Significant progress is being made concerning the development of oligonucleotides as therapeutic agents. Studies with antisense, siRNA, and other forms of oligonucleotides have shown promise in ...cellular and animal models and in some clinical studies. Nonetheless, our understanding of how oligonucleotides function in cells and tissues is really quite limited. One major issue concerns the modes of uptake and intracellular trafficking of oligonucleotides, whether as "free" molecules or linked to various delivery moieties such as nanoparticles or targeting ligands. In this review, we examine the recent literature on oligonucleotide internalization and subcellular trafficking in the context of current insights into the basic machinery for endocytosis and intracellular vesicular traffic.
Short DNA or RNA oligonucleotides have tremendous potential as therapeutic agents. Because of their ability to engage in Watson-Crick base pairing, they can interact with mRNA or pre-mRNA targets ...with high selectivity. As a result, they could precisely manipulate gene expression. This possibility has engendered extensive efforts to develop oligonucleotides as drugs, and many candidates are already in clinical trials. However, a major impediment to the maturation of this field of oligonucleotide-based therapeutics remains: these relatively large and often highly charged molecules don't easily cross cellular membranes, making it difficult for them to reach their sites of action in the cytosol or nucleus. In this Account, we summarize some basic features of the biology of antisense and siRNA oligonucleotides. We then discuss chemical conjugation as an approach to improving the intracellular delivery and therapeutic potential of these agents. Instead of focusing on the details of conjugation chemistry, we emphasize the pharmacological ramifications of oligonucleotide conjugates. In one important approach to improving delivery and efficacy, researchers have conjugated oligonucleotides with ligands designed to bind to particular receptors and thus provide specific interactions with cells. In another strategy, researchers have coupled antisense or siRNA with agents such as cell penetrating peptides that are designed to provoke escape of the conjugate from intracellular vesicular compartments. Although both of these strategies have had some success, further research is needed before oligonucleotide conjugates can find an important place in human therapeutics.
A basic helix‐loop‐helix transcription factor Hey2 is expressed in the ventricular myocardium and endocardium of mouse embryos, and Hey2 null mice die perinatally showing ventricular septal defect, ...dysplastic tricuspid valve and hypoplastic right ventricle. In order to understand region‐specific roles of Hey2 during cardiac morphogenesis, we generated Hey2 conditional knockout (cKO) mice using Mef2c‐AHF‐Cre, which was active in the anterior part of the second heart field and the right ventricle and outflow tract of the heart. Hey2 cKO neonates reproduced three anomalies commonly observed in Hey2 null mice. An earliest morphological defect was the lack of right ventricular extension along the apico‐basal axis at midgestational stages. Underdevelopment of the right ventricle was present in all cKO neonates including those without apparent atresia of right‐sided atrioventricular connection. RNA sequencing analysis of cKO embryos identified that the gene expression of a non‐chamber T‐box factor Tbx2 was ectopically induced in the chamber myocardium of the right ventricle. Consistently, mRNA expression of the Mycn transcription factor, which was a cell cycle regulator transcriptionally repressed by Tbx2, was down regulated, and the number of S‐phase cells was significantly decreased in the right ventricle of cKO heart. These results suggest that Hey2 plays an important role in right ventricle development during cardiac morphogenesis, at least in part, through mitigating Tbx2‐dependent inhibition of Mycn expression.
Hey2 plays an important role in right ventricle development during cardiac morphogenesis, at least in part, through mitigating Tbx2‐dependent inhibition of Mycn expression and cell proliferation.
Development of multi-chambered heart is associated with spatio-temporal regulation of gene expression. A basic helix-loop-helix transcription factor Hey2 is specifically expressed in the embryonic ...mouse ventricles and is indispensable for ventricular myocyte differentiation, compartment identity and morphogenesis of the heart. However, how Hey2 transcription is precisely regulated in the heart remains unclear. In this study, we identified a distal Hey2 enhancer conserved in the mouse and human to possess specific transcriptional activity in ventricular free wall myocytes at the looping stage of cardiac development. Deletion of the enhancer significantly decreased endogenous Hey2 expression in the ventricular myocardium but not in other tissues of mouse embryos. Mutation/deletion of the conserved binding sites for T-box and Gata proteins, but not NK-2 proteins, abolished the enhancer activity, and Tbx20 null mice completely lost the enhancer activity in the embryonic ventricles. Luciferase reporter analysis suggested that the ventricular enhancer activity was controlled by Tbx20 through its DNA binding and cooperative function with cardiac Gata proteins. These results delineate a regulatory mechanism of ventricular Hey2 expression and help fully understand molecular cascades in myocardial cell differentiation and cardiac morphogenesis during embryonic development.
•A mechanism of ventricular expression of Hey2 transcription factor in embryonic heart.•Identification of a distal enhancer for Hey2 expression in ventricular myocytes.•CRISPR/Cas9-based deletion analysis to prove importance of Hey2 enhancer in vivo.•Structural and functional conservation of Hey2 enhancer in the mouse and human.•Regulation of ventricular Hey2 enhancer activity by Tbx20 and cardiac Gata proteins.
