Conversion of RNA Aptamer into Modified DNA Aptamers Provides for Prolonged Stability and Enhanced Antitumor Activity.

Aptamers, synthetic single-strand oligonucleotides that are similar in function to antibodies, are promising as therapeutics because of their minimal side effects. However, the stability and bioavailability of the aptamers pose a challenge. We developed aptamers converted from RNA aptamer to modified DNA aptamers that target phospho-AXL with improved stability and bioavailability. On the basis of the comparative analysis of a library of 17 converted modified DNA aptamers, we selected aptamer candidates, GLB-G25 and GLB-A04, that exhibited the highest bioavailability, stability, and robust antitumor effect in in vitro experiments. Backbone modifications such as thiophosphate or dithiophosphate and a covalent modification of the 5'-end of the aptamer with polyethylene glycol optimized the pharmacokinetic properties, improved the stability of the aptamers in vivo by reducing nuclease hydrolysis and renal clearance, and achieved high and sustained inhibition of AXL at a very low dose. Treatment with these modified aptamers in ovarian cancer orthotopic mouse models significantly reduced tumor growth and the number of metastases. This effective silencing of the phospho-AXL target thus demonstrated that aptamer specificity and bioavailability can be improved by the chemical modification of existing aptamers for phospho-AXL. These results lay the foundation for the translation of these aptamer candidates and companion biomarkers to the clinic.

Selection and Characterization of Vimentin-Binding Aptamer Motifs for Ovarian Cancer.

The application of aptamers in biomedicine is emerging as an essential technology in the field of cancer research. As small single-stranded DNA or RNA ligands with high specificity and low immunogenicity for their targets, aptamers provide many advantages in cancer therapeutics over protein-based molecules, such as antibodies. Vimentin is an intermediate filament protein that is overexpressed in endothelial cells of cancerous tissue. High expression levels of vimentin have been associated with increased capacity for migration and invasion of the tumor cells. We have selected and identified thioated aptamers with high specificity for vimentin using human ovarian cancer tissues. Tentative binding motifs were chosen for two vimentin aptamers based on predicted secondary structures. Each of these shorter, tentative binding motifs was synthesized, purified, and characterized via cell binding assays. Two vimentin binding motifs with high fidelity binding were selected and further characterized via cell and tissue binding assays, as well as flow cytometric analysis. The equilibrium binding constants of these small thioated aptamer constructs were also determined. Future applications for the vimentin binding aptamer motifs include conjugation of the aptamers to synthetic dyes for use in targeted imaging and therapy, and ultimately more detailed and precise monitoring of treatment response and tumor progression in ovarian pathology.

Bioinformatics service center projects go beyond service.

To probe the complexity of modern diseases, multidisciplinary approaches are increasingly applied. Typically underpinning such studies are collaborations between wet bench experimentalists and dry lab bioinformaticians. Despite the need, bioinformatics collaborators remain difficult to find. Therefore, we undertook a study to understand the nature of this research, so that we may better understand how to meet the needs of future multidisciplinary projects. To accomplish this, we have performed a retrospective study of data from three years of projects performed by the UTHealth Bioinformatics Service Center. Based on this, we found that the bioinformatics in these collaborative projects are extremely diverse and require a high degree of intellectual engagement, while requiring only a small amount of publishable methods development. Very few of the specific skills, the strength of a service core, could be recycled across projects, which were generally exploratory and open-ended and required cycles of biological hypothesis development and (in silico) testing. We find that biomedical research requires bioinformaticians that are highly trained, having the ability to think biologically, but investigating using computational rather than bench experiments. This is in contrast to the activities that are typically the basis for an independent career in biomedical informatics, namely developing new software and algorithms. These findings suggest that to foster team-based multidisciplinary research, institutions must adopt policies that recognize contributions to research by applied bioinformatics scientists.

DNA Thioaptamer with Homing Specificity to Lymphoma Bone Marrow Involvement.

