Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers

The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8–15 nucleotides and by the incorporation of high affinity RNA binders such as 2′, 4′-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2′,4′-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4–5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.     

Male germ cell-specific protein Trs4 binds to multiple proteins

Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners.     

Identification of Cargo Proteins Specific for Importin-β with Importin-α Applying a Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based in Vitro Transport System

The human importin (Imp)-β family consists of 21 nucleocytoplasmic transport carrier proteins, which transport thousands of proteins (cargoes) across the nuclear envelope through nuclear pores in specific directions. To understand the nucleocytoplasmic transport in a physiological context, the specificity of cargoes for their cognate carriers should be determined; however, only a limited number of nuclear proteins have been linked to specific carriers. To address this biological question, we recently developed a novel method to identify carrier-specific cargoes. This method includes the following three steps: (i) the cells are labeled by stable isotope labeling by amino acids in cell culture (SILAC); (ii) the labeled cells are permeabilized, and proteins in the unlabeled cell extracts are transported into the nuclei of the permeabilized cells by a particular carrier; and (iii) the proteins in the nuclei are quantitatively identified by LC-MS/MS. The effectiveness of this method was demonstrated by the identification of transportin (Trn)-specific cargoes. Here, we applied this method to identify cargo proteins specific for Imp-β, which is a predominant carrier that exclusively utilizes Imp-α as an adapter for cargo binding. We identified candidate cargoes, which included previously reported and potentially novel Imp-β cargoes. In in vitro binding assays, most of the candidate cargoes bound to Imp-β in one of three binding modes: directly, via Imp-α, or via other cargoes. Thus, our method is effective for identifying a variety of Imp-β cargoes. The identified Imp-β and Trn cargoes were compared, ensuring the carrier specificity of the method and illustrating the complexity of these transport pathways.     

Functional binding of hexanucleotides to 3C protease of hepatitis A virus

Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C^pro). Inhibition assays in vitro identified the hexanucleotide 5′-GGGGGT-3′ (G₅T) as a 3C^pro protease inhibitor. Using ¹H NMR spectroscopy, G₅T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of ¹H, ¹⁵N-HSQC experiments the binding site for G₅T was located to the C-terminal β-barrel of HAV 3C^pro. Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G₅T-binding site, nor does G₅T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid–protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.     

MiR-30b is involved in methylglyoxal-induced epithelial-mesenchymal transition of peritoneal mesothelial cells in rats

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMC) is a major contributor to the pathogenesis of peritoneal fibrosis. EMT is at least in part caused by repeated exposure to glucose degradation products (GDPs), such as methylglyoxal (MGO). MiRNA contributes greatly to the EMT of PMCs. In this study, we tried to profile whether differences exist between the peritoneal membrane (PM) miRNA expression seen in control rats and that seen in rats injected intraperitoneally with MGO. We assessed whether miR-30b has a possible role in MGO-induced EMT of PMCs in rats. Comparative miRNA expression array and real-time PCR analyses were conducted for the control group at the start of the experiment and for the MGO group after 1 and 2 weeks. During the second week, the MGO rats were treated with: a chemically modified antisense RNA oligonucleotide (ASO) complementary to the mature miR-30b (ASO group); an miR-30b mismatch control sequence (MIS group); or a citrate buffer (EMT group). Bioinformatic analyses indicated that the 3′ untranslated region (3′-UTR) of bone morphogenetic protein 7 (BMP7) mRNA did contain a putative binding site for miR-30b. We also tried to investigate whether miR-30b targeted BMP7 in vitro by transfection. Of the upregulated miRNAs, miR-30b expression demonstrated the greatest increase. The administration of miR-30b ASO for two weeks significantly reduced α-SMA excretion and upregulated E-cadherin and BMP-7 expression. Our in vitro study showed that miR-30b directly targeted and inhibited BMP7 by binding to its 3’-UTR. Our results revealed that miR-30b is involved in MGO-induced EMT of PMCs in rats.     

