Ubiquitinated histone H2B as gatekeeper of the nucleosome acidic patch.

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.

A comparative analysis of cell surface targeting aptamers.

Aptamers represent a potentially important class of ligands for the development of diagnostics and therapeutics. However, it is often difficult to compare the function and specificity of many of these molecules as assay formats and conditions vary greatly. Here, with an interest in developing aptamer targeted therapeutics that could effectively deliver cargoes to cells, we chemically synthesize 15 aptamers that have been reported to target cell surface receptors or cells. Using standardized assay conditions, we assess each aptamer's binding properties on a panel of 11 different cancer cell lines, correlate aptamer binding to antibody controls and use siRNA transfection to validate each aptamer's binding to reported target receptors. Using a subset of these molecules known to be expressed on prostate cancers, we use near-infrared in vivo imaging to assess the tumor localization following intravenous injection. Our data demonstrate some surprising differences in the reported specificity and function for many of these molecules and raise concerns regarding their cell targeting capabilities. They also identify an anti-human transferrin aptamer, Waz, as a robust candidate for targeting prostate cancers and for future development of aptamer-based therapeutics.

AptCompare: optimized de novo motif discovery of RNA aptamers via HTS-SELEX.

SUMMARY: High-throughput sequencing can enhance the analysis of aptamer libraries generated by the Systematic Evolution of Ligands by EXponential enrichment. Robust analysis of the resulting sequenced rounds is best implemented by determining a ranked consensus of reads following the processing by multiple aptamer detection algorithms. While several such approaches have been developed to this end, their installation and implementation is problematic. We developed AptCompare, a cross-platform program that combines six of the most widely used analytical approaches for the identification of RNA aptamer motifs and uses a simple weighted ranking to order the candidate aptamers, all driven within the same GUI-enabled environment. We demonstrate AptCompare's performance by identifying the top-ranked candidate aptamers from a previously published selection experiment in our laboratory, with follow-up bench assays demonstrating good correspondence between the sequences' rankings and their binding affinities. AVAILABILITY AND IMPLEMENTATION: The source code and pre-built virtual machine images are freely available at https://bitbucket.org/shiehk/aptcompare. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Covalently tethering siRNA to hydrogels for localized, controlled release and gene silencing.

Small interfering RNA (siRNA) has found many applications in tissue regeneration and disease therapeutics. Effective and localized siRNA delivery remains challenging, reducing its therapeutic potential. Here, we report a strategy to control and prolong siRNA release by directly tethering transfection-capable siRNA to photocrosslinked dextran hydrogels. siRNA release is governed via the hydrolytic degradation of ester and/or disulfide linkages between the siRNA and hydrogels, which is independent of hydrogel degradation rate. The released siRNA is shown to be bioactive by inhibiting protein expression in green fluorescent protein-expressing HeLa cells without the need of a transfection agent. This strategy provides an excellent platform for controlling nucleic acid delivery through covalent bonds with a biomaterial and regulating cellular gene expression, which has promising potential in many biomedical applications.

To PEGylate or Not To PEGylate Therapeutics?

PEGylation is a common modulator of pharmacokinetics for therapeutic agents in vivo. In this issue of Cell Chemical Biology, Moreno et al. (2019) show anti-PEG antibodies may directly inhibit the activity of a PEGylated aptamer, RB006, both in vitro and in vivo, and the issues surrounding anti-PEG antibodies are discussed.

The role of microsporidian polar tube protein 4 (PTP4) in host cell infection.

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

A New Transferrin Receptor Aptamer Inhibits New World Hemorrhagic Fever Mammarenavirus Entry.

Pathogenic New World hemorrhagic fever mammarenaviruses (NWM) utilize Glycoprotein 1 (GP1) to target the apical domain of the human transferrin receptor (hTfR) for facilitating cell entry. However, the conservation between their GP1s is low. Considering this and the slow evolutionary progression of mammals compared to viruses, therapeutic targeting of hTfR provides an attractive avenue for cross-strain inhibition and diminishing the likelihood of escape mutants. Aptamers present unique advantages for the development of inhibitors to vial entry, including ease of synthesis, lack of immunogenicity, and potentially cold-chain breaking solutions to diseases endemic to South America. Here, recognizing that in vivo competition with the natural ligand, transferrin (Tf), likely drove the evolution of GP1 to recognize the apical domain, we performed competitive in vitro selections against hTfR-expressing cells with supplemented Tf. The resultant minimized aptamer, Waz, binds the apical domain of the receptor and inhibits infection of human cells by recombinant NWM in culture (EC50 ~400 nmol/l). Aptamer multimerization further enhanced inhibition >10-fold (EC50 ~30 nmol/l). Together, our results highlight the ability to use a competitor to bias the outcome of a selection and demonstrate how avidity effects can be leveraged to enhance both aptamer binding and the potency of viral inhibition.

Improved Synthesis and In Vitro Evaluation of an Aptamer Ribosomal Toxin Conjugate.

