Margaret J. Lange

High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase

Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs and in clusters of RNAs with nearly identical sequences that share common ancestry. Both of these features informed inferences of the secondary structures of enriched RNAs, their minimal structural requirements and their stabilities in RT-aptamer complexes. Monitoring population dynamics in response to increasing selection pressure revealed RNA inhibitors of RT that are more potent than the previously identified pseudoknots. Improved potency was observed for inhibition of both purified RT in enzymatic assays and viral replication in cell-based assays. Structural and functional details of converged motifs that are obscured by simple consensus descriptions are also revealed by the HTS analysis. The approach presented here can readily be generalized for the efficient and systematic post-SELEX development of aptamers for down-stream applications.

Toll-like receptors in tonsillar epithelial cells

OBJECTIVE The Waldeyers ring, comprised of the nasopharyngeal tonsil, the paired tubal tonsils, the paired palatine tonsils, and the lingual tonsil, is arranged in a circular orientation around the wall of the throat. The location of the palatine tonsils, specifically, enables these structures to come in direct contact with potentially harmful inhaled and ingested material that exist in their native form since digestive enzymes are not present in the oral cavity. Thus, the tonsillar epithelium must not only serve a protective role but it must also function in an antigen-sampling role. Previous studies involving the tissues of the Waldeyers ring have been focused on the adaptive immune system, with little consideration toward the innate immune system. Studies have demonstrated that the tonsils are capable of producing proinflammatory and antiviral cytokines and chemokines. In addition, other studies have highlighted the importance of epithelial cells in this response. Therefore, we postulate that toll-like receptors (TLRs), which recognize components of pathogenic organisms, may play a key role in the innate immune response in tonsillar epithelial cells. TLRs are innate pattern recognition receptors, which produce proinflammatory cytokines and chemokines upon ligation. In this study, we examine the expression and function of TLRs in the tonsillar epithelial cell lines, UT-SCC-60A and UT-SCC-60B. Additionally, we demonstrate successful isolation of primary tonsillar epithelial cells and examine TLR expression in these cells. METHODS We utilized endpoint RT-PCR, real time RT-PCR, and flow cytometric analysis to determine TLR expression. To assess TLR function, cells were stimulated with TLR ligands and supernatants were assayed for secretion of cytokines. RESULTS UT-SCC-60A and UTSCC-60B express TLR mRNA and TLR protein, and the observed responses to the TLR ligands, Pam3Cys and Poly I:C suggest that TLR2 and TLR3 are functional in these cells. Additionally, primary tonsillar epithelial cells express TLRs. CONCLUSIONS TLRs are expressed in human tonsillar epithelial cells and may play a vital role in the immunological outcomes in this tissue.

RNA–protein interactions govern antiviral specificity and encapsidation of broad spectrum anti-HIV reverse transcriptase aptamers

Abstract RNA aptamers that bind HIV-1 reverse transcriptase (RT) inhibit HIV-1 replication, but little is known about potential aptamer-specific viral resistance. During replication, RT interacts with diverse nucleic acids. Thus, the genetic threshold for eliciting resistance may be high for aptamers that make numerous contacts with RT. To evaluate the impact of RT–aptamer binding specificity on replication, we engineered proviral plasmids encoding diverse RTs within the backbone of HIV-1 strain NL4-3. Viruses inhibited by pseudoknot aptamers were rendered insensitive by a naturally occurring R277K variant, providing the first demonstration of aptamer-specific resistance in cell culture. Naturally occurring, pseudoknot-insensitive viruses were rendered sensitive by the inverse K277R mutation, establishing RT as the genetic locus for aptamer-mediated HIV-1 inhibition. Non-pseudoknot RNA aptamers exhibited broad-spectrum inhibition. Inhibition was observed only when virus was produced in aptamer-expressing cells, indicating that encapsidation is required. HIV-1 suppression magnitude correlated with the number of encapsidated aptamer transcripts per virion, with saturation occurring around 1:1 stoichiometry with packaged RT. Encapsidation specificity suggests that aptamers may encounter dimerized GagPol in the cytosol during viral assembly. This study provides new insights into HIV-1s capacity to escape aptamer-mediated inhibition, the potential utility of broad-spectrum aptamers to overcome resistance, and molecular interactions that occur during viral assembly.

Screening Inhibitory Potential of Anti-HIV RT RNA Aptamers

Aptamers targeted to HIV reverse transcriptase (RT) have been demonstrated to inhibit RT in biochemical assays and as in cell culture. However, methods employed to date to evaluate viral suppression utilize time-consuming serial passage of infectious HIV in aptamer-expressing stable cell lines. We have established a rapid, transfection-based assay system to effectively examine the inhibitory potential of anti-HIV RT aptamers expressed between two catalytically inactive hammerhead ribozymes. Our system can be altered and optimized for a variety of cloning schemes, and addition of sequences of interest to the cassette is simple and straightforward. When paired with methods to analyze aptamer RNA accumulation and localization in cells and as packaging into pseudotyped virions, the method has a very high level of success in predicting good inhibitors.

