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PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject's pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1-2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data., (© 2022. Springer Nature Limited.)

NF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB-related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders., (© 2020 by The American Society of Hematology.)

The clinical application of siRNA is limited largely by the lack of efficient, cell-specific delivery systems. Antibodies are attractive delivery vehicles for targeted therapy due to their high specificity. In this study we describe the use of a humanized monoclonal antibody (mAb), hu3S193, against Lewis-Y (Le(y)), as a delivery vehicle for STAT3 siRNA. This mAb is rapidly internalized into Le(y)-expressing cancer cells via antigen recognition, and when coupled to STAT3 siRNA, a potentially powerful molecularly targeted delivery agent is created. Selective silencing of STAT3 is associated with tumor suppression. Two hu3S193 based siRNA delivery systems using STAT3 siRNA as a prototype were developed and tested in Le(y)-positive cancer cells: (a) a covalent construct based on a reductive disulfide linker that is expected to undergo cleavage within cells and (b) a noncovalent construct based on (d-arginine)(9) (9r) modified hu3S193. Le(y)-specific binding and internalization of both the covalent and noncovalent constructs were confirmed by flow cytometry and confocal microscopy. Both the covalent and the noncovalent system led to efficient STAT3 silencing in Le(y)-positive cancer cells (A431) but not in Le(y)-negative cancer cells (MDA-MB-435). The covalent construct, however, required co-treatment with reagents such as chloroquine or 9r that facilitate the escape of the siRNA from endosomes to achieve significant gene silencing. The 9r modified noncovalent construct induced ∼70% STAT3 knockdown at submicromolar siRNA concentrations when used at an optimal vehicle-to-siRNA ratio of 5:1. The STAT3 knockdown also led to ∼50% inhibition of cell proliferation of Le(y)-positive cells. Noncovalent linked STAT3 siRNA-hu3S193 has great promise for targeted knockdown of STAT3 in tumor cells.

Ubiquitin-like modifications are important mechanisms in cellular regulation, and are carried out through several steps with reaction intermediates being thioester conjugates of ubiquitin-like proteins with E1, E2, and sometimes E3. Despite their importance, a thorough characterization of the intrinsic stability of these thioester intermediates has been lacking. In this study, we investigated the intrinsic stability by using a model compound and the Ubc9 approximately SUMO-1 thioester conjugate. The Ubc9 approximately SUMO-1 thioester intermediate has a half life of approximately 3.6 h (hydrolysis rate k = 5.33 +/- 2.8 x10(-5) s(-1)), and the stability decreased slightly under denaturing conditions (k = 12.5 +/- 1.8 x 10(-5) s(-1)), indicating a moderate effect of the three-dimensional structural context on the stability of these intermediates. Binding to active and inactive E3, (RanBP2) also has only a moderate effect on the hydrolysis rate (13.8 +/- 0.8 x 10(-5) s(-1) for active E3 versus 7.38 +/- 0.7 x 10(-5) s(-1) for inactive E3). The intrinsically high stability of these intermediates suggests that unwanted thioester intermediates may be eliminated enzymatically, such as by thioesterases, to regulate their intracellular abundance and trafficking in the control of ubiquitin-like modifications.

Objective: The reactions of carbohydrate- or lipid-derived intermediates with proteins lead to the formation of Maillard reaction products, which subsequently leads to the formation of advanced glycation/lipoxidation end products (AGE/ALEs). Levels of AGE/ALEs are increased in diseases like diabetes. Unlike AGEs, very little is known about ALE effects in vitro. We hypothesized that ALEs can have proinflammatory effects in monocytes., Research Design and Methods: In a profiling approach, conditioned media from THP-1 cells either cultured in normal glucose (5.5 mmol/l) or treated with MDA-Lys or MDA alone were hybridized to arrays containing antibodies to 120 known human cytokines/chemokines. Pathway analyses with bioinformatics software were used to identify signalling networks., Results: Synthetic ALE (malondialdehyde-lysine [MDA-Lys]) (50 micromol/l) could induce oxidant stress and also activate the transcriptional factor nuclear factor-kappaB (NF-kappaB) in THP-1 monocytes. MDA-Lys also significantly increased the expression of key candidate proinflammatory genes, interferon-gamma-inducible protein-10, beta1- and beta2-integrins, cyclooxygenase-2 (COX-2), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 and -8, and inducible nitric-oxide synthase, which are also associated with monocyte dysfunction. Several key target proinflammatory proteins were significantly induced by MDA-Lys relative to normal glucose or MDA alone, including MCP-1; tumor necrosis factor ligand superfamily member-14; chemokine CC motif ligand-11 (CCL11); growth-related oncogene-alpha, -beta, and -gamma; and chemokine CXC motif ligand-13. Bioinformatics analyses identified a network of chemokine signaling among MDA-Lys-regulated genes. MDA-Lys also increased monocyte binding to vascular smooth muscle and endothelial cells. Furthermore, plasma from diabetic rats showed significantly higher levels of MDA-Lys and CCL11., Conclusions: These new results suggest that ALEs can promote monocyte activation and vascular complications via induction of inflammatory pathways and networks.