Delivery of toxins, such as the ricin A chain, Pseudomonas exotoxin, and gelonin, using antibodies has had some success in inducing specific toxicity in cancer treatments. However, these antibody-toxin conjugates, called immunotoxins, can be bulky, difficult to express, and may induce an immune response upon in vivo administration. We previously reported delivery of a recombinant variant of gelonin (rGel) by the full-length prostate-specific membrane antigen (PSMA) binding aptamer, A9, to potentially circumvent some of these problems. Here, we report a streamlined approach to generating aptamer-rGel conjugates utilizing a chemically synthesized minimized form of the A9 aptamer. Unlike the full-length A9 aptamer, this minimized variant can be chemically synthesized with a 5' terminal thiol. This facilitates the large scale synthesis and generation of aptamer toxin conjugates linked by a reducible disulfide linkage. Using this approach, we generated aptamer-toxin conjugates and evaluated their binding specificity and toxicity. On PSMA(+) LNCaP prostate cancer cells, the A9.min-rGel conjugate demonstrated an IC50 of ∼60 nM. Additionally, we performed a stability analysis of this conjugate in mouse serum where the conjugate displayed a t1/2 of ∼4 h, paving the way for future in vivo experiments.

From selection hits to clinical leads: progress in aptamer discovery.

Aptamers were discovered more than 25 years ago, yet only one has been approved by the US Food and Drug Administration to date. With some noteworthy advances in their chemical design and the enzymes we use to make them, aptamers and aptamer-based therapeutics have seen a resurgence in interest. New aptamer drugs are being approved for clinical evaluation, and it is certain that we will see increasingly more aptamers and aptamer-like drugs in the future. In this review, we will discuss the production of aptamers with an emphasis on the advances and modifications that enabled early aptamers to succeed in clinical trials as well as those that are likely to be important for future generations of these drugs.

Delivery of siRNAs to Dendritic Cells Using DEC205-Targeted Lipid Nanoparticles to Inhibit Immune Responses.

Due to their ability to knock down the expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases, including those involving "undruggable" targets. However, the therapeutic potential of siRNAs remains severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed for hepatic siRNA delivery. However, their suitability for delivery to other cell types has not been determined. We have modified LNPs for preferential targeting to dendritic cells (DCs), central regulators of immune responses. To achieve directed delivery, we coated LNPs with a single-chain antibody (scFv; DEC-LNPs), specific to murine DEC205, which is highly expressed on distinct DC subsets. Here we show that injection of siRNAs encapsulated in DEC-LNPs are preferentially delivered to DEC205(+) DCs. Gene knockdown following uptake of DEC-LNPs containing siRNAs specific for the costimulatory molecules CD40, CD80, and CD86 dramatically decreases gene expression levels. We demonstrate the functionality of this knockdown with a mixed lymphocyte response (MLR). Overall, we report that injection of LNPs modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the MLR.

A nanoparticle delivery vehicle for S-nitroso-N-acetyl cysteine: sustained vascular response.

Interest in the development of nitric oxide (NO) based therapeutics has grown exponentially due to its well elucidated and established biological functions. In line with this surge, S-nitroso thiol (RSNO) therapeutics are also receiving more attention in recent years both as potential stable sources of NO as well as for their ability to serve as S-nitrosating agents; S-nitrosation of protein thiols is implicated in many physiological processes. We describe two hydrogel based RSNO containing nanoparticle platforms. In one platform the SNO groups are covalently attached to the particles (SNO-np) and the other contains S-nitroso-N-acetyl cysteine encapsulated within the particles (NAC-SNO-np). Both platforms function as vehicles for sustained activity as trans-S-nitrosating agents. NAC-SNO-np exhibited higher efficiency for generating GSNO from GSH and maintained higher levels of GSNO concentration for longer time (24 h) as compared to SNO-np as well as a previously characterized nitric oxide releasing platform, NO-np (nitric oxide releasing nanoparticles). In vivo, intravenous infusion of the NAC-SNO-np and NO-np resulted in sustained decreases in mean arterial pressure, though NAC-SNO-np induced longer vasodilatory effects as compared to the NO-np. Serum chemistries following infusion demonstrated no toxicity in both treatment groups. Together, these data suggest that the NAC-SNO-np represents a novel means to both study the biologic effects of nitrosothiols and effectively capitalize on its therapeutic potential.

An RNA alternative to human transferrin: a new tool for targeting human cells.

The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery.

Identification of antibiotics using small molecule variable ligand display on gold nanoparticles.

Here we describe the use of simple 1-pot thiol exchange reactions to generate a library of mixed ligand-coated gold nanoparticles that was screened for antibiotic activity. A library of 120 nanoparticle conjugates was assembled and antibiotic activity toward E. coli was determined and found to depend upon the combination of thiols assembled onto the nanoparticles. The most active conjugate displayed 99.9% growth inhibition at 0.5 µM.