Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis,Characterization, and Evaluation

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; M_W 6–9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.     

Sequence,Chromatin and Evolution of Satellite DNA

Satellite DNA consists of abundant tandem repeats that play important roles in cellular processes, including chromosome segregation, genome organization and chromosome end protection. Most satellite DNA repeat units are either of nucleosomal length or 5–10 bp long and occupy centromeric, pericentromeric or telomeric regions. Due to high repetitiveness, satellite DNA sequences have largely been absent from genome assemblies. Although few conserved satellite-specific sequence motifs have been identified, DNA curvature, dyad symmetries and inverted repeats are features of various satellite DNAs in several organisms. Satellite DNA sequences are either embedded in highly compact gene-poor heterochromatin or specialized chromatin that is distinct from euchromatin. Nevertheless, some satellite DNAs are transcribed into non-coding RNAs that may play important roles in satellite DNA function. Intriguingly, satellite DNAs are among the most rapidly evolving genomic elements, such that a large fraction is species-specific in most organisms. Here we describe the different classes of satellite DNA sequences, their satellite-specific chromatin features, and how these features may contribute to satellite DNA biology and evolution. We also discuss how the evolution of functional satellite DNA classes may contribute to speciation in plants and animals.     

Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5’-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.     

Efficient Linear dsDNA Tagging Using Deoxyuridine Excision**

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3’ ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3’ overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5’ overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.     

Characterization of New DNA Aptamers for Anti-HIV-1 Reverse Transcriptase

HIV-1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti-HIV-1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild-type (WT) HIV-1 RT and gave a KD value of 75.10±0.29 nM and an IC₅₀ value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV-1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV-1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RTs at the connection domain.     

多表位DNA壳聚糖微球疫苗的体液免疫应答

目的研究多表位DNA壳聚糖微球疫苗的体液免疫应答。方法制备多表位DNA壳聚糖微球疫苗pcD-NA3.1-HME-3C3d,经鼻腔免疫小鼠,蛋白检测微孔试剂盒检测小鼠特异性IgG抗体水平。结果经免疫的小鼠均能产生针对各表位的特异性IgG抗体,DNA壳聚糖微球疫苗的抗体水平明显高于DNA疫苗。结论壳聚糖微球疫苗投递系统可提高多表位DNA疫苗的免疫应答效果。     

聚甲基丙烯酰氧乙基二甲基丁烷基溴化铵共振光散射法测定核酸

通过光谱法研究聚甲基丙烯酰氧乙基二甲基丁烷基溴化铵(PMDB)和核酸的作用。结果表明通过共振光散射法用PMDB检测微量的DNA可靠、准确和简单。     

丙型肝炎病毒多表位基因核酸疫苗的构建及其免疫原性

目的构建丙型肝炎病毒(HCV)多表位基因的真核表达载体pcDNA3.1(-)-CtEm,用该重组质粒免疫BALB/c小鼠,并检测其免疫原性。方法用BamHⅠ和HindⅢ双酶切含有HCVC区、E2区模拟表位和NS3~NS5区细胞表位串联基因的原核表达载体pQE30-CtEm,克隆入真核表达载体pcDNA3.1(-),脂质体瞬时转染CHO细胞,并检测其表达。以100μg重组质粒免疫BALB/c小鼠,检测其体液免疫和细胞免疫效果。结果所构建的真核表达载体pcDNA3.1(-)-CtEm在CHO细胞中能获得有效表达。该质粒免疫小鼠后可诱导高水平的抗体,特异性淋巴细胞增殖反应、IFN-γ水平及CTL活性均明显高于空载体和生理盐水对照组。结论已成功构建HCV多表位基因的真核表达载体pcDNA3.1(-)-CtEm,免疫小鼠后可诱导特异性体液免疫和细胞免疫应答。