Selective drug accumulation in the malignant tissue is a prerequisite for effective cancer treatment. However, most drug molecules and their formulated particles are blocked en route to the destiny tissue due to the existence of multiple biological and physical barriers including the tumor microvessel endothelium. Since the endothelial cells on the surface of the microvessel wall can be modulated by inflammatory cytokines and chemokines secreted by the tumor or stromal cells, an effective drug delivery approach is to enhance interaction between the drug particles and the unique spectrum of surface proteins on the tumor endothelium. In this study, we performed in vivo screening for thioaptamers that bind to the bone marrow endothelium with specificity in a murine model of lymphoma with bone marrow involvement (BMI). The R1 thioaptamer was isolated based on its high homing potency to bones with BMI, and 40-60% less efficiency in accumulation to healthy bones. In cell culture, R1 binds to human umbilical vein endothelial cells (HUVEC) with a high affinity ( Kd ≈ 3 nM), and the binding affinity can be further enhanced when cells were treated with a mixture of lymphoma cell and bone marrow cell conditioned media. Cellular uptake of R1 is through clathrin-mediated endocytosis. Conjugating R1 on to the surface of liposomal doxorubicin nanoparticles resulted in 2-3-fold increase in drug accumulation in lymphoma BMI. Taking together, we have successfully identified a thioaptamer that preferentially binds to the endothelium of lymphoma BMI. It can serve as an affinity moiety for targeted delivery of drug particles to the disease organ.

A Unique and Simple Approach to Improve Sensitivity in 15N-NMR Relaxation Measurements for NH3⁺ Groups: Application to a Protein-DNA Complex.

NMR spectroscopy is a powerful tool for research on protein dynamics. In the past decade, there has been significant progress in the development of NMR methods for studying charged side chains. In particular, NMR methods for lysine side-chain NH3⁺ groups have been proven to be powerful for investigating the dynamics of hydrogen bonds or ion pairs that play important roles in biological processes. However, relatively low sensitivity has been a major practical issue in NMR experiments on NH3⁺ groups. In this paper, we present a unique and simple approach to improve sensitivity in 15N relaxation measurements for NH3⁺ groups. In this approach, the efficiency of coherence transfers for the desired components are maximized, whereas undesired anti-phase or multi-spin order components are purged through pulse schemes and rapid relaxation. For lysine side-chain NH3⁺ groups of a protein-DNA complex, we compared the data obtained with the previous and new pulse sequences under the same conditions and confirmed that the 15N relaxation parameters were consistent for these datasets. While retaining accuracy in measuring 15N relaxation, our new pulse sequences for NH3⁺ groups allowed an 82% increase in detection sensitivity of 15N longitudinal and transverse relaxation measurements.

Safety evaluation of intravenously administered mono-thioated aptamer against E-selectin in mice.

The medical applications of aptamers have recently emerged. We developed an antagonistic thioaptamer (ESTA) against E-selectin. Previously, we showed that a single injection of ESTA at a dose of 100µg inhibits breast cancer metastasis in mice through the functional blockade of E-selectin. In the present study, we evaluated the safety of different doses of intravenously administered ESTA in single-dose acute and repeat-dose subacute studies in ICR mice. Our data indicated that intravenous administration of up to 500µg ESTA did not result in hematologic abnormality in either study. Additionally, intravenous injection of ESTA did not affect the levels of plasma cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α) or complement split products (C3a and C5a) in either study. However, repeated injections of ESTA slightly increased plasma ALT and AST activities, in accordance with the appearance of small necrotic areas in the liver. In conclusion, our data demonstrated that intravenous administration of ESTA does not cause overt hematologic, organs, and immunologic responses under the experimental conditions.

Aptaligner: automated software for aligning pseudorandom DNA X-aptamers from next-generation sequencing data.

Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provides this feature as well as length error and noise level cutoff features, is parallelized to run on multiple central processing units (cores), and sorts sequences from a single chip into projects and subprojects.

Thioaptamers targeting dengue virus type-2 envelope protein domain III.

Thioaptamers targeting the dengue-2 virus (DENV-2) envelope protein domain III (EDIII) were developed. EDIII, which contains epitopes for binding neutralizing antibodies, is the putative host-receptor binding domain and is thus an attractive target for development of vaccines, anti-viral therapeutic and diagnostic agents. Thioaptamer DENTA-1 bound to DENV-2 EDIII adjacent to a known neutralizing antibody binding site with a dissociation constant of 154nM.

Aggregation behavior of ibuprofen, cholic acid and dodecylphosphocholine micelles.

