IntroductionAn enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques.
Synthetic oligonucleotides and their analogs have attracted considerable interest recently. These compounds may lead to highly specific therapeutic agents, as well as to powerful diagnostic tools. Here, we present the synthesis of uniformly modified oligodeoxyribonucleotide N3' -> P5' phosphoramidates containing 3'-NHP(O)(0-)0-5' internucleoside linkages and the study of Synthetic oligonucleotides have a great potential to become a new type of rationally designed therapeutic agent. These compounds could interfere with the expression of selected genes through a specific interaction with RNA or DNA regions of interest (1). Several modifications of the natural phosphodiester internucleoside bond have been introduced to improve hybridization properties of oligonucleotides as well as their cellular membrane penetration and stability and distribution inside cells (2-4). Unfortunately, the vast majority of these more hydrolytically stable analogs show reduced binding with RNA or DNA via duplex or triplex formation (5).In the present report we describe the synthesis and hybridization properties of the new analogs of nucleic acidsuniformly modified oligonucleotide N3' -* P5' phosphoramidates (Fig. 1). These compounds possess some very attractive features for therapeutic and diagnostic applications, such as a negatively charged and achiral phosphorus, good solubility in water, and phosphodiesterase digestion resistance, buttressed by a high affinity for natural RNA and DNA. MATERIALS AND METHODSOligonucleotide Synthesis. Oligodeoxyribonucleotides and oligoribonucleotides were prepared on an ABI 380B synthesizer using standard DNA or RNA assembly protocols via the phosphoramidite method. Oligonucleotide N3' -> P5' phosphoramidates were synthesized on 1 ,umol scale using an ABI 394 synthesizer and 5'-4,4'-dimethoxytrityl (DMT)-N-acyl-3'-amino-2',3'-dideoxynucleosides, as described (6). Oligonucleotides were purified by ion exchange HPLC on a Pharmacia Mono Q 10/10 column at pH 12 (10 mM NaOH) with a 1%The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. per min gradient of 1.5 M NaCl in 10 mM NaOH and a flow rate of 2 ml/min. Oligonucleotides were desalted immediately after purification on Pharmacia NAP-5 gel filtration columns and then lyophilized in vacuo.Thermal Dissociation Experiments. A Cary-Varian 1E spectrophotometer equipped with a temperature programmer and data processor from Varian was used according to the reported procedure for thermal dissociation experiments (7). Buffer concentrations and compositions are listed in the legends to Tables 1-3. Absorbance values at 260 nm were obtained at 1-min intervals with a temperature increase of 1.0°C/min. The reported tm (°C) is the temperature at the midpoint of the slope of a plot of absorbance vs. temperature.Thermodynamic Parameters Determination. Thermodynamic parameters for the dodecamers 11-13 (see T...
CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at “off-target” sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.
Transforming growth factor-β-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-β-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.