Artificially ordered protein arrays provide a facile approach to a variety of problems in biology and nanoscience. Current demonstration systems use either nucleic acid tethers or methyltransferase fusions in order to target proteins or peptides of interest to nucleic acid scaffolds. These demonstrations point to the large number of useful devices and assemblies that can be envisioned using this approach, including smart biological probes and drug delivery systems. In principle, these systems are now capable of imitating the earliest forms of prebiotic organisms and can be expected to reach the complexity of a small virus in the near future. Third-generation methyltransferase inhibitors provide an example of a smart chemotherapeutics that can be constructed with this approach. We describe the use of mechanistic enzymology, computer-aided design, and microfluidic chip-based capillary electrophoresis in assessing the final assembly and testing of designs of this type.

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized. Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms. Homologues of UVDE have been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway. After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 endonuclease V-incised DNA was processed in the same way. Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1. The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.

The formation of DNA photoproducts by ultraviolet (UV) light is responsible for the induction of mutations and the development of skin cancer. Cis-syn cyclobutane pyrimidine dimers (pyrimidine dimers) are the most frequent lesions produced in DNA by UV irradiation. Besides being mutagenic, pyrimidine dimers may interfere with other important DNA-dependent processes. To analyze the effects of pyrimidine dimers on the ability of DNA sequences to be recognized by trans-acting factors, we have incorporated site-specific T-T dimers into oligonucleotides containing the recognition sequences of the sequence-specific transcription factors E2F, NF-Y, AP-1, NF kappa B, and p53. In each case, presence of the photodimer strongly inhibited binding of the respective transcription factor complex. Reduction of binding varied between 11- and 60-fold. The results indicate that the most common UV-induced DNA lesion can interfere severely with binding of several important cell cycle regulatory and DNA damage responsive transcription factors. We suggest that inhibition of transcription factor binding may be a major biological effect of UV radiation since promoter regions are known to be repaired inefficiently and since UV damage can deregulate the function of a large number of different factors.

The use of a reverse-phase polystyrene resin for ion-pair HPLC purification of large amounts of synthetic chimeric DNA-RNA oligomers that is faster and more reliable than previously used techniques has been developed. The preparation of synthetic oligomers containing RNA requires the use of tetrabutylammonium fluoride in the final step, the cleavage of the tert-butyl-dimethyl silyl protecting group from the ribonucleotides. Cleavage is accompanied by the serendipitous formation of ion pairs between tetrabutylammonium cations and the oligomer phosphates. The formation of these ion pairs retards the elution of the oligomer during HPLC, which allows rapid removal of excess tetrabutylammonium fluoride and the concomitant purification of chimeric ribozymes. This technique is based on a correlation between the length of ion-paired oligomers and their retardation during HPLC. The advantages of reverse-phase ion-pair HPLC on polystyrene resin for the fast purification of oligoribonucleotides are discussed and illustrated through the examples of synthesized chimeric ribozymes.

Oligonucleotides containing the nonbase residues 1,3-propanediol or 1,4-anhydro-2-deoxy-D-ribitol were synthesized and used as primers for the polymerase chain reaction (PCR). Since these residues cannot be replicated by a DNA polymerase, the resulting PCR products have protruding 5' ends. Primers were designed with three regions, a 3' region complementary to the desired template, a 5' region complementary to a preselected nucleotide sequence, and a nonreplicable element interposed between these two containing 1-3 of the nonbase residues. The primers were used in a PCR and the products hybridized without denaturation to a solid support containing an immobilized preselected nucleotide sequence. Studies are reported showing the effects of the nonreplicable elements in primer extension reactions and the application to the capture of PCR products.