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID-BA or NSAID-BA-PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA-IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA-IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA-IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA-IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.

Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most widely consumed pharmaceuticals, yet both the mechanisms involved in their therapeutic actions and side-effects, notably gastrointestinal (GI) ulceration/bleeding, have not been clearly defined. In this study, we have used a number of biochemical, structural, computational and biological systems including; Fourier Transform InfraRed (FTIR). Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) spectroscopy, and cell culture using a specific fluorescent membrane probe, to demonstrate that NSAIDs have a strong affinity to form ionic and hydrophobic associations with zwitterionic phospholipids, and specifically phosphatidylcholine (PC), that are reversible and non-covalent in nature. We propose that the pH-dependent partition of these potent anti-inflammatory drugs into the phospholipid bilayer, and possibly extracellular mono/multilayers present on the luminal interface of the mucus gel layer, may result in profound changes in the hydrophobicity, fluidity, permeability, biomechanical properties and stability of these membranes and barriers. These changes may not only provide an explanation of how NSAIDs induce surface injury to the GI mucosa as a component in the pathogenic mechanism leading to peptic ulceration and bleeding, but potentially an explanation for a number of (COX-independent) biological actions of this family of pharmaceuticals. This insight also has proven useful in the design and development of a novel class of PC-associated NSAIDs that have reduced GI toxicity while maintaining their essential therapeutic efficacy to inhibit pain and inflammation.

X-aptamers: a bead-based selection method for random incorporation of druglike moieties onto next-generation aptamers for enhanced binding.

By combining pseudorandom bead-based aptamer libraries with conjugation chemistry, we have created next-generation aptamers, X-aptamers (XAs). Several X-ligands can be added in a directed or random fashion to the aptamers to further enhance their binding affinities for the target proteins. Here we describe the addition of a drug (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid), demonstrated to bind to CD44-HABD, to a complete monothioate backbone-substituted aptamer to increase its binding affinity for the target protein by up to 23-fold, while increasing the drug's level of binding 1-million fold.

NMR structure of human thymosin alpha-1.

800 MHz NMR structure of the 28-residue peptide thymosin alpha-1 in 40% TFE/60% water (v/v) has been determined. Restrained molecular dynamic simulations with an explicit solvent box containing 40% TFE/60% TIP3P water (v/v) were used, in order to get the 3D model of the NMR structure. We found that the peptide adopts a structured conformation having two stable regions: an alpha-helix region from residues 14 to 26 and two double ß-turns in the N-terminal twelve residues which form a distorted helical structure.

¹H and ³¹P NMR lipidome of ethanol-induced fatty liver.

BACKGROUND: Hepatic steatosis (fatty liver), an early and reversible stage of alcoholic liver disease, is characterized by triglyceride deposition in hepatocytes, which can advance to steatohepatitis, fibrosis, cirrhosis, and ultimately to hepatocellular carcinoma. In the present work, we studied altered plasma and hepatic lipid metabolome (lipidome) to understand the mechanisms and lipid pattern of early-stage alcohol-induced-fatty liver. METHODS: Male Fischer 344 rats were fed 5% alcohol in a Lieber-DeCarli diet. Control rats were pair-fed an equivalent amount of maltose-dextrin. After 1 month, animals were killed and plasma collected. Livers were excised for morphological, immunohistochemical, and biochemical studies. The lipids from plasma and livers were extracted with methyl-tert-butyl ether and analyzed by 750/800 MHz proton nuclear magnetic resonance (¹H NMR) and phosphorus (³¹P) NMR spectroscopy on a 600 MHz spectrometer. The NMR data were then subjected to multivariate statistical analysis. RESULTS: Hematoxylin and Eosin and Oil Red O stained liver sections showed significant fatty infiltration. Immunohistochemical analysis of liver sections from ethanol-fed rats showed no inflammation (absence of CD3 positive cells) or oxidative stress (absence of malondialdehyde reactivity or 4-hydroxynonenal positive staining). Cluster analysis and principal component analysis of ¹H NMR data of lipid extracts of both plasma and livers showed a significant difference in the lipid metabolome of ethanol-fed versus control rats. ³¹P NMR data of liver lipid extracts showed significant changes in phospholipids similar to ¹H NMR data. ¹H NMR data of plasma and liver reflected several changes, while comparison of ¹H NMR and ³¹P NMR data offered a correlation among the phospholipids. CONCLUSIONS: Our results show that alcohol consumption alters metabolism of cholesterol, triglycerides, and phospholipids that could contribute to the development of fatty liver. These studies also indicate that fatty liver precedes oxidative stress and inflammation. The similarities observed in plasma and liver lipid profiles offer a potential methodology for detecting early-stage alcohol-induced fatty liver disease by analyzing the plasma lipid profile.

Functional Blockade of E-Selectin in Tumor-Associated Vessels Enhances Anti-Tumor Effect of Doxorubicin in Breast Cancer.

Chemotherapy is a mainstay of treatment for solid tumors. However, little is known about how therapy-induced immune cell infiltration may affect therapy response. We found substantial CD45+ immune cell density adjacent to E-selectin expressing inflamed vessels in doxorubicin (DOX)-treated residual human breast tumors. While CD45 level was significantly elevated in DOX-treated wildtype mice, it remained unchanged in DOX-treated tumors from E-selectin null mice. Similarly, intravenous administration of anti-E-selectin aptamer (ESTA) resulted in a significant reduction in CD45+ immune cell density in DOX-treated residual tumors, which coincided with a delay in tumor growth and lung metastasis in MMTV-pyMT mice. Additionally, both tumor infiltrating T-lymphocytes and tumor associated-macrophages were skewed towards TH2 in DOX-treated residual breast tumors; however, ESTA suppressed these changes. This study suggests that DOX treatment instigates de novo intratumoral infiltration of immune cells through E-selectin, and functional blockade of E-selectin may reduce residual tumor burden as well as metastasis through suppression of TH2 shift.

Investigation of chemometric instrumental transfer methods for high-resolution NMR.

The implementation of direct standardization (DS), piecewise direct standardization (PDS), and double-window piecewise direct standardization (DWPDS) instrumental transfer techniques for high-resolution (1)H NMR spectral data was explored. The ability to transfer a multivariate calibration model developed for a "master or target" NMR instrument configuration to seven different ("secondary") NMR instrument configurations was measured. Partial least-squares (PLS) calibration of glucose, glycine, and citrate metabolite relative concentrations in model mixtures following mapping of the secondary instrumental configurations using DS, PDS, or DWPDS instrumental transfer allowed the performance of the different transfer methods to be assessed. Results from these studies suggest that DS and PDS transfer techniques produce similar improvements in the error of prediction compared to each other and provide a significant improvement over standard spectral preprocessing techniques including reference deconvolution and spectral binning. The DS instrumental transfer method produced the largest percent improvement in the predictions of concentrations for these model mixtures but, in general, required that additional transfer calibration standards be used. Limitations of the different instrumental transfer methods with respect to sample subset selection are also discussed.

X-Aptamer Technology Identifies C4A and ApoB in Blood as Potential Markers for Schizophrenia.

The field of proteomics is rapidly gaining territory as a promising alternative to genomic approaches in the efforts to unravel the complex molecular mechanisms underlying schizophrenia and other psychiatric disorders. X-aptamer tech-nology has emerged as a novel proteomic approach for high-sensitivity analyses, and we hypothesized that this technology would identify unique molecular signatures in plasma samples from schizophrenia patients (n = 60) compared to controls (n = 20). Using a combinatorial library of X-aptamer beads, we developed a two-color flow cytometer-based approach to identify specific X-aptamers that bound with high specificity to each target group. Based on this, we synthesized two unique X-aptamer sequences, and specific proteins pulled down from the patient and control groups by these X-aptamers were identified by mass spectrometry. We identified two protein biomarkers, complement component C4A and ApoB, upregulated in plasma samples from schizophrenia patients. ELISA validation suggested that the observed differences in C4 levels in patients are likely due to the presence of the illness itself, while ApoB may be a marker of antipsychotic-induced alterations. These studies highlight the utility of the X-aptamer technology in the identification of biomarkers for schizophrenia that will advance our understanding of the pathophysiological mechanisms of this disorder.

Tracking Biodistribution of Myeloid-Derived Cells in Murine Models of Breast Cancer.