Suppression of tumor growth using antisense oligonucleotide against survivin in an orthotopic transplant model of human hepatocellular carcinoma in nude mice

Survivin, an inhibitor of apoptosis protein, deserves attention as a selective target for cancer therapy because it is overexpressed in many cancers, including human hepatocellular carcinoma (HCC). Here, we report a novel antisense oligonucleotide (ASO) against survivin for its effectiveness against tumor growth both in vitro and in vivo, and providing evidence in treatment for HCC. Initially, transfection of liver tumor cells HepG2 with ASO resulted in significant cells growth inhibition and reduction expression of survivin mRNA and protein, in a dose-dependent manner. Using caspase-3 protease activation assays, we observed that ASO has induced significantly greater apoptosis rate compared to control oligonucleotides. Furthermore, we used an orthotopic transplant model of HCC in nude mice to investigate the effect of ASO on tumor growth in vivo, and ASO reagents were delivered by intravenous injection. Interestingly, this systemic treatment also resulted in significant inhibition in tumor growth. Tumor growth in mice treated with ASO (50 and 75 mg/kg per day) was significantly inhibited (45.31% and 60.94%, respectively) compared with saline-injected group (p < 0.01), in a dose-dependent manner, and the effect of ASO on tumor growth was associated with downregulation of survivin in tumor xenografts. Moreover, the level of serum alpha-fetoprotein in ASO-treated groups was also decreased in a dose-dependent manner. Taken together, these data suggest that the usefulness of survivin ASO could potentially be a promising gene therapy approach to treatment of HCC.     

Regulation of CaMKII by phospho-Thr253 or phospho-Thr286 sensitive targeting alters cellular function

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is an important mediator of synaptic function that is regulated by multi-site phosphorylation and targeting through interactions with proteins. A new phosphorylation site at Thr253 has been identified in vivo, that does not alter CaMKII activity, but does alter CaMKII function through interactions with binding proteins. To identify these proteins, as well as to examine the specific effects following Thr253 or Thr286 phosphorylation on these interactions, we developed an in vitro overlay binding assay. We demonstrated that the interaction between CaMKII and its binding proteins was altered by the phosphorylation state of both the CaMKII and the partner, and identified a CaMKII-specific sequence that was responsible for the interaction between CaMKII and two interacting proteins. By comparing CaMKII binding profiles in tissue and cell extracts, we demonstrated that the CaMKII binding profiles varied with cell type, and also showed that overexpression of a CaMKII Thr253 phospho-mimic mutant in human neuroblastoma and breast cancer cells dramatically altered the morphology and growth rates when compared to overexpression of non-phosphorylated CaMKII. This data highlights the importance of the microenvironment in regulating CaMKII function, and describes a potentially new mechanism by which the functions of CaMKII can be regulated.     

Identification of protein binding partners of ALK-5 kinase inhibitors

We have investigated the binding characteristics of a potent member of the bis-ortho-substituted five-membered nitrogen heterocycle class of ALK-5 kinase inhibitors with lysates of cultured HEK-293 cells to identify protein binding partners of potential biological significance. An affinity chromatographic resin containing an immobilized ALK-5 kinase inhibitor, 2-phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine, was used to capture specific proteins from the cell lysate. The soluble inhibitor was then used to specifically elute the proteins which selectively bound to the pharmacophore ligand structure. Application of 2-D SDS–PAGE analysis with positive and negative controls demonstrated the inhibitor bound several different proteins via selective molecular recognition processes. The structural features of the specifically eluted proteins were identified by peptide mass fingerprinting (PMF) methods and included proteins with structural, metabolic and chaperone functions. Furthermore, these PMF results identified the therapeutic target in various cancer treatment studies, HSP-70, as a potential high-affinity binding partner. These observations warrant examination of bis-ortho-substituted five-membered nitrogen heterocycles as dual ALK-5/HSP-70 inhibitors for anti-cancer drug development.     

Fine mapping of interactions between eEF1α protein and 3′UTR of metallothionein-1 mRNA

The localization of metallothionein-1 (MT-1) mRNA to the perinuclear cytoskeleton is determined by a signal in the 3′untranslated region (3′UTR) and trans-acting binding proteins. The present study carried out detailed mapping of this signal and further characterized the binding to elongation factor 1 alpha (eEF1α) and other interacting proteins. Electrophoresis mobility shift assays demonstrated that shortening of a stem region proximal to nucleotides 66-76 abrogated binding. Full length recombinant rat eEF1α, and independently domains I and III, formed complexes with the mRNA. Proteins binding to biotinylated MT-1 3′UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3′UTR binding proteins. We conclude that a 5-bp internal stem in the MT-1 3′UTR is critical for binding of eEF1α and histidine-tRNA ligase, and that binding of eEF1α is facilitated through domains I and III.     

