Synthesis and biological properties of chemically modified siRNAs bearing 1-deoxy-D-ribofuranose in their 3'-overhang region

To elucidate the role of the sugar moiety in the two natural nucleotides of the 3'-overhang region of small interfering RNA (siRNA), we synthesized siRNAs that incorporated two abasic nucleosides, 1-deoxy-D-ribofuranose (R(H)). We improved the method for preparing an O-protected abasic nucleoside, 1-deoxy-2,3,5-tri-O-benzoyl-β-D-ribofuranose, via the reductive cleavage of the anomeric position of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose. To incorporate R(H) into oligonucleotides by the standard phosphoramidite solid phase method, R(H) was converted into its phosphoramidite derivative and the solid support linked to a controlled pore glass resin. Chemically modified RNAs possessing R(H) at the 3'-overhang region were easily prepared in good yields. siRNAs containing R(H) showed moderate nuclease-resistance and a desirable knockdown effect.     

Synthesis and silencing properties of siRNAs possessing lipophilic groups at their 3'-termini

Short-interfering RNAs (siRNAs) conjugated with lipophilic groups at their 3'-termini were synthesized. The properties of the synthesized siRNAs were examined in detail, and it was found that at low concentrations, their silencing abilities were dependent on the positions of the modifications and the types of organic molecules attached. Although the modification of siRNAs with palmitic acid or oleic acid at the 3'-end slightly reduced their silencing activities, siRNAs had enough abilities to induce RNAi at 10 nM concentrations. On the other hand, the modification of siRNAs with cholesterol at the 3'-end of the passenger strand was tolerated; however, the modification at the guide strand significantly reduces its silencing activity. The siRNAs modified with the lipophilic groups did not possess ability to penetrate the plasma membranes of HT-1080 cells without the transfection reagent. However, the results described in this report will aid in designing novel siRNAs with cell membrane-permeable molecules.     

In vivo activity of nuclease-resistant siRNAs

Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2'-modified siRNAs in mammals. We demonstrate that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs. Moreover, we show that the 2'-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2'-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.     

Synthesis of modified siRNA bearing C-5 polyamine-substituted pyrimidine nucleoside in their 3'-overhang regions and its RNAi activity

Short interfering RNA (siRNA) induces specific gene silencing by the RNA interference (RNAi) pathway. Nucleosides in the 3′-overhang regions of siRNAs were replaced with 5-bis(aminoethyl)aminoethylcarbamoylmethyl-2′-deoxyuridine or thymidine. siRNA bearing modified nucleoside was more active in silencing the gene expression of hepatocyte nuclear factor 4α (HNF4α) compared with siRNA bearing thymidine.     

Synthesis of novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions and their ability to suppress human RNase L protein expression

In order to examine the effect of modifications at the 3' overhang regions of short interfering RNAs (siRNAs) on their gene-silencing activities, we designed and synthesized novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions. Suppression of human RNase L protein expression by these siRNAs was analyzed by immunoblot with RNase L-specific antibody. It was found that, at 24 h post-transfection, the modified siRNAs having the thymidine dimers with the carbamate and urea linkage suppress the protein expression 78 and 37 times more efficiently than that with the natural phosphodiester linkage, respectively. Furthermore, the siRNA containing the carbamate linkage was 37 times more resistant to nucleolytic degradation by snake venom phosphodiesterase than the siRNA consisting of the natural phosphodiester linkage. Thus, the RNA duplexes having the thymidine dimers with the carbamate or urea linkage at their 3' overhang regions will be promising candidates for novel siRNA molecules to down-regulate protein expression.     

Synthesis of 2'-O-guanidinopropyl-modified nucleoside phosphoramidites and their incorporation into siRNAs targeting hepatitis B virus

Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approximately 21 bp with 2 nt overhangs at the 3' ends. To improve efficacy of siRNAs, chemical modification at the 2'-OH group of ribose has been employed. Enhanced stability, gene silencing and attenuated immunostimulation have been demonstrated using this approach. Although promising, efficient and controlled delivery of highly negatively charged nucleic acid gene silencers remains problematic. To assess the potential utility of introducing positively charged groups at the 2' position, our investigations aimed at assessing efficacy of novel siRNAs containing 2'-O-guanidinopropyl (GP) moieties. We describe the formation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acrylonitrile followed by Raney-Ni reduction and guanidinylation. These precursors were used successfully to generate antihepatitis B virus (HBV) siRNAs. Testing in a cell culture model of viral replication demonstrated that the GP modifications improved silencing. Moreover, thermodynamic stability was not affected by the GP moieties and their introduction into each position of the seed region of the siRNA guide strand did not alter the silencing efficacy of the intended HBV target. These results demonstrate that modification of siRNAs with GP groups confers properties that may be useful for advancing therapeutic application of synthetic RNAi activators.     