Modular cell-internalizing aptamer nanostructure enables targeted delivery of large functional RNAs in cancer cell lines

Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19–21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50–80 kDa, 175–250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs.Large RNAs and ribonucleoprotein complexes have shown potential as novel therapeutic agents, but their targeted delivery to cells is still challenging. Here the authors present a modular aptamer nanostructure for intracellular delivery of RNAs up to 250 nucleotides to cancer cells.

Poly-Target Selection Identifies RNA Broad-Spectrum Inhibitors of HIV Reverse Transcriptases

Reverse transcriptase (RT) inhibitors are core components of antiretroviral therapies; however, HIV’s high mutation rate prompts the emergence of drug resistance and necessitates new inhibitors with high genetic barriers to resistance. RNA aptamers that have been selected to bind RT exhibit potent RT inhibition and suppress viral replication when targeting the strain-specific RT that they were originally selected to bind, but some of these same, otherwise potent aptamers fail against both single-point mutant and phylogenetically-diverse RTs. We hypothesized that a subset of the total aptamer population in libraries pre-enriched against a single RT may exhibit broad-spectrum RT binding and inhibition, and we devised a multiplexed Poly-Target selection approach to elicit those phenotypes against a panel of diverse lentiviral RTs. High-throughput sequencing of starting, negative, and final libraries, followed by coenrichment analysis of parallel and duplicate selection trajectories, narrowed the list of candidate aptamers by orders of magnitude. Biochemical characterization of candidates identified a novel aptamer motif and several rare and unobserved variants of previously-known motifs that inhibited recombinant RTs from HIV-1, HIV-2 and SIV to varying degrees. These broad-spectrum aptamers also suppressed replication of viruses carrying phylogenetically-diverse RTs. The Poly-Target selection and coenrichment approach described herein is a generalizable strategy for identifying broad-spectrum behavior and cross-reactivity among related targets from combinatorial libraries.Aptamer selections often yield distinct subpopulations, each with unique phenotypes that can be leveraged for specialized applications. RNA aptamers that bind HIV-1 reverse transcriptase (RT) exhibit potent RT inhibition and suppress viral replication when targeting the strain-specific RT that they were originally selected to bind, but some of these same aptamers fail against single-point mutant and phylogenetically-diverse RTs. We hypothesized that a subset of the total aptamer population in libraries pre-enriched against a single RT may exhibit broad-spectrum RT binding and inhibition, and we devised a multiplexed Poly-Target selection approach to elicit those phenotypes against a panel of diverse primate lentiviral RTs. High-throughput sequencing of starting, negative, and final libraries, followed by analysis of coenrichment and codepletion in parallel and duplicate selection trajectories, narrowed the list of candidate aptamers by orders of magnitude. Biochemical characterization of candidates identified a novel aptamer motif and several rare and unobserved variants of previously-known motifs that inhibited recombinant RTs from HIV-1, HIV-2 and SIV to varying degrees. These broad-spectrum aptamers also suppressed replication of viral constructs carrying phylogenetically-diverse RTs. The Poly-Target selection and coenrichment approach described herein is a generalizable strategy for identifying broad-spectrum behavior and cross-reactivity among related targets from combinatorial libraries.

Activation of innate immune responses by a CpG oligonucleotide sequence composed entirely of threose nucleic acid.

Recent advances in synthetic biology have led to the development of nucleic acid polymers with backbone structures distinct from those found in nature, termed xeno-nucleic acids (XNAs). Several unique properties of XNAs make them attractive as nucleic acid therapeutics, most notably their high resistance to serum nucleases and ability to form Watson-Crick base-pairing with DNA and RNA. The ability of XNAs to induce immune responses has not been investigated. Threose nucleic acid (TNA), a type of XNA, is recalcitrant to nuclease digestion and capable of undergoing Darwinian evolution to produce high affinity aptamers; thus, TNA is an attractive candidate for diverse applications, including nucleic acid therapeutics. Here, we evaluated a TNA oligonucleotide derived from a CpG oligonucleotide sequence known to activate TLR9-dependent immune signaling in B cell lines. We observed a slight induction of relevant mRNA signals, robust B cell line activation, and negligible effects on cellular proliferation.

Poly-Target Selection Identifies Broad-Spectrum RNA Aptamers

Aptamer selections often yield distinct subpopulations, each with unique phenotypes that can be leveraged for specialized applications. Although most selections aim to attain ever higher specificity, we sought to identify aptamers that recognize increasingly divergent primate lentiviral reverse transcriptases (RTs). We hypothesized that aptamer subpopulations in libraries pre-enriched against a single RT may exhibit broad-spectrum binding and inhibition, and we devised a multiplexed poly-target selection to elicit those phenotypes against a panel of primate lentiviral RTs. High-throughput sequencing and coenrichment/codepletion analysis of parallel and duplicate selection trajectories rapidly narrowed the list of candidate aptamers by orders of magnitude and identified dozens of priority candidates for further screening. Biochemical characterization validated a novel aptamer motif and several rare and unobserved variants of previously known motifs that inhibited recombinant RTs to varying degrees. These broad-spectrum aptamers also suppressed replication of viral constructs carrying phylogenetically diverse RTs. The poly-target selection and coenrichment/codepletion approach described herein is a generalizable strategy for identifying cross-reactivity among related targets from combinatorial libraries.