A growing tumor is constantly secreting inflammatory chemokines and cytokines that induce release of immature myeloid cells, including myeloid-derived suppressor cells (MDSCs) and macrophages, from the bone marrow. These cells not only promote tumor growth, but also prepare distant organs for tumor metastasis. On the other hand, the myeloid-derived cells also have phagocytic potential, and can serve as vehicles for drug delivery. We have previously identified thioaptamers that bind a subset of MDSCs with high affinity and specificity. In the current study, we applied one of the thioaptamers as a probe to track myeloid cell distribution in the bone, liver, spleen and tumor in multiple murine models of breast cancer including the 4T1 syngeneic model and MDA-MB-231 and SUM159 xenograft models. Information generated from this study will facilitate further understanding of tumor growth and metastasis, and predict biodistribution patterns of cell-mediated drug delivery.

Base-pairing properties of the oxidized cytosine derivative, 5-hydroxy uracil.

The most abundant base-substitution mutation resulting from oxidative damage to DNA is the GC to AT transition mutation. 5-hydroxyuracil (5-OHU), produced by the oxidative deamination of cystosine, has been established as the major chemical precursor for this most abundant transition mutation. Results from NMR spectroscopy and UV melting experiments show that 5-OHU would form the most stable pair with G, and the least stable pair with C. The hydroxyl group in the 5th position of the 5-OHU residue may play a role in increasing the stability of the 5-OHU:G pair over the normal Watson-Crick pair, the 5-OHU:A. The 5-OHU:C base pair would be least stable, and would destabilize the base-stacking in the duplex. Our results explain why certain DNA polymerases preferentially incorporate G opposite to 5-OHU over A and why C does not get incorporated against 5-OHU during DNA replication in vivo.

Aptamer Therapeutics in Cancer: Current and Future.

Aptamer-related technologies represent a revolutionary advancement in the capacity to rapidly develop new classes of targeting ligands. Structurally distinct RNA and DNA oligonucleotides, aptamers mimic small, protein-binding molecules and exhibit high binding affinity and selectivity. Although their molecular weight is relatively small-approximately one-tenth that of monoclonal antibodies-their complex tertiary folded structures create sufficient recognition surface area for tight interaction with target molecules. Additionally, unlike antibodies, aptamers can be readily chemically synthesized and modified. In addition, aptamers' long storage period and low immunogenicity are favorable properties for clinical utility. Due to their flexibility of chemical modification, aptamers are conjugated to other chemical entities including chemotherapeutic agents, siRNA, nanoparticles, and solid phase surfaces for therapeutic and diagnostic applications. However, as relatively small sized oligonucleotides, aptamers present several challenges for successful clinical translation. Their short plasma half-lives due to nuclease degradation and rapid renal excretion necessitate further structural modification of aptamers for clinical application. Since the US Food and Drug Administration (FDA) approval of the first aptamer drug, Macugen® (pegaptanib), which treats wet-age-related macular degeneration, several aptamer therapeutics for oncology have followed and shown promise in pre-clinical models as well as clinical trials. This review discusses the advantages and challenges of aptamers and introduces therapeutic aptamers under investigation and in clinical trials for cancer treatments.

A Novel DNA Aptamer for Dual Targeting of Polymorphonuclear Myeloid-derived Suppressor Cells and Tumor Cells.

Aptamers have the potential to be used as targeting ligands for cancer treatment as they form unique spatial structures. Methods: In this study, a DNA aptamer (T1) that accumulates in the tumor microenvironment was identified through in vivo selection and validation in breast cancer models. The use of T1 as a targeting ligand was evaluated by conjugating the aptamer to liposomal doxorubicin. Results: T1 exhibited a high affinity for both tumor cells and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Treatment with T1 targeted doxorubicin liposomes triggered apoptosis of breast cancer cells and PMN-MDSCs. Suppression of PMN-MDSCs, which serve an immunosuppressive function, leads to increased intratumoral infiltration of cytotoxic T cells. Conclusion: The cytotoxic and immunomodulatory effects of T1-liposomes resulted in superior therapeutic efficacy compared to treatment with untargeted liposomes, highlighting the promise of T1 as a targeting ligand in cancer therapy.