The T-box transcription factor TBX5 plays essential roles in cardiac and limb development. Various mutations in the TBX5 gene have been identified in patients with Holt-Oram syndrome, which is ...characterized by congenital defects in the heart and upper extremities. In this study, we identified a WW-domain-containing transcriptional regulator TAZ as a potent TBX5 coactivator. TAZ directly associates with TBX5 and markedly stimulates TBX5-dependent promoters by interacting with the histone acetyltransferases p300 and PCAF. YAP, a TAZ-related protein with conserved functional domains, also stimulates TBX5-dependent transcription, possibly by forming a heterodimer with TAZ. TBX5 lacks a PY motif, which mediates the association of other proteins with TAZ, and interacts with TAZ through multiple domains including its carboxyl-terminal structure. Truncation mutants of TBX5 identified in patients with Holt-Oram syndrome were markedly impaired in their ability to associate with and be stimulated by TAZ. These findings reveal key roles for TAZ and YAP in the control of TBX5-dependent transcription and suggest the involvement of these coactivators in cardiac and limb development.
The 2′‐O,4′‐C‐methylene‐bridged or locked nucleic acid (2′,4′‐BNA/LNA), with an N‐type sugar conformation, effectively improves duplex‐forming ability. 2′,4′‐BNA/LNA is widely used to improve gene ...knockdown in nucleic acid based therapies and is used in gene diagnosis. Metal‐mediated base pairs (MMBPs), such as thymine (T)‐HgII‐T and cytosine (C)‐AgI‐C have been developed and used as attractive tools in DNA nanotechnology studies. This study aimed to investigate the application of 2′,4′‐BNA/LNA in the field of MMBPs. 2′,4′‐BNA/LNA with 5‐methylcytosine stabilized the MMBP of C with AgI ions. Moreover, the 2′,4′‐BNA/LNA sugar significantly improved the duplex‐forming ability of the DNA/DNA complex, relative to that by the unmodified sugar. These results suggest that the sugar conformation is important for improving the stability of duplex‐containing MMBPs. The results indicate that 2′,4′‐BNA/LNA can be applied not only to nucleic acid based therapies, but also to MMBP technologies.
Sugar, sugar! 2′‐O,4′‐C‐methylene‐bridged or locked nucleic acid (2′,4′‐BNA/LNA) bearing 5‐methylcytosine forms a metal‐mediated base pair with AgI ions. The 2′,4′‐BNA/LNA sugar significantly improves the duplex‐forming ability of the DNA/DNA complex compared with that of the unmodified sugar.
1,3‐Diaza‐2‐oxophenoxazine (“phenoxazine”), a tricyclic cytosine analogue, can strongly bind to guanine moieties and improve π–π stacking effects with adjacent bases in a duplex. Phenoxazine has been ...widely used for improving duplex‐forming abilities. In this study, we have investigated whether phenoxazine and its analogue, 1,3,9‐triaza‐2‐oxophenoxazine (9‐TAP), could improve triplex‐forming abilities. A triplex‐forming oligonucleotide (TFO) incorporating a phenoxazine component was found to show considerably decreased binding affinity with homopurine/homopyrimidine double‐stranded DNA, so the phenoxazine system was considered not to function as either a protonated cytosine or thymine analogue. Alternatively, a 9‐TAP‐containing artificial nucleobase developed by us earlier as a new phenoxazine analogue functioned as a thymine analogue with respect to AT base pairs in a parallel triplex DNA motif. The fluorescence of the 9‐TAP moiety was maintained even in triplex (9‐TAP:AT) formation, so 9‐TAP might be useful as an imaging tool for various oligonucleotide nanotechnologies requiring triplex formation.
Preferring T: The 1,3,9‐triaza‐2‐oxophenoxazine‐based artificial nucleobase 9‐TAP, developed as a phenoxazine analogue, functioned as a thymine analogue with adenine–thymine base pairs in a parallel motif triplex DNA. Its fluorescence was maintained even in triplex (9‐TAP:AT) formation. 9‐TAP might be useful as an imaging tool for various oligonucleotide technologies involving triplex formation.
Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic ...helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre–mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.
Silver turns up the A–C: In the presence of AgI ions, a DNA polymerase incorporated deoxyadenosine (from dATP) at the site opposite cytosine in the template strand to afford the full‐length product ...(see scheme), meaning that DNA polymerases prefer a C‐AgI‐A base pair to the more thermodynamically stable C‐AgI‐C base pair.
Serum/glucocorticoid‐regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. ...However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5’ region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500 kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS‐regulated enhancers located in the proximal and distal regions.
Serum/glucocorticoid‐regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. The results in this study suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS‐regulated enhancers located in the proximal and distal regions.
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