptamers as potent antivirals. Curr. Drug Targets Infect. Disord. 2003, 3, 383–400. [Google Scholar] [CrossRef]
58. Scherer, L.; Rossi, J.J.; Weinberg, M.S. Progress and prospects: RNA-based therapies for treatment of HIV infection. Gene Ther. 2007, 14, 1057–1064. [Google Scholar] [CrossRef]
59. Held, D.M.; Kissel, J.D.; Patterson, J.T.; Nickens, D.G.; Burke, D.H. HIV-1 inactivation by nucleic acid aptamers. Front. Biosci. 2006, 11, 89–112. [Google Scholar] [CrossRef]
60. Jager, J.; Pata, J.D. Getting a grip: Polymerases and their substrate complexes. Curr. Opin. Struct. Biol. 1999, 9, 21–28. [Google Scholar] [CrossRef]
61. De Clercq, E. Chemotherapeutic approaches to the treatment of the acquired immune deficiency syndrome (AIDS). J. Med. Chem. 1986, 29, 1561–1569. [Google Scholar] [CrossRef]
62. De Clercq, E. Perspectives of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the therapy of HIV-1 infection. Farmaco 1999, 54, 26–45. [Google Scholar] [CrossRef]
63. Kensch, O.; Connolly, B.A.; Steinhoff, H.J.; McGregor, A.; Goody, R.S.; Restle, T. HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity. J. Biol. Chem. 2000, 275, 18271–18278. [Google Scholar]
64. Chaloin, L.; Lehmann, M.J.; Sczakiel, G.; Restle, T. Endogenous expression of a high-affinity pseudoknot RNA aptamer suppresses replication of HIV-1. Nucleic Acids Res. 2002, 30, 4001–4008. [Google Scholar] [CrossRef]
65. Li, N.; Wang, Y.; Pothukuchy, A.; Syrett, A.; Husain, N.; Gopalakrisha, S.; Kosaraju, P.; Ellington, A.D. Aptamers that recognize drug-resistant HIV-1 reverse transcriptase. Nucleic Acids Res. 2008, 36, 6739–6751. [Google Scholar] [CrossRef]
66. Andreola, M.L.; Pileur, F.; Calmels, C.; Ventura, M.; Tarrago-Litvak, L.; Toulme, J.J.; Litvak, S. DNA aptamers selected against the HIV-1 RNase H display in vitro antiviral activity. Biochemistry 2001, 40, 10087–10094. [Google Scholar] [CrossRef]
67. Somasunderam, A.; Ferguson, M.R.; Rojo, D.R.; Thiviyanathan, V.; Li, X.; O’Brien, W.A.; Gorenstein, D.G. Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase. Biochemistry 2005, 44, 10388–10395. [Google Scholar] [CrossRef]
68. DeStefano, J.J.; Nair, G.R. Novel aptamer inhibitors of human immunodeficiency virus reverse transcriptase. Oligonucleotides 2008, 18, 133–144. [Google Scholar] [CrossRef]
69. Schneider, D.J.; Feigon, J.; Hostomsky, Z.; Gold, L. High-affinity ssDNA inhibitors of the reverse transcriptase of type 1 human immunodeficiency virus. Biochemistry 1995, 34, 9599–9610. [Google Scholar] [CrossRef]
70. Kissel, J.D.; Held, D.M.; Hardy, R.W.; Burke, D.H. Single-stranded DNA aptamer RT1t49 inhibits RT polymerase and RNase H functions of HIV type 1, HIV type 2, and SIVCPZ RTs. AIDS Res. Hum. Retroviruses 2007, 23, 699–708. [Google Scholar] [CrossRef]
71. Lai, Y.T.; DeStefano, J.J. DNA aptamers to human immunodeficiency virus reverse transcriptase selected by a primer-free SELEX method: Characterization and comparison with other aptamers. Nucleic Acid Ther. 2012, 22, 162–176. [Google Scholar]
72. Whatley, A.S.; Ditzler, M.A.; Lange, M.J.; Biondi, E.; Sawyer, A.W.; Chang, J.L.; Franken, J.D.; Burke, D.H. Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, “UCAA-motif” RNA Aptamers. Mol. Ther. Nucleic Acids 2013, 2, e71. [Google Scholar] [CrossRef]
73. Ditzler, M.A.; Lange, M.J.; Bose, D.; Bottoms, C.A.; Virkler, K.F.; Sawyer, A.W.; Whatley, A.S.; Spollen, W.; Givan, S.A.; Burke, D.H. High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase. Nucleic Acids Res. 2013, 41, 1873–1884. [Google Scholar] [CrossRef]
74. De Soultrait, V.R.; Lozach, P.Y.; Altmeyer, R.; Tarrago-Litvak, L.; Litvak, S.; Andreola, M.L. DNA aptamers derived from HIV-1 RNase H inhibitors are strong anti-integrase agents. J. Mol. Biol. 2002, 324, 195–203. [Google Scholar] [CrossRef]
75. Faure-Perraud, A.; Metifiot, M.; Reigadas, S.; Recordon-Pinson, P.; Parissi, V.; Ventura, M.; Andreola, M.L. The guanine-quadruplex aptamer 93del inhibits HIV-1 replication ex vivo by interfering with viral entry, reverse transcription and integration. Antivir. Ther. 2011, 16, 383–394. [Google Scholar] [CrossRef]
76. Jing, N.; Hogan, M.E. Structure-activity of tetrad-forming oligonucleotides as a potent anti-HIV therapeutic drug. J. Biol. Chem. 1998, 273, 34992–34999. [Google Scholar] [CrossRef]
77. Ramalingam, D.; Duclair, S.; Datta, S.A.; Ellington, A.; Rein, A.; Prasad, V.R. RNA aptamers directed to human immunodeficiency virus type 1 Gag polyprotein bind to the matrix and nucleocapsid domains and inhibit virus production. J. Virol. 2011, 85, 305–314. [Google Scholar] [CrossRef]
78. Kim, S.J.; Kim, M.Y.; Lee, J.H.; You, J.C.; Jeong, S. Selection and stabilization of the RNA aptamers against the human immunodeficiency virus type-1 nucleocapsid protein. Biochem. Biophys. Res. Commun. 2002, 291, 925–931. [Google Scholar] [CrossRef]
79. Kim, M.Y.; Jeong, S. RNA aptamers that bind the nucleocapsid protein contain pseudoknots. Mol. Cells 2003, 16, 413–417. [Google Scholar]
80. Sayer, N.; Ibrahim, J.; Turner, K.; Tahiri-Alaoui, A.; James, W. Structural characterization of a 2'F-RNA aptamer that binds a HIV-1 SU glycoprotein, gp120. Biochem. Biophys. Res. Commun. 2002, 293, 924–931. [Google Scholar] [CrossRef]
81. Mufhandu, H.T.; Gray, E.S.; Madiga, M.C.; Tumba, N.; Alexandre, K.B.; Khoza, T.; Wibmer, C.K.; Moore, P.L.; Morris, L.; Khati, M. UCLA1, a synthetic derivative of a gp120 RNA aptamer, inhibits entry of human immunodeficiency virus type 1 subtype C. J. Virol. 2012, 86, 4989–4999. [Google Scholar] [CrossRef]
82. Zhou, J.; Neff, C.P.; Liu, X.; Zhang, J.; Li, H.; Smith, D.D.; Swiderski, P.; Aboellail, T.; Huang, Y.; Du, Q.; et al. Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently suppresses HIV-1 infection in humanized mice. Mol. Ther. 2011, 19, 2228–2238. [Google Scholar] [CrossRef]
83. Zhou, J.; Swiderski, P.; Li, H.; Zhang, J.; Neff, C.P.; Akkina, R.; Rossi, J.J. Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells. Nucleic Acids Res. 2009, 37, 3094–3109. [Google Scholar] [CrossRef]
84. Yamamoto, R.; Katahira, M.; Nishikawa, S.; Baba, T.; Taira, K.; Kumar, P.K. A novel RNA motif that binds efficiently and specifically to the Ttat protein of HIV and inhibits the trans-activation by Tat of transcription in vitro and in vivo. Genes Cells 2000, 5, 371–388. [Google Scholar] [CrossRef]
85. Jensen, K.B.; Green, L.; MacDougal-Waugh, S.; Tuerk, C. Characterization of an in vitro-selected RNA ligand to the HIV-1 Rev protein. J. Mol. Biol. 1994, 235, 237–247. [Google Scholar] [CrossRef]
86. Boiziau, C.; Dausse, E.; Yurchenko, L.; Toulme, J.J. DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes. J. Biol. Chem. 1999, 274, 12730–12737. [Google Scholar] [CrossRef]
87. Duconge, F.; Toulme, J.J. In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA 1999, 5, 1605–1614. [Google Scholar] [CrossRef]
88. Watrin, M.; von Pelchrzim, F.; Dausse, E.; Schroeder, R.; Toulme, J.J. In vitro selection of RNA aptamers derived from a genomic human library against the TAR RNA element of HIV-1. Biochemistry 2009, 48, 6278–6284. [Google Scholar] [CrossRef]
89. Sekkai, D.; Dausse, E.; di Primo, C.; Darfeuille, F.; Boiziau, C.; Toulme, J.J. In vitro selection of DNA aptamers against the HIV-1 TAR RNA hairpin. Antisense Nucleic Acid Drug Dev. 2002, 12, 265–274. [Google Scholar] [CrossRef]
90. Kumar, P.K.; Machida, K.; Urvil, P.T.; Kakiuchi, N.; Vishnuvardhan, D.; Shimotohno, K.; Taira, K.; Nishikawa, S. Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA. Virology 1997, 237, 270–282. [Google Scholar] [CrossRef]
91. Fukuda, K.; Vishnuvardhan, D.; Sekiya, S.; Hwang, J.; Kakiuchi, N.; Taira, K.; Shimotohno, K.; Kumar, P.K.; Nishikawa, S. Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease. Eur. J. Biochem. 2000, 267, 3685–3694. [Google Scholar] [CrossRef]
92. Nishikawa, F.; Funaji, K.; Fukuda, K.; Nishikawa, S. In vitro selection of RNA aptamers against the HCV NS3 helicase domain. Oligonucleotides 2004, 14, 114–129. [Google Scholar] [CrossRef]
93. Biroccio, A.; Hamm, J.; Incitti, I.; de Francesco, R.; Tomei, L. Selection of RNA aptamers that are specific and high-affinity ligands of the hepatitis C virus RNA-dependent RNA polymerase. J. Virol. 2002, 76, 3688–3696. [Google Scholar] [CrossRef]
94. Bellecave, P.; Cazenave, C.; Rumi, J.; Staedel, C.; Cosnefroy, O.; Andreola, M.L.; Ventura, M.; Tarrago-Litvak, L.; Astier-Gin, T. Inhibition of hepatitis C virus (HCV) RNA polymerase by DNA aptamers: Mechanism of inhibition of in vitro RNA synthesis and effect on HCV-infected cells. Antimicrob. Agents Chemother. 2008, 52, 2097–2110. [Google Scholar] [CrossRef]
95. Lee, C.H.; Lee, Y.J.; Kim, J.H.; Lim, J.H.; Kim, J.H.; Han, W.; Lee, S.H.; Noh, G.J.; Lee, S.W. Inhibition of hepatitis C virus (HCV) replication by specific RNA aptamers against HCV NS5B RNA replicase. J. Virol. 2013, 87, 7064–7074. [Google Scholar] [CrossRef]
96. Marton, S.; Romero-Lopez, C.; Berzal-Herranz, A. RNA aptamer-mediated interference of HCV replication by targeting the CRE-5BSL3.2 domain. J. Viral Hepat. 2013, 20, 103–112. [Google Scholar] [CrossRef]
97. Kikuchi, K.; Umehara, T.; Fukuda, K.; Hwang, J.; Kuno, A.; Hasegawa, T.; Nishikawa, S. RNA aptamers targeted to domain II of hepatitis C virus IRES that bind to its apical loop region. J. Biochem. 2003, 133, 263–270. [Google Scholar] [CrossRef]
98. Kikuchi, K.; Umehara, T.; Fukuda, K.; Kuno, A.; Hasegawa, T.; Nishikawa, S. A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III-IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId. Nucleic Acids Res. 2005, 33, 683–692. [Google Scholar] [CrossRef]
99. Romero-Lopez, C.; Barroso-delJesus, A.; Puerta-Fernandez, E.; Berzal-Herranz, A. Interfering with hepatitis C virus IRES activity using RNA molecules identified by a novel in vitro selection method. Biol. Chem. 2005, 386, 183–190. [Google Scholar]
100. Romero-Lopez, C.; Diaz-Gonzalez, R.; Barroso-delJesus, A.; Berzal-Herranz, A. Inhibition of hepatitis C virus replication and internal ribosome entry site-dependent translation by an RNA molecule. J. Gen. Virol. 2009, 90, 1659–1669. [Google Scholar] [CrossRef]
101. Liu, J.; Yang, Y.; Hu, B.; Ma, Z.Y.; Huang, H.P.; Yu, Y.; Liu, S.P.; Lu, M.J.; Yang, D.L. Development of HBsAg-binding aptamers that bind HepG2.2.15 cells via HBV surface antigen. Virol. Sin. 2010, 25, 27–35. [Google Scholar] [CrossRef]
102. Feng, H.; Beck, J.; Nassal, M.; Hu, K.H. A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication. PLoS One 2011, 6, e27862. [Google Scholar] [CrossRef]
103. Shum, K.T.; Tanner, J.A. Differential inhibitory activities and stabilisation of DNA aptamers against the SARS coronavirus helicase. ChemBioChem 2008, 9, 3037–3045. [Google Scholar] [CrossRef]
104. Jang, K.J.; Lee, N.R.; Yeo, W.S.; Jeong, Y.J.; Kim, D.E. Isolation of inhibitory RNA aptamers against severe acute respiratory syndrome (SARS) coronavirus NTPase/Helicase. Biochem. Biophys. Res. Commun. 2008, 366, 738–744. [Google Scholar] [CrossRef]
105. Jeon, S.H.; Kayhan, B.; Ben-Yedidia, T.; Arnon, R. A DNA aptamer prevents influenza infection by blocking the receptor binding region of the viral hemagglutinin. J. Biol. Chem. 2004, 279, 48410–48419. [Google Scholar] [CrossRef]
106. Cheng, C.; Dong, J.; Yao, L.; Chen, A.; Jia, R.; Huan, L.; Guo, J.; Shu, Y.; Zhang, Z. Potent inhibition of human influenza H5N1 virus by oligonucleotides derived by SELEX. Biochem. Biophys. Res. Commun. 2008, 366, 670–674. [Google Scholar] [CrossRef]
107. Gopinath, S.C.; Misono, T.S.; Kawasaki, K.; Mizuno, T.; Imai, M.; Odagiri, T.; Kumar, P.K. An RNA aptamer that distinguishes between closely related human influenza viruses and inhibits haemagglutinin-mediated membrane fusion. J. Gen. Virol. 2006, 87, 479–487. [Google Scholar] [CrossRef]
108. Misono, T.S.; Kumar, P.K. Selection of RNA aptamers against human influenza virus hemagglutinin using surface plasmon resonance. Anal. Biochem. 2005, 342, 312–317. [Google Scholar] [CrossRef]
109. Wongphatcharachai, M.; Wang, P.; Enomoto, S.; Webby, R.J.; Gramer, M.R.; Amonsin, A.; Sreevatsan, S. Neutralizing DNA aptamers against swine influenza H3N2 viruses. J. Clin. Microbiol. 2013, 51, 46–54. [Google Scholar] [CrossRef]
110. Choi, S.K.; Lee, C.; Lee, K.S.; Choe, S.Y.; Mo, I.P.; Seong, R.H.; Hong, S.; Jeon, S.H. DNA aptamers against the receptor binding region of hemagglutinin prevent avian influenza viral infection. Mol. Cells 2011, 32, 527–533. [Google Scholar] [CrossRef]
111. Park, S.Y.; Kim, S.; Yoon, H.; Kim, K.B.; Kalme, S.S.; Oh, S.; Song, C.S.; Kim, D.E. Selection of an antiviral RNA aptamer against hemagglutinin of the subtype H5 avian influenza virus. Nucleic Acid Ther. 2011, 21, 395–402. [Google Scholar] [CrossRef]
112. Gopinath, S.C.; Sakamaki, Y.; Kawasaki, K.; Kumar, P.K. An efficient RNA aptamer against human influenza B virus hemagglutinin. J. Biochem. 2006, 139, 837–846. [Google Scholar] [CrossRef]
113. Liang, H.R.; Hu, G.Q.; Zhang, T.; Yang, Y.J.; Zhao, L.L.; Qi, Y.L.; Wang, H.L.; Gao, Y.W.; Yang, S.T.; Xia, X.Z. Isolation of ssDNA aptamers that inhibit rabies virus. Int. Immunopharmacol. 2012, 14, 341–347. [Google Scholar] [CrossRef]
114. Liang, H.R.; Liu, Q.; Zheng, X.X.; Gai, W.W.; Xue, X.H.; Hu, G.Q.; Wu, H.X.; Wang, H.L.; Yang, S.T.; Xia, X.Z. Aptamers targeting rabies virus-infected cells inhibit viral replication both in vitro and in vivo. Virus Res. 2013, 173, 398–403. [Google Scholar] [CrossRef]
115. Toscano-Garibay, J.D.; Benitez-Hess, M.L.; Alvarez-Salas, L.M. Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein. Arch. Med. Res. 2011, 42, 88–96. [Google Scholar] [CrossRef]
116. Graham, J.C.; Zarbl, H. Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells. PLoS One 2012, 7, e36103. [Google Scholar] [CrossRef]
117. Nicol, C.; Cesur, O.; Forrest, S.; Belyaeva, T.A.; Bunka, D.H.; Blair, G.E.; Stonehouse, N.J. An RNA aptamer provides a novel approach for the induction of apoptosis by targeting the HPV16 E7 oncoprotein. PLoS One 2013, 8, e64781. [Google Scholar]
118. Nicol, C.; Bunka, D.H.; Blair, G.E.; Stonehouse, N.J. Effects of single nucleotide changes on the binding and activity of RNA aptamers to human papillomavirus 16 E7 oncoprotein. Biochem. Biophys. Res. Commun. 2011, 405, 417–421. [Google Scholar] [CrossRef]
119. Gopinath, S.C.; Hayashi, K.; Kumar, P.K. Aptamer that binds to the gD protein of herpes simplex virus 1 and efficiently inhibits viral entry. J. Virol. 2012, 86, 6732–6744. [Google Scholar] [CrossRef]
120. Moore, M.D.; Bunka, D.H.; Forzan, M.; Spear, P.G.; Stockley, P.G.; McGowan, I.; James, W. Generation of neutralizing aptamers against herpes simplex virus type 2: Potential components of multivalent microbicides. J. Gen. Virol. 2011, 92, 1493–1499. [Google Scholar] [CrossRef]
121. Nickens, D.G.; Patterson, J.T.; Burke, D.H. Inhibition of HIV-1 reverse transcriptase by RNA aptamers in Escherichia coli. RNA 2003, 9, 1029–1033. [Google Scholar] [CrossRef]
122. Joshi, P.; Prasad, V.R. Potent inhibition of human immunodeficiency virus type 1 replication by template analog reverse transcriptase inhibitors derived by SELEX (systematic evolution of ligands by exponential enrichment). J. Virol. 2002, 76, 6545–6557. [Google Scholar] [CrossRef]
123. Lange, M.J.; Sharma, T.K.; Whatley, A.S.; Landon, L.A.; Tempesta, M.A.; Johnson, M.C.; Burke, D.H. Robust suppression of HIV replication by intracellularly expressed reverse transcriptase aptamers is independent of ribozyme processing. Mol. Ther. 2012, 20, 2304–2314. [Google Scholar] [CrossRef]
124. Asante-Appiah, E.; Skalka, A.M. Molecular mechanisms in retrovirus DNA integration. Antiviral Res. 1997, 36, 139–156. [Google Scholar] [CrossRef]
125. Li, T.; Shi, L.; Wang, E.; Dong, S. Multifunctional G-quadruplex aptamers and their application to protein detection. Chemistry 2009, 15, 1036–1042. [Google Scholar] [CrossRef]
126. Phan, A.T.; Kuryavyi, V.; Ma, J.B.; Faure, A.; Andreola, M.L.; Patel, D.J. An interlocked dimeric parallel-stranded DNA quadruplex: A potent inhibitor of HIV-1 integrase. Proc. Natl. Acad. Sci. USA 2005, 102, 634–639. [Google Scholar]
127. Chou, S.H.; Chin, K.H.; Wang, A.H. DNA aptamers as potential anti-HIV agents. Trends Biochem. Sci. 2005, 30, 231–234. [Google Scholar] [CrossRef]
128. Mazumder, A.; Neamati, N.; Ojwang, J.O.; Sunder, S.; Rando, R.F.; Pommier, Y. Inhibition of the human immunodeficiency virus type 1 integrase by guanosine quartet structures. Biochemistry 1996, 35, 13762–13771. [Google Scholar] [CrossRef]
129. Magbanua, E.; Zivkovic, T.; Hansen, B.; Beschorner, N.; Meyer, C.; Lorenzen, I.; Grotzinger, J.; Hauber, J.; Torda, A.E.; Mayer, G.; et al. d(GGGT) 4 and r(GGGU) 4 are both HIV-1 inhibitors and interleukin-6 receptor aptamers. RNA Biol. 2013, 10, 216–227. [Google Scholar] [CrossRef]
130. Gheysen, D.; Jacobs, E.; de Foresta, F.; Thiriart, C.; Francotte, M.; Thines, D.; de Wilde, M. Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell 1989, 59, 103–112. [Google Scholar]
131. Wills, J.W.; Craven, R.C. Form, function, and use of retroviral gag proteins. AIDS 1991, 5, 639–654. [Google Scholar] [CrossRef]
132. Demirov, D.G.; Freed, E.O. Retrovirus budding. Virus Res. 2004, 106, 87–102. [Google Scholar] [CrossRef]
133. Rein, A.; Henderson, L.E.; Levin, J.G. Nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: Significance for viral replication. Trends Biochem. Sci. 1998, 23, 297–301. [Google Scholar] [CrossRef]
134. De Guzman, R.N.; Wu, Z.R.; Stalling, C.C.; Pappalardo, L.; Borer, P.N.; Summers, M.F. Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science 1998, 279, 384–388. [Google Scholar] [CrossRef]
135. Kim, M.Y.; Jeong, S. Inhibition of the functions of the nucleocapsid protein of human immunodeficiency virus-1 by an RNA aptamer. Biochem. Biophys. Res. Commun. 2004, 320, 1181–1186. [Google Scholar] [CrossRef]
136. Kwong, P.D.; Wyatt, R.; Robinson, J.; Sweet, R.W.; Sodroski, J.; Hendrickson, W.A. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 1998, 393, 648–659. [Google Scholar] [CrossRef]
137. Sattentau, Q.J.; Moore, J.P. The role of CD4 in HIV binding and entry. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1993, 342, 59–66. [Google Scholar] [CrossRef]
138. Ugolini, S.; Mondor, I.; Sattentau, Q.J. HIV-1 attachment: Another look. Trends Microbiol. 1999, 7, 144–149. [Google Scholar] [CrossRef]
139. Khati, M.; Schuman, M.; Ibrahim, J.; Sattentau, Q.; Gordon, S.; James, W. Neutralization of infectivity of diverse R5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2'F-RNA aptamers. J. Virol. 2003, 77, 12692–12698. [Google Scholar] [CrossRef]
140. Dey, A.K.; Griffiths, C.; Lea, S.M.; James, W. Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1. RNA 2005, 11, 873–884. [Google Scholar] [CrossRef]
141. Dey, A.K.; Khati, M.; Tang, M.; Wyatt, R.; Lea, S.M.; James, W. An aptamer that neutralizes R5 strains of human immunodeficiency virus type 1 blocks gp120-CCR5 interaction. J. Virol. 2005, 79, 13806–13810. [Google Scholar] [CrossRef]
142. Cohen, C.; Forzan, M.; Sproat, B.; Pantophlet, R.; McGowan, I.; Burton, D.; James, W. An aptamer that neutralizes R5 strains of HIV-1 binds to core residues of gp120 in the CCR5 binding site. Virology 2008, 381, 46–54. [Google Scholar] [CrossRef]
143. Zhou, J.; Li, H.; Li, S.; Zaia, J.; Rossi, J.J. Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy. Mol. Ther. 2008, 16, 1481–1489. [Google Scholar] [CrossRef]
144. Zhou, J.; Neff, C.P.; Swiderski, P.; Li, H.; Smith, D.D.; Aboellail, T.; Remling-Mulder, L.; Akkina, R.; Rossi, J.J. Functional in vivo delivery of multiplexed anti-HIV-1 siRNAs via a chemically synthesized aptamer with a sticky bridge. Mol. Ther. 2013, 21, 192–200. [Google Scholar] [CrossRef]
145. Arya, S.K.; Guo, C.; Josephs, S.F.; Wong-Staal, F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 1985, 229, 69–73. [Google Scholar]
146. Berkhout, B.; Silverman, R.H.; Jeang, K.T. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell 1989, 59, 273–282. [Google Scholar] [CrossRef]
147. Ensoli, B.; Barillari, G.; Salahuddin, S.Z.; Gallo, R.C.; Wong-Staal, F. Tat protein of HIV-1 stimulates growth of cells derived from Kaposi’s sarcoma lesions of AIDS patients. Nature 1990, 345, 84–86. [Google Scholar] [CrossRef]
148. Cullen, B.R.; Greene, W.C. Regulatory pathways governing HIV-1 replication. Cell 1989, 58, 423–426. [Google Scholar] [CrossRef]
149. Rosen, C.A.; Pavlakis, G.N. Tat and Rev: Positive regulators of HIV gene expression. AIDS 1990, 4, 499–509. [Google Scholar] [CrossRef]
150. Kjems, J.; Frankel, A.D.; Sharp, P.A. Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev. Cell 1991, 67, 169–178. [Google Scholar] [CrossRef]
151. Konopka, K.; Lee, N.S.; Rossi, J.; Duzgunes, N. Rev-binding aptamer and CMV promoter act as decoys to inhibit HIV replication. Gene 2000, 255, 235–244. [Google Scholar] [CrossRef]
152. Gait, M.J.; Karn, J. RNA recognition by the human immunodeficiency virus Tat and Rev proteins. Trends Biochem. Sci. 1993, 18, 255–259. [Google Scholar] [CrossRef]
153. Gatignol, A.; Duarte, M.; Daviet, L.; Chang, Y.N.; Jeang, K.T. Sequential steps in Tat trans-activation of HIV-1 mediated through cellular DNA, RNA, and protein binding factors. Gene Expr. 1996, 5, 217–228. [Google Scholar]
154. Zimmermann, B.; Bilusic, I.; Lorenz, C.; Schroeder, R. Genomic SELEX: A discovery tool for genomic aptamers. Methods 2010, 52, 125–132. [Google Scholar] [CrossRef]
155. Li, M.; Rossi, J.J. Lentiviral vector delivery of siRNA and shRNA encoding genes into cultured and primary hematopoietic cells. Methods Mol. Biol. 2005, 309, 261–272. [Google Scholar]
156. DiGiusto, D.L.; Krishnan, A.; Li, L.; Li, H.; Li, S.; Rao, A.; Mi, S.; Yam, P.; Stinson, S.; Kalos, M.; et al. RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma. Sci. Transl. Med. 2010, 2, 36–ra43. [Google Scholar]
157. Wasley, A.; Alter, M.J. Epidemiology of hepatitis C: Geographic differences and temporal trends. Semin. Liver Dis. 2000, 20, 1–16. [Google Scholar] [CrossRef]
158. Hanazaki, K. Antiviral therapy for chronic hepatitis B: A review. Curr. Drug Targets Inflamm. Allergy 2004, 3, 63–70. [Google Scholar] [CrossRef]
159. Kato, N. Genome of human hepatitis C virus (HCV): Gene organization, sequence diversity, and variation. Microb. Comp. Genomics 2000, 5, 129–151. [Google Scholar]
160. Failla, C.; Tomei, L.; de Francesco, R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J. Virol. 1994, 68, 3753–3760. [Google Scholar]
161. Kim, D.W.; Gwack, Y.; Han, J.H.; Choe, J. C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem. Biophys. Res. Commun. 1995, 215, 160–166. [Google Scholar] [CrossRef]
162. Nishikawa, F.; Kakiuchi, N.; Funaji, K.; Fukuda, K.; Sekiya, S.; Nishikawa, S. Inhibition of HCV NS3 protease by RNA aptamers in cells. Nucleic Acids Res. 2003, 31, 1935–1943. [Google Scholar] [CrossRef]
163. Fukuda, K.; Umehara, T.; Sekiya, S.; Kunio, K.; Hasegawa, T.; Nishikawa, S. An RNA ligand inhibits hepatitis C virus NS3 protease and helicase activities. Biochem. Biophys. Res. Commun. 2004, 325, 670–675. [Google Scholar] [CrossRef]
164. Umehara, T.; Fukuda, K.; Nishikawa, F.; Kohara, M.; Hasegawa, T.; Nishikawa, S. Rational design of dual-functional aptamers that inhibit the protease and helicase activities of HCV NS3. J. Biochem. 2005, 137, 339–347. [Google Scholar] [CrossRef]
165. Varshney, J.; Sharma, P.K.; Sharma, A. A review on an update of NS5B polymerase hepatitis C virus inhibitors. Eur. Rev. Med. Pharmacol. Sci. 2012, 16, 667–671. [Google Scholar]
166. Tsukiyama-Kohara, K.; Iizuka, N.; Kohara, M.; Nomoto, A. Internal ribosome entry site within hepatitis C virus RNA. J. Virol. 1992, 66, 1476–1483. [Google Scholar]
167. Gallego, J.; Varani, G. The hepatitis C virus internal ribosome-entry site: A new target for antiviral research. Biochem. Soc. Trans. 2002, 30, 140–145. [Google Scholar] [CrossRef]
168. Kikuchi, K.; Umehara, T.; Nishikawa, F.; Fukuda, K.; Hasegawa, T.; Nishikawa, S. Increased inhibitory ability of conjugated RNA aptamers against the HCV IRES. Biochem. Biophys. Res. Commun. 2009, 386, 118–123. [Google Scholar] [CrossRef]
169. Chen, Y.; Cheng, G.; Mahato, R.I. RNAi for treating hepatitis B viral infection. Pharm. Res. 2008, 25, 72–86. [Google Scholar] [CrossRef]
170. Zoulim, F. Hepatitis B virus resistance to antiviral drugs: Where are we going? Liver Int. 2011, 31, 111–116. [Google Scholar] [CrossRef]
171. Seeger, C.; Mason, W.S. Hepatitis B virus biology. Microbiol. Mol. Biol. Rev. 2000, 64, 51–68. [Google Scholar] [CrossRef]
