DNA Methylation and Histone H1 Jointly Repress Transposable Elements and Aberrant Intragenic Transcripts

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Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.     

Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.     

Retracted: Long noncoding RNA HAGLROS regulates apoptosis and autophagy in colorectal cancer cells via sponging miR-100 to target ATG5 expression

The aim of this study was to explore the relationship between the expression of HOXD antisense growth-associated long noncoding RNA (HAGLROS) and prognosis of patients with colorectal cancer (CRC), as well as the roles and regulatory mechanism of HAGLROS in CRC development. The HAGLROS expression in CRC tissues and cells was detected. The correlation between HAGLROS expression and survival time of CRC patients was investigated. Moreover, HAGLROS was overexpressed and suppressed in HCT-116 cells, followed by detection of cell viability, apoptosis, and the expression of apoptosis-related proteins and autophagy markers. Furthermore, the association between HAGLROS and miR-100 and the potential targets of miR-100 were investigated. Besides, the regulatory relationship between HAGLROS and PI3K/AKT/mTOR pathway was elucidated. The results showed that HAGLROS was highly expressed in CRC tissues and cells. Highly expression of HAGLROS correlated with a shorter survival time of CRC patients. Moreover, knockdown of HAGLROS in HCT-116 cells induced apoptosis by increasing the expression of Bax/Bcl-2 ratio, cleaved-caspase-3, and cleaved-caspase-9, and inhibited autophagy by decreasing the expression of LC3II/LC3I and Beclin-1 and increasing P62 expression. Furthermore, HAGLROS negatively regulated the expression of miR-100, and HAGLROS controlled HCT-116 cell apoptosis and autophagy through negatively regulation of miR-100. Autophagy related 5 (ATG5) was verified as a functional target of miR-100 and miR-100 regulated HCT-116 cell apoptosis and autophagy through targeting ATG5. Besides, HAGLROS overexpression activated phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. In conclusion, a highly expression of HAGLROS correlated with shorter survival time of CRC patients. Downregulation of HAGLROS may induce apoptosis and inhibit autophagy in CRC cells by regulation of miR-100/ATG5 axis and PI3K/AKT/mTOR pathway.     

Novel Biotin Phosphoramidites with Super-long Tethering Arms

Abstract

A number of novel biotin phosphoramidites, possessing exceptionally long and uncharged tethering arms, were synthesized from methoxyoxalamido (MOX) and succinimido (SUC) precursors. Included among these monomers is a uridine derivative with the biotin moiety attached through the 2′-position. Some of these phosphoramidites were used to make 5′-biotinylated primers, which were applied in direct sequencing of genomic DNA and capture of Sanger fragment pools.     

An in vitro system to silence mitochondrial gene expression

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Specific Lipoplex-Mediated Antisense Against Bcl-2 in Breast Cancer Cells: A Comparison between Different Formulations

G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3β[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms — either large unilamellar vesicles (～100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.     

白桦反义CCoAOMT基因调控木质素生物合成

白桦是我国北方重要的造林树种,但其中的高木质素含量严重制约了它作为造纸资源植物的开发利用。本文利用RACE技术获得了白桦咖啡酰辅酶A-3-O-甲基转移酶（CCoAOMT）基因全长ORF序列,并构建了白桦CCoAOMT基因的反义表达载体,通过农杆菌介导法将其导入到白桦中。PCR检测表明反义CCoAOMT基因已整合到白桦的基因组中。对转化植株的半定量PCR检测显示转基因株系的CCoAOMT基因表达量下降;Wiesner染色发现,与野生型相比,转基因植株木质素含量有所下降。对七年生的转基因白桦和野生型对照进行了化学成分分析,结果表明转基因白桦的苯醇抽提物和Klason木质素显著减少,聚戊糖含量升高。上述结果暗示BpCCoAOMT基因参与白桦木质素的合成,反义表达该基因后木质素含量减少,更易于去除。白桦CCoAOMT基因对木质素的合成起重要作用,这为培育低木质素含量的制浆新品种白桦奠定了基础。     

Towards Metal-Mediated G-Quartet Analogues: 1,2,4-Triazole Nucleotides

We proposed that metal-coordinating nucleotides could be used to control the assembly of G-quadruplexes through the formation of an artificial metal-centered quartet. Several guanine-rich DNA sequences containing 1,2,4-triazole-functionalized nucleotides were investigated. These oligonucleotides were designed to form quartets mediated by metal-triazole bonding both on the surface of and within the G-quadruplex core. In contrast to duplex studies in which 1,2,4-triazole nucleosides serve as a mimic of Watson-Crick base-pairs, our results show that these nucleosides are not suitable components of an artificial metal-centered quartet.