Synthesis and modification of triterpenoids with two lupan backbones

The first derivatives containing two lupan backbones were synthesized by the interaction of betulonic acid chloride with diols (ethylene glycol and diethylene glycol) and monoethanolamine. A modification of ring A of ethylene-1,2-bis(betulonnate) led to its bis(3β-aminopropyloxy) and bis(3,4-seco-2-cyano) derivatives.     

Synthesis,RNAi activity and nuclease-resistant properties of apolar carbohydrates siRNA conjugates

Oligoribonucleotide conjugates carrying apolar carbohydrates at the 5′-end and the corresponding siRNA duplexes have been prepared using phosphoramidite chemistry. All the carbohydrate-siRNA derivatives were compatible with RNA interference machinery if transfected with oligofectamine. In the absence of a transfection agent, some of them exerted certain reduction of gene expression. Double-tailed permethylated glucose conjugated to siRNA through a long spacer inhibited gene expression up to 26% compared to the scrambled duplex. Such modifications contribute positively to the stability of oligoribonucleotides against 5′-exonuclease degradation.     

The length of telomeric G-rich strand 3'-overhang measured by oligonucleotide ligation assay

A typical G-rich telomeric DNA strand, which runs 5'-->3' toward the chromosome ends, protrudes by several nucleotides in lower eukaryotes. In human chromosomes long G-rich 3'-overhangs have been found. Apart from the standard G-rich tail, several non-canonical terminal structures have been proposed. However, the mechanism of long-tail formation, the presence and the role of these structures in telomere maintenance or shortening are not completely understood. In a search for a simple method to accurately measure the 3'-overhang we have established a protocol based on the ligation of telomeric oligonucleotide hybridized to non-denatured DNA under stringent conditions (oligonucleotide ligation assay with telomeric repeat oligonucleotide). This method enabled us to detect a large proportion of G-rich single-stranded telomeric DNA that was as short as 24 nt. Nevertheless, we showed G-tails longer than 400 nt. In all tested cells the lengths ranging from 108 to 270 nt represented only 37% of the whole molecule population, while 56-62% were <90 nt. Our protocol provides a simple and sensitive method for measuring the length of naturally occurring unpaired repeated DNA.     

Telomere shortening is proportional to the size of the G-rich telomeric 3'-overhang

Most normal diploid human cells do not express telomerase activity and are unable to maintain telomere length with ongoing cell divisions. We show that the length of the single-stranded G-rich telomeric 3'-overhang is proportional to the rate of shortening in four human cell types that exhibit different rates of telomere shortening in culture. These results provide direct evidence that the size of the G-rich overhang is not fixed but subject to regulation. The potential ability to manipulate this rate has profound implications both for slowing the rate of replicative aging in normal cells and for accelerating the rate of telomere loss in cancer cells in combination with anti-telomerase therapies.     

Synthesis and activity of tetrapeptidic HTLV-I protease inhibitors possessing different P3-cap moieties

The causative agent behind adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy is the human T-cell leukemia virus type 1 (HTLV-I). Tetrapeptidic HTLV-I protease inhibitors were designed on a previously reported potent inhibitor KNI-10516, with modifications at the P(3)-cap moieties. All the inhibitors showed high HIV-1 protease inhibitory activity (over 98% inhibition at 50nM) and most exhibited highly potent inhibition against HTLV-I protease (IC(50) values were less than 100nM).     

Synthesis and properties of myelopeptides possessing differentiating activity

Bone marrow peptides Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro (MP-4) and Val-Asp-Pro-Pro (MP-6) have been synthesized by the classical and solid-phase methods of peptide chemistry, and their differentiating activity has been studied on leukemia cell lines HL-60 and K-562. It has been shown that both peptides induce the terminal differentiation of leukemic blasts; however, their mechanisms of action are different.     

Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-3'-overhang

Telomeres shorten in human somatic cells with each round of DNA replication, and this shortening is thought to ultimately trigger replicative