Palmitic acid conjugation enhances potency of tricyclo-DNA splice switching oligonucleotides

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.     

A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis

BackgroundTelomeres are chromosome end structures important in the maintenance of genome homeostasis. They are replenished by the action of telomerase and associated proteins, such as the OB (oligonucleotide/oligosaccharide-binding)-fold containing telomere-end binding proteins (TEBP) which plays an essential role in telomere maintenance and protection. The nature of TEBPs is well known in higher and some primitive eukaryotes, but it remains undetermined in trypanosomatids. Previous in silico searches have shown that there are no homologs of the classical TEPBs in trypanosomatids, including Leishmania sp. However, Replication Protein A subunit 1 (RPA-1), an OB-fold containing DNA-binding protein, was found co-localized with trypanosomatids telomeres and showed a high preference for the telomeric G-rich strand.Methods and resultsWe predicted the absence of structural homologs of OB-fold containing TEBPs in the Leishmania sp. genome using structural comparisons. We demonstrated by molecular docking that the ssDNA binding mode of LaRPA-1 shares features with the higher eukaryotes POT1 and RPA-1 crystal structures ssDNA binding mode. Using fluorescence spectroscopy, protein-DNA interaction assays, and FRET, we respectively show that LaRPA-1 shares some telomeric functions with the classical TEBPs since it can bind at least one telomeric repeat, protect the telomeric G-rich DNA from 3′-5′ Exonuclease I digestion, and unfold telomeric G-quadruplex.ConclusionsOur results suggest that RPA-1 emerges as a TEBP in trypanosomatids, and in this context, we present two possible evolutionary landscapes of trypanosomatids RPA-1 that could reflect upon the evolution of OB-fold containing TEBPs from all eukaryotes.     

Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation

Eukaryotic DNA replication initiates at origins of replication by the assembly of the highly conserved pre-replicative complex (pre-RC). However, exact sequences for pre-RC binding still remain unknown. By chromatin immunoprecipitation we identified in vivo a pre-RC-binding site within the origin of bidirectional replication in the murine rDNA locus. At this sequence, ORC1, -2, -4 and -5 are bound in G1 phase and at the G1/S transition. During S phase, ORC1 is released. An ATP-dependent and site-specific assembly of the pre-RC at origin DNA was demonstrated in vitro using partially purified murine pre-RC proteins in electrophoretic mobility shift assays. By deletion experiments the sequence required for pre-RC binding was confined to 119 bp. Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus. During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses. ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.     

Determinants of RNA Binding and Translational Repression by the Bicaudal-C Regulatory Protein

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.     

In Vivo Mutational Characterization of DndE Involved in DNA Phosphorothioate Modification

DNA phosphorothioate (PT) modification is a recently identified epigenetic modification that occurs in the sugar-phosphate backbone of prokaryotic DNA. Previous studies have demonstrated that DNA PT modification is governed by the five DndABCDE proteins in a sequence-selective and R _P stereo-specific manner. Bacteria may have acquired this physiological modification along with dndFGH as a restriction-modification system. However, little is known about the biological function of Dnd proteins, especially the smallest protein, DndE, in the PT modification pathway. DndE was reported to be a DNA-binding protein with a preference for nicked dsDNA in vitro; the binding of DndE to DNA occurs via six positively charged lysine residues on its surface. The substitution of these key lysine residues significantly decreased the DNA binding affinities of DndE proteins to undetectable levels. In this study, we conducted site-directed mutagenesis of dndE on a plasmid and measured DNA PT modifications under physiological conditions by mass spectrometry. We observed distinctive differences from the in vitro binding assays. Several mutants with lysine residues mutated to alanine decreased the total frequency of PT modifications, but none of the mutants completely eliminated PT modification. Our results suggest that the nicked dsDNA-binding capacity of DndE may not be crucial for PT modification and/or that DndE may have other biological functions in addition to binding to dsDNA.     

Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides

The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5′-terminus of well-characterised 2′,4′-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.     

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay

We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1 °C for (dT)₅ to a maximum of 9 °C with oligomers ?10 nucleotides, with an apparent K_d of <1 μM for (dT)₂₀. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9 °C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner.