Mapping RNA with Nuclease S1.

This protocol provides details for nuclease S1 mapping of mRNA using a uniformly labeled, single-stranded DNA probe. DNA-RNA hybrids are generated, which are subsequently digested with nuclease S1. The digestion products are separated using gel electrophoresis and analyzed by radiography.

Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA Probes.

In this protocol a randomly labeled single-stranded RNA probe is prepared and then hybridized to a population of mRNA molecules. The RNAs are digested with a mixture of RNase A and RNase T1. The hybrid molecules, which are resistant to the RNases, are separated and analyzed using gel electrophoresis and radiography.

Mapping RNA.

Details of the fine structure of a particular transcript may be assessed by S1 mapping or ribonuclease protection. The use of either of these techniques allows the detection of the 5' and 3' ends of a particular mRNA, as well as the splice junctions, precursors, and processing intermediates of mRNA. Primer extension provides a measure of the amount of a particular mRNA species and allows an exact determination of the 5' end of the mRNA. These three methods are introduced here.

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins.

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia-axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

Detection of the dinoflagellate, Cochlodinium polykrikoides, that forms algal blooms using sandwich hybridization integrated with nuclease protection assay.

OBJECTIVES: To detect Cochlodinum polykrikoides in long-term monitoring and high-throughput sampling projects using an integrated sandwich hybridization and nuclease protection assay (NPA-SH). RESULTS: The specificity of the probes was verified with individual and mixed cultures as well as field collection, and the quantity of C. polykrikoides determined by NPA-SH analysis showed a good correlation with that determined by cell-counting with a light microscope. In addition a standard curve for C. polykrikoides was established to represent the correlation between optical absorbance in the NPA-SH assay and cell density. CONCLUSIONS: This approach provides an efficient alternative to traditional, morphology-based methods for the rapid identification and quantification of harmful algal species and could be used to monitor phytoplankton in field surveys.

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1ß, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1ß, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin 1 Beta 2: a novel interleukin 1 expressed by the elongating pig conceptus.

Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin 1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent, and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. The gene encoding IL1B has duplicated in the pig, resulting in a novel gene. Preliminary observations indicate that the novel IL1B is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B was cloned from mRNA isolated from Day 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBLs). The pig conceptuses, but not the PBLs, expressed a novel IL1B, referred to here as interleukin 1 beta 2 (IL1B2). Porcine endometrium was treated with recombinant porcine interleukin 1 beta 1 (IL1B1), the prototypical cytokine, and IL1B2 proteins. Immunohistochemistry and real-time RT-PCR were used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts, respectively, within the endometrium. Both IL1B1 and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lower in endometrium treated with IL1B2, suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. In conclusion, the peri-implantation pig conceptus expresses a novel IL1B that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig.

Gene delivery potential of biofunctional carbonate apatite nanoparticles in lungs.

Existing nonviral gene delivery systems to lungs are inefficient and associated with dose limiting toxicity in mammalian cells. Therefore, carbonate apatite (CO3Ap) nanoparticles were examined as an alternative strategy for effective gene delivery to the lungs. This study aimed to (1) assess the gene delivery efficiency of CO3Ap in vitro and in mouse lungs, (2) evaluate the cytotoxicity effect of CO3Ap/pDNA in vitro, and (3) characterize the CO3Ap/pDNA complex formulations. A significantly high level of reporter gene expression was detected from the lung cell line transfected with CO3Ap/pDNA complex prepared in both serum and serum-free medium. Cytotoxicity analysis revealed that the percentage of the viable cells treated with CO3Ap to be almost similar to the untreated cells. Characterization analyses showed that the CO3Ap/pDNA complexes are in a nanometer range with aggregated spherical structures and tended to be more negatively charged. In the lung of mice, highest level of transgene expression was observed when CO3Ap (8 µL) was complexed with 40 µg of pDNA at day 1 after administration. Although massive reduction of gene expression was seen beyond day 1 post administration, the level of expression remained significant throughout the study period.

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation.

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

Dom34 rescues ribosomes in 3' untranslated regions.

Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here, we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3' UTRs. These ribosomes appear to gain access to the 3' UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them.

Combination of dendrimer-nanovector-mediated small interfering RNA delivery to target Akt with the clinical anticancer drug paclitaxel for effective and potent anticancer activity in treating ovarian cancer.

The recently discovered small interfering RNA (siRNA) holds great promise in cancer therapy. However, efficient and safe delivery systems are required for the development of new therapeutic paradigms. Ovarian cancer has the highest mortality of all gynecologic tumors, and there is an urgent need for specific and effective therapies. The phosphatidylinositol 3-kinase/Akt pathway, which is strongly implicated in the biology of ovarian cancer, constitutes an attractive therapeutic target. In this study, we describe a triethanolamine-core poly(amidoamine) dendrimer which forms stable nanoparticles with the Akt siRNA, protects siRNA against RNase digestion, and is highly effective for initiating Akt target-gene silencing both in vitro and in vivo, while being minimally toxic. Most importantly, it could potentiate the antitumor effect of the anticancer drug paclitaxel. These results represent the proof-of-concept, demonstrating that dendrimer-mediated Akt siRNA delivery, in combination with a chemotherapeutic regimen, may constitute a promising nanomedicine approach in cancer therapy.

Sequence-based design of bioactive small molecules that target precursor microRNAs.

Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.

Selective blocking of primary amines in branched polyethylenimine with biocompatible ligand alleviates cytotoxicity and augments gene delivery efficacy in mammalian cells.

Recently, polyethylenimines (PEIs) have emerged as efficient vectors for nucleic acids delivery. However, inherent cytotoxicity has limited their in vivo applications. To address this concern as well as to incorporate hydrophobic domains for improving interactions with the lipid bilayers in the cell membranes, we have tethered varying amounts of amphiphilic pyridoxyl moieties onto bPEI to generate a small series of pyridoxyl-PEI (PyP) polymers. Spectroscopic characterization confirms the formation of PyP polymers, which subsequently form stable complexes with pDNA in nanometric range with positive surface charge. The projected modification not only accounts for a decrease in the density of 1° amines but also allows formation of relatively loose complexes with pDNA (cf. bPEI). Alleviation of the cytotoxicity, efficient interaction with cell membranes and easy disassembly of the pDNA complexes have led to the remarkable enhancement in the transfection efficiency of PyP/pDNA complexes in mammalian cells with one of the formulations, PyP-3/pDNA complex, showing transfection in ∼68% cells compared to ∼16% cells by Lipofectamine/pDNA complex. Further, the efficacy of PyP-3 vector has been established by delivering GFP-specific siRNA resulting in ∼88% suppression of the target gene expression. These results demonstrate the efficacy of the projected carriers that can be used in future gene therapy applications.

Molecular features of neural stem cells enable their enrichment using pharmacological inhibitors of survival-promoting kinases.

Isolating a pure population of neural stem cells (NSCs) has been difficult since no exclusive surface markers have been identified for panning or FACS purification. Moreover, additional refinements for maintaining NSCs in culture are required, since NSCs generate a variety of neural precursors (NPs) as they proliferate. Here, we demonstrate that post-natal rat NPs express low levels of pro-apoptotic molecules and resist phosphatidylinositol 3'OH kinase and extracellular regulated kinase 1/2 inhibition as compared to late oligodendrocyte progenitors. Furthermore, maintaining subventricular zone precursors in LY294002 and PD98059, inhibitors of PI3K and ERK1/2 signaling, eliminated lineage-restricted precursors as revealed by enrichment for Nestin(+)/SOX-2(+) cells. The cells that survived formed neurospheres and 89% of these neurospheres were tripotential, generating neurons, astrocytes, and oligodendrocytes. Without this enrichment step, less than 50% of the NPs were Nestin(+)/SOX-2(+) and 42% of the neurospheres were tripotential. In addition, neurospheres enriched using this procedure produced 3-times more secondary neurospheres, supporting the conclusion that this procedure enriches for NSCs. A number of genes that enhance survival were more highly expressed in neurospheres compared to late oligodendrocyte progenitors. Altogether, these studies demonstrate that primitive neural precursors can be enriched using a relatively simple and inexpensive means that will facilitate cell replacement strategies using stem cells as well as other studies whose goal is to reveal the fundamental properties of primitive neural precursors.

Efficient targeted pDNA/siRNA delivery with folate-low-molecular-weight polyethyleneimine-modified pullulan as non-viral carrier.

Folate receptor (FR)-mediated gene/short interfering RNA (siRNA) targeting shows advantage for the delivery of gene/siRNA into specific FR-overexpressing cancer cells. In this study, the non-targeted gene vector P-PEI was synthesized by grafting low-molecular-weight (1kDa) branched polyethyleneimine (PEI) to succinylated pullulan, and the targeted gene vector P-PEI-FA was synthesized by coupling the carboxyl of folate (FA) to the amino of PEI. Gel electrophoresis retardation assay demonstrated that both P-PEI and P-PEI-FA can efficiently wrap pDNA and siRNA with electrostatic interaction at N/P ratios higher than 1.56 and can protect pDNA from degradation by DNase I and serum. Compared with PEI/pDNA, P-PEI/pDNA and P-PEI-FA/pDNA showed lower cytotoxicity against different cells. Under serum-containing conditions, compared with Lipofamine 2000/DNA and Lipofamine2000/siRNA, P-PEI-FA/DNA at N/P ratio of 6.25 displayed higher gene transfection efficiency, whereas P-PEI-FA/siRNA at N/P ratio of 12.5 demonstrated better enhanced gene silencing effect. P-PEI-FA/siRNA can also deliver FAM-labeled siRNA to endosomes and escape. Moreover, the gene transfection and silencing effects of P-PEI-FA were higher than those of P-PEI, and were dependent on the dose of FA in FR(+) HeLa cells. Thus, P-PEI-FA can assist DNA or siRNA targeting to FR-overexpressing cells, and the uptake pathway of P-PEI-FA/siRNA was FR-mediated endocytosis. These results indicate that P-PEI-FA is a potential candidate for safe and targeted gene delivery applications.

SMG6 cleavage generates metastable decay intermediates from nonsense-containing ß-globin mRNA.

mRNAs targeted by endonuclease decay generally disappear without detectable decay intermediates. The exception to this is nonsense-containing human ß-globin mRNA, where the destabilization of full-length mRNA is accompanied by the cytoplasmic accumulation of 5'-truncated transcripts in erythroid cells of transgenic mice and in transfected erythroid cell lines. The relationship of the shortened RNAs to the decay process was characterized using an inducible erythroid cell system and an assay for quantifying full-length mRNA and a truncated RNA missing 169 nucleotides from the 5' end. In cells knocked down for Upf1 a reciprocal increase in full-length and decrease in shortened RNA confirmed the role of NMD in this process. Kinetic analysis demonstrated that the 5'-truncated RNAs are metastable intermediates generated during the decay process. SMG6 previously was identified as an endonuclease involved in NMD. Consistent with involvement of SMG6 in the decay process full-length nonsense-containing ß-globin mRNA was increased and the Δ169 decay intermediate was decreased in cells knocked down for SMG6. This was reversed by complementation with siRNA-resistant SMG6, but not by SMG6 with inactivating PIN domain mutations. Importantly, none of these altered the phosphorylation state of Upf1. These data provide the first proof for accumulation of stable NMD products by SMG6 endonuclease cleavage.

Explanatory chapter: nuclease protection assays.

Nuclease protection assays are a highly sensitive, solution-based technique used to detect and quantify specific RNA targets from complex RNA mixtures. Today, this technique is frequently performed using kits, and the following chapter will explain the principles of how these kits work and some considerations to keep in mind when using them.

Vibrio cholerae classical biotype is converted to the viable non-culturable state when cultured with the El Tor biotype.

A unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysis of the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strains.

Multimodal analysis in acute and chronic experimental autoimmune encephalomyelitis.

Different experimental autoimmune encephalomyelitis models (EAE) have been developed. However, due to the different experimental conditions applied, observations simultaneously considering different pathological targets are still scarce. Using EAE induced in Dark Agouti rats with syngenic whole spinal cord homogenate suspended in incomplete Freund's adjuvant, we here analyze neurosteroidogenic machinery, cytokine levels, microglial cells, infiltration of inflammatory cells, myelin proteins and Na(+), K(+)-ATPase pump activity in the spinal cord. Data obtained in the acute phase of the disease confirmed that neurological signs were accompanied by the presence of perivascular infiltrating T cells (CD3(+) cells) and activated monocytic/microglial cells (ED1(+) and MHC-II(+)) in the spinal cord. In particular, the number of MHC-II(+) cells was significantly increased in association with increased expression of pro- (i.e., TNF-α, IL-1ß) and anti-inflammatory (i.e., TGF-ß) cytokines as well as with decreased expression of proteolipid protein and myelin basic protein. During the chronic phase of the disease, the number of MHC-II(+) cells was still increased, although less than in the acute phase. Changes in the number of MHC-II(+) cells were associated with decreased Na(+),K(+)-ATPase enzymatic activity. A general decrease in the levels of neuroactive steroids, with the exception of an increase in tetrahydroprogesterone and 17ß-estradiol, was detected in the acute phase. These changes were maintained or reverted in the chronic phase of EAE. In conclusion, we report that modifications in the neuroimmune response in the acute and chronic phases of EAE are associated with specific changes in myelin proteins, Na(+),K(+)-ATPase pump and in the levels of neuroactive steroids.