141. Combinatorial Aptamer Transcripts (CATs) for HIV RT Suppression

The Human Immunodeficiency Virus (HIV) Reverse Transcriptase (RT) is a DNA polymerase encoded by the viral genome and it is preferentially targeted by current therapeutics due to its critical role in the viral life cycle. High affinity RNA aptamers that bind RT out-compete viral genome for access to the active site and thereby inhibit replication. Aptamer binding to RT during viral formation is believed to drive aptamer encapsidation into the budding virus, which leads to significantly reduced infectivity. We reasoned that co-transcribing multiple aptamer modules as combinatorial transcripts would increase avidity and packaging, resulting in greater net viral suppression. To test this hypothesis, we built a series of Combinatorial Aptamer Transcripts (CATs) carrying multiple, co-transcribed aptamer modules and compared their inhibitory capabilities as a function of valency. Specifically, aptamers from two different structural types (representing (6/5)AL and UCAA structural motifs) were combined into homodimeric and homotrimeric CATs.Two designs were explored for increasing valency; in the first we generated threads of homo-aptamer modules. In general, transcripts with more modules exhibited moderately increased RT inhibition in Primer Extension Assays and increased net binding affinity in Electrophoretic Mobility Shift Assays (EMSAs). The EMSAs do not show evidence of binding multiple RT per transcript, and preliminary cell-based assays do not indicate that this approach to multivalency improves net viral suppression. In contrast, an alternative design appears to be more promising, in which individual aptamer modules are incorporated into a stable3-Way Junction (3WJ) to increase modularity. The 3WJ structural core is expected both to isolate the individual aptamer structures from each other (and thereby reduce misfolding) and to allow sufficient separation to prevent steric hindrance in the binding of multiple RT. Other design features simplify the operational requirements of replacing individual modules with other aptamers or with libraries of aptamers. Initial data based on this design are encouraging.Combinatorial transcripts are expected to block evolutionary escape by forcing the virus to acquire multiple simultaneous mutations, especially for designs that include hetero-multimers. It is well established that combinations of small molecule drugs or of siRNA improves their targeting, delivery, and potency, while reducing their susceptibility to escape mutations. By demonstrating the benefits of multimerization we will inform downstream utilization of CATs as a therapeutic design in the treatment of HIV.

137. Viral Hitchhiking to Increase Therapeutic Potential: Autonomously Packaged Elements (APEs)

Human Immunodeficiency Virus (HIV) preferentially packages its own RNA genome and various RNA transcripts (tRNA-Lys3, 7SL, aptamers, etc.) with a broad range of consequences. For example, genome and tRNA are required for viral life cycle (positive impact on viral fitness), various cellular RNAs such as 7SL appear to “hitchhike” with no obvious essential roles (neutral), and aptamers that bind RT exert their influence by co-packaging with the viral proteins (inhibitory). While the mechanisms that promote selective incorporation of these transcripts have not been fully described, it is generally accepted that RNA secondary structures allow the RNA to associate with viral proteins during packaging. Identification of these RNA secondary structures would allow for exploitation of a range of packaging mechanisms to drive RNA incorporation for therapeutic purposes. We therefore applied a novel in vivo selection to identify RNAs specifically associated with incorporation into virus particles.We have previously demonstrated that multiple RNA aptamers selected against HIV Reverse Transcriptase (RT) are incorporated into the budding virus– potentially through interactions with the RT– and that they effectively reduce infectivity. To identify conserved features of highly efficient aptamer packagers, we have developed a cell-based protocol for the selection of Autonomously Packaging Elements (APEs). Pre-enriched RNA aptamer libraries were transfected into virus producing cells where we exploit the intracellular viral life cycles to perform the selection of APEs. We inserted an aptamer library downstream of a CMV promoter and co-transfected the library with HIV proviral plasmids. This generated pseudotyped virus particles containing the packaged RNA aptamers, while the cells retain the non-packaged RNA. By reverse transcribing the packaged RNA component, then amplifying and re-inserting it into the expression vector and iteratively transfecting fresh producer cells, we increase packaging competition and aptamer specificity. Subsequent High-Throughput Sequencing and computational analysis will allow us to probe the sequence and structural requirements of APEs. Individual aptamers will be evaluated for their packaging affinity and ability to inhibit viral replication in cell culture. Expanding on this selection scheme by swapping out the RT in the transfection with a panel of divergent RTs is expected to identify aptamer motifs which target evolutionarily conserved RT residues. Application of the APE selection system to an extended viral panel utilizing a range of starting libraries allows for nearly limitless variability in selection output.