Aptamers in neuro-oncology: An emerging therapeutic modality.

Despite recent advances in the understanding of brain tumor pathophysiology, challenges associated with tumor location and characteristics have prevented significant improvement in neuro-oncology therapies. Aptamers are short, single-stranded DNA or RNA oligonucleotides that fold into sequence-specific, 3-dimensional shapes that, like protein antibodies, interact with targeted ligands with high affinity and specificity. Aptamer technology has recently been applied to neuro-oncology as a potential approach to innovative therapy. Preclinical research has demonstrated the ability of aptamers to overcome some obstacles that have traditionally rendered neuro-oncology therapies ineffective. Potential aptamer advantages include their small size, ability in some cases to penetrate the blood-brain barrier, inherent lack of immunogenicity, and applicability for discovering novel biomarkers. Herein, we review recent reports of aptamer applications in neuro-oncology including aptamers found by cell- and in vivo- Systematic Evolution of Ligands by Exponential Enrichment approaches, aptamer-targeted therapeutic delivery modalities, and aptamers in diagnostics and imaging. We further identify crucial future directions for the field that will be important to advance aptamer-based drugs or tools to clinical application in neuro-oncology.

A non-rational approach to optimized targeting of CRISPR-Cas9 complexes.

In this issue of Cell Chemical Biology, Bush et al.1 report an in vitro selection method for optimizing CRISPR-Cas9 single-guide RNAs. This approach may be useful in targeting previously intractable genomic sequences. The results also provide insights into which positions in single-guide RNAs are most amenable to modification.

Peroxidase proximity selection to identify aptamers targeting a subcellular location.

The efficient and specific delivery of functional cargos such as small-molecule drugs, proteins, or nucleic acids across lipid membranes and into subcellular compartments is a significant unmet need in nanomedicine and molecular biology. Systematic Evolution of Ligands by EXponential enrichment (SELEX) exploits vast combinatorial nucleic acid libraries to identify short, nonimmunogenic single-stranded DNA molecules (aptamers) capable of recognizing specific targets based on their 3D structures and molecular interactions. While SELEX has previously been applied to identify aptamers that bind specific cell types or gain cellular uptake, selection of aptamers capable of carrying cargos to specific subcellular compartments is challenging. Here, we describe peroxidase proximity selection (PPS), a generalizable subcellular SELEX approach. We implement local expression of engineered ascorbate peroxidase APEX2 to biotinylate naked DNA aptamers capable of gaining access to the cytoplasm of living cells without assistance. We discovered DNA aptamers that are preferentially taken up into endosomes by macropinocytosis, with a fraction apparently accessing APEX2 in the cytoplasm. One of these selected aptamers is capable of endosomal delivery of an IgG antibody.

DNA Modifications Enabling Proximity Biotinylation.

Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" that react with phenoxy radicals or biotinoyl-5'-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.

A suppressor of dioxygenase inhibition in a yeast model of SDH deficiency.

A fascinating class of familial paraganglioma (PGL) neuroendocrine tumors is driven by the loss of the tricarboxylic acid (TCA) cycle enzyme succinate dehydrogenase (SDH) resulting in succinate accumulation as an oncometabolite and other metabolic derangements. Here, we exploit a Saccharomyces cerevisiae yeast model of SDH loss where accumulating succinate, and possibly reactive oxygen species, poison a dioxygenase enzyme required for sulfur scavenging. Using this model, we performed a chemical suppression screen for compounds that relieve dioxygenase inhibition. After testing 1280 pharmaceutically active compounds, we identified meclofenoxate HCl and its hydrolysis product, dimethylaminoethanol (DMAE), as suppressors of dioxygenase intoxication in SDH-loss yeast cells. We show that DMAE acts to alter metabolism so as to normalize the succinate:2-ketoglutarate ratio, improving dioxygenase function. This study raises the possibility that oncometabolite effects might be therapeutically suppressed by drugs that rewire metabolism to reduce the flux of carbon into pathological metabolic pathways.

Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.

Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H2 O2 is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.

Remyelination-Promoting DNA Aptamer Conjugate Myaptavin-3064 Binds to Adult Oligodendrocytes In Vitro.

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.

An in Vitro Selection Strategy Identifying Naked DNA That Localizes to Cell Nuclei.

Combinatorial chemistry drives the biological generation of protein structural diversity in antibodies and T-cell receptors. When applied to nucleic acids, vast engineered random libraries of DNA and RNA strands allow selection of affinity reagents ("aptamers") against molecular targets. Selection involves cycles rewarding target binding affinity with amplification. Despite the success of this approach, delivery of selected aptamers across cell membranes and to specific subcellular compartments is an unmet need in chemical biology. Here, we address this challenge, demonstrating in vitro selection of DNA aptamers capable of homing to nuclei of cultured cells without transfection agents or viral transduction. Selection of such folded karyophilic DNA aptamers (∼100 nucleotides) is achieved by a biosensor strategy that rewards exposure to nuclear DNA ligase. Identified DNA molecules are preferentially delivered to cell nuclei within minutes. Related strategies can be envisioned to select aptamers that home to other subcellular compartments.

Glial cells as therapeutic targets in progressive multiple sclerosis.