172. Summers, J.; Mason, W.S. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 1982, 29, 403–415. [Google Scholar] [CrossRef]
173. Wang, G.H.; Seeger, C. The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis. Cell 1992, 71, 663–670. [Google Scholar] [CrossRef]
174. Hirsch, R.C.; Lavine, J.E.; Chang, L.J.; Varmus, H.E.; Ganem, D. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature 1990, 344, 552–555. [Google Scholar] [CrossRef]
175. Flodell, S.; Petersen, M.; Girard, F.; Zdunek, J.; Kidd-Ljunggren, K.; Schleucher, J.; Wijmenga, S. Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal. Nucleic Acids Res. 2006, 34, 4449–4457. [Google Scholar] [CrossRef]
176. Peiris, J.S.; Lai, S.T.; Poon, L.L.; Guan, Y.; Yam, L.Y.; Lim, W.; Nicholls, J.; Yee, W.K.; Yan, W.W.; Cheung, M.T.; et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003, 361, 1319–1325. [Google Scholar] [CrossRef]
177. Stadler, K.; Masignani, V.; Eickmann, M.; Becker, S.; Abrignani, S.; Klenk, H.D.; Rappuoli, R. SARS—beginning to understand a new virus. Nat. Rev. Microbiol. 2003, 1, 209–218. [Google Scholar] [CrossRef]
178. Tanner, J.A.; Watt, R.M.; Chai, Y.B.; Lu, L.Y.; Lin, M.C.; Peiris, J.S.; Poon, L.L.; Kung, H.F.; Huang, J.D. The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases. J. Biol. Chem. 2003, 278, 39578–39582. [Google Scholar] [CrossRef]
179. Zaki, A.M.; van Boheemen, S.; Bestebroer, T.M.; Osterhaus, A.D.; Fouchier, R.A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 2012, 367, 1814–1820. [Google Scholar] [CrossRef]
180. Van Boheemen, S.; de Graaf, M.; Lauber, C.; Bestebroer, T.M.; Raj, V.S.; Zaki, A.M.; Osterhaus, A.D.; Haagmans, B.L.; Gorbalenya, A.E.; Snijder, E.J.; et al. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio 2012. [Google Scholar] [CrossRef]
181. Josset, L.; Menachery, V.D.; Gralinski, L.E.; Agnihothram, S.; Sova, P.; Carter, V.S.; Yount, B.L.; Graham, R.L.; Baric, R.S.; Katze, M.G. Cell host response to infection with novel human coronavirus EMC predicts potential antivirals and important differences with SARS coronavirus. mBio 2013. [Google Scholar] [CrossRef]
182. Elderfield, R.; Barclay, W. Influenza pandemics. Adv. Exp. Med. Biol. 2011, 719, 81–103. [Google Scholar] [CrossRef]
183. Webster, R.G. 1918 Spanish influenza: The secrets remain elusive. Proc. Natl. Acad. Sci. USA 1999, 96, 1164–1166. [Google Scholar] [CrossRef]
184. Bai, T.; Zhou, J.; Shu, Y. Serologic study for influenza A (H7N9) among high-risk groups in China. N. Engl. J. Med. 2013, 368, 2339–2340. [Google Scholar] [CrossRef]
185. Watanabe, Y.; Ibrahim, M.S.; Suzuki, Y.; Ikuta, K. The changing nature of avian influenza A virus (H5N1). Trends Microbiol. 2012, 20, 11–20. [Google Scholar] [CrossRef]
186. Gultyaev, A.P.; Fouchier, R.A.; Olsthoorn, R.C. Influenza virus RNA structure: Unique and common features. Int. Rev. Immunol. 2010, 29, 533–556. [Google Scholar] [CrossRef]
187. Saladino, R.; Barontini, M.; Crucianelli, M.; Nencioni, L.; Sgarbanti, R.; Palamara, A.T. Current advances in anti-influenza therapy. Curr. Med. Chem. 2010, 17, 2101–2140. [Google Scholar] [CrossRef]
188. Stropkovska, A.; Janulikova, J.; Vareckova, E. Trends in development of the influenza vaccine with broader cross-protection. Acta Virol. 2010, 54, 7–19. [Google Scholar] [CrossRef]
189. Burton, D.R.; Poignard, P.; Stanfield, R.L.; Wilson, I.A. Broadly neutralizing antibodies present new prospects to counter highly antigenically diverse viruses. Science 2012, 337, 183–186. [Google Scholar] [CrossRef]
190. Skehel, J.J.; Wiley, D.C. Receptor binding and membrane fusion in virus entry: The influenza hemagglutinin. Annu. Rev. Biochem. 2000, 69, 531–569. [Google Scholar] [CrossRef]
191. Nicholson, K.G.; Wood, J.M.; Zambon, M. Influenza. Lancet 2003, 362, 1733–1745. [Google Scholar] [CrossRef]
192. Yousaf, M.Z.; Qasim, M.; Zia, S.; Khan, M.; Ashfaq, U.A.; Khan, S. Rabies molecular virology, diagnosis, prevention and treatment. Virol. J. 2012, 9, 50. [Google Scholar] [CrossRef]
193. Dietzschold, B.; Schnell, M.; Koprowski, H. Pathogenesis of rabies. Curr. Top. Microbiol. Immunol. 2005, 292, 45–56. [Google Scholar] [CrossRef]