Introduction: Multiple sclerosis is a serious demyelinating disease of the central nervous system (CNS) with treatments generally restricted to immunosuppression to reduce attack rate and for symptom management. Glial cells may be useful targets for future CNS regenerative therapies to reverse disease. Areas covered: In this review, the authors cover currently available multiple sclerosis treatments and examine potential upcoming therapies targeting glial cells. The potential for new therapeutic approaches in the treatment of progressive multiple sclerosis is examined. Expert opinion: Microglia, astrocytes, and oligodendrocytes are each promising targets for the disease-altering treatment of multiple sclerosis. Though challenging, the opportunities presented have great potential for CNS regeneration and further investigation of glial cells in therapy is warranted. Patient-specific combinatorial therapy targeting the three glial cell types is expected to be the future of MS treatment.

Optimization of a 40-mer Antimyelin DNA Aptamer Identifies a 20-mer with Enhanced Properties for Potential Multiple Sclerosis Therapy.

We previously reported the in vitro selection and characterization of a DNA aptamer capable of stimulating remyelination in a mouse model of multiple sclerosis. This aptamer was selected for its ability to bind to suspensions of crude murine myelin in vitro. Our initial studies in vitro and in vivo involved a 40-nucleotide derivative (LJM-3064) of the original 100-nucleotide aptamer. LJM-3064 retained robust myelin-binding properties. Structural characterization of LJM-3064 revealed that the guanosine-rich 5' half of the sequence forms different G-quadruplex-type structures that are variably stable in the presence of physiologically relevant ions. We hypothesized that this structured domain is sufficient for myelin binding. In this study, we confirm that a 20-nucleotide DNA, corresponding to the 5' half of LJM-3064, retains myelin-binding properties. We then optimize this minimal myelin-binding aptamer via systematic evolution of ligands by exponential enrichment after sparse rerandomization. We report a sequence variant (LJM-5708) of the 20-nucleotide myelin-binding aptamer with enhanced myelin-binding properties and the ability to bind cultured human oligodendroglioma cells in vitro, providing the first evidence of cross-species reactivity of this myelin-binding aptamer. As our formulation of DNA aptamers for in vivo remyelination therapy involves conjugation to streptavidin, we verified that the myelin-binding properties of LJM-5708 were retained in conjugates to avidin, streptavidin, and neutravidin. DNA aptamer LJM-5708 is a lead for further preclinical development of remyelinating aptamer technologies.

An Assay that Predicts In Vivo Efficacy for DNA Aptamers that Stimulate Remyelination in a Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a debilitating disease for which regenerative therapies are sought. We have previously described human antibodies and DNA aptamer-streptavidin conjugates that promote remyelination after systemic injection into mice infected by Theiler's murine encephalomyelitis virus. Here, we report an in vitro assay of myelin binding with results that correlate with remyelination outcome in vivo, as shown for data from a set of DNA aptamer complexes of different size and formulation. This in vitro assay will be valuable for future screening of MS regenerative therapies targeting remyelination.

A Prototype for Modular Cell Engineering.

When aiming to produce a target chemical at high yield, titer, and productivity, various combinations of genetic parts available to build the target pathway can generate a large number of strains for characterization. This engineering approach will become increasingly laborious and expensive when seeking to develop desirable strains for optimal production of a large space of biochemicals due to extensive screening. Our recent theoretical development of modular cell (MODCELL) design principles can offer a promising solution for rapid generation of optimal strains by coupling a modular cell with exchangeable production modules in a plug-and-play fashion. In this study, we experimentally validated some design properties of MODCELL by demonstrating the following: (i) a modular (chassis) cell is required to couple with a production module, a heterologous ethanol pathway, as a testbed, (ii) degree of coupling between the modular cell and production modules can be modulated to enhance growth and product synthesis, (iii) a modular cell can be used as a host to select an optimal pyruvate decarboxylase (PDC) of the ethanol production module and to help identify a hypothetical PDC protein, and (iv) adaptive laboratory evolution based on growth selection of the modular cell can enhance growth and product synthesis rates. We envision that the MODCELL design provides a powerful prototype for modular cell engineering to rapidly create optimal strains for synthesis of a large space of biochemicals.

Comprehensive characterization of toxicity of fermentative metabolites on microbial growth.

BACKGROUND: Volatile carboxylic acids, alcohols, and esters are natural fermentative products, typically derived from anaerobic digestion. These metabolites have important functional roles to regulate cellular metabolisms and broad use as food supplements, flavors and fragrances, solvents, and fuels. Comprehensive characterization of toxic effects of these metabolites on microbial growth under similar conditions is very limited. RESULTS: We characterized a comprehensive list of thirty-two short-chain carboxylic acids, alcohols, and esters on microbial growth of Escherichia coli MG1655 under anaerobic conditions. We analyzed toxic effects of these metabolites on E. coli health, quantified by growth rate and cell mass, as a function of metabolite types, concentrations, and physiochemical properties including carbon number, chemical functional group, chain branching feature, energy density, total surface area, and hydrophobicity. Strain characterization revealed that these metabolites exert distinct toxic effects on E. coli health. We found that higher concentrations and/or carbon numbers of metabolites cause more severe growth inhibition. For the same carbon numbers and metabolite concentrations, we discovered that branched chain metabolites are less toxic than the linear chain ones. Remarkably, shorter alkyl esters (e.g., ethyl butyrate) appear less toxic than longer alkyl esters (e.g., butyl acetate). Regardless of metabolites, hydrophobicity of a metabolite, governed by its physiochemical properties, strongly correlates with the metabolite's toxic effect on E. coli health. CONCLUSIONS: Short-chain alcohols, acids, and esters exhibit distinctive toxic effects on E. coli health. Hydrophobicity is a quantitative predictor to evaluate the toxic effect of a metabolite. This study sheds light on degrees of toxicity of fermentative metabolites on microbial health and further helps in the selection of desirable metabolites and hosts for industrial fermentation to overproduce them.