194. Stanley, M. Pathology and epidemiology of HPV infection in females. Gynecol. Oncol. 2010, 117, S5–S10. [Google Scholar] [CrossRef]
195. McLaughlin-Drubin, M.E.; Meyers, J.; Munger, K. Cancer associated human papillomaviruses. Curr. Opin. Virol. 2012, 2, 459–466. [Google Scholar] [CrossRef]
196. Wilczynski, S.P.; Bergen, S.; Walker, J.; Liao, S.Y.; Pearlman, L.F. Human papillomaviruses and cervical cancer: Analysis of histopathologic features associated with different viral types. Hum. Pathol. 1988, 19, 697–704. [Google Scholar] [CrossRef]
197. Thomas, M.; Narayan, N.; Pim, D.; Tomaic, V.; Massimi, P.; Nagasaka, K.; Kranjec, C.; Gammoh, N.; Banks, L. Human papillomaviruses, cervical cancer and cell polarity. Oncogene 2008, 27, 7018–7030. [Google Scholar] [CrossRef]
198. DiMaio, D.; Liao, J.B. Human papillomaviruses and cervical cancer. Adv. Virus Res. 2006, 66, 125–159. [Google Scholar] [CrossRef]
199. Mineta, H.; Ogino, T.; Amano, H.M.; Ohkawa, Y.; Araki, K.; Takebayashi, S.; Miura, K. Human papilloma virus (HPV) type 16 and 18 detected in head and neck squamous cell carcinoma. Anticancer Res. 1998, 18, 4765–4768. [Google Scholar]
200. Schiller, J.T.; Lowy, D.R. Understanding and learning from the success of prophylactic human papillomavirus vaccines. Nat. Rev. Microbiol. 2012, 10, 681–692. [Google Scholar] [CrossRef]
201. Ghittoni, R.; Accardi, R.; Hasan, U.; Gheit, T.; Sylla, B.; Tommasino, M. The biological properties of E6 and E7 oncoproteins from human papillomaviruses. Virus Genes 2010, 40, 1–13. [Google Scholar] [CrossRef]
202. Nauenburg, S.; Zwerschke, W.; Jansen-Durr, P. Induction of apoptosis in cervical carcinoma cells by peptide aptamers that bind to the HPV-16 E7 oncoprotein. FASEB J. 2001, 15, 592–594. [Google Scholar]
203. Finzer, P.; Aguilar-Lemarroy, A.; Rosl, F. The role of human papillomavirus oncoproteins E6 and E7 in apoptosis. Cancer Lett. 2002, 188, 15–24. [Google Scholar] [CrossRef]
204. Maldonado, E.; Cabrejos, M.E.; Banks, L.; Allende, J.E. Human papillomavirus-16 E7 protein inhibits the DNA interaction of the TATA binding transcription factor. J. Cell. Biochem. 2002, 85, 663–669. [Google Scholar] [CrossRef]
205. Bernat, A.; Avvakumov, N.; Mymryk, J.S.; Banks, L. Interaction between the HPV E7 oncoprotein and the transcriptional coactivator p300. Oncogene 2003, 22, 7871–7881. [Google Scholar] [CrossRef]
206. Draper, K.G.; Frink, R.J.; Devi, G.B.; Swain, M.; Galloway, D.; Wagner, E.K. Herpes simplex virus types 1 and 2 homology in the region between 0.58 and 0.68 map units. J. Virol. 1984, 52, 615–623. [Google Scholar]
207. Rozenberg, F.; Deback, C.; Agut, H. Herpes simplex encephalitis: From virus to therapy. Infect. Disord. Drug Targets 2011, 11, 235–250. [Google Scholar] [CrossRef]
208. Reske, A.; Pollara, G.; Krummenacher, C.; Chain, B.M.; Katz, D.R. Understanding HSV-1 entry glycoproteins. Rev. Med. Virol. 2007, 17, 205–215. [Google Scholar] [CrossRef]
209. Lasky, L.A.; Dowbenko, D.J. DNA sequence analysis of the type-common glycoprotein-D genes of herpes simplex virus types 1 and 2. DNA 1984, 3, 23–29. [Google Scholar] [CrossRef]
210. Zhou, J.; Shum, K.T.; Burnett, J.C.; Rossi, J.J. Nanoparticle-based delivery of RNAi therapeutics: Progress and challenges. Pharmaceuticals 2013, 6, 85–107. [Google Scholar] [CrossRef]
211. James, W. Aptamers in the virologists’ toolkit. J. Gen. Virol. 2007, 88, 351–364. [Google Scholar] [CrossRef]
212. Moore, M.D.; Cookson, J.; Coventry, V.K.; Sproat, B.; Rabe, L.; Cranston, R.D.; McGowan, I.; James, W. Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: Implications for RNA-based therapeutics. J. Biol. Chem. 2011, 286, 2526–2535. [Google Scholar] [CrossRef]
213. Wheeler, L.A.; Vrbanac, V.; Trifonova, R.; Brehm, M.A.; Gilboa-Geffen, A.; Tanno, S.; Greiner, D.L.; Luster, A.D.; Tager, A.M.; Lieberman, J. Durable knockdown and protection from HIV transmission in humanized mice treated with gel-formulated CD4 aptamer-siRNA chimeras. Mol. Ther. 2013, 21, 1378–1389. [Google Scholar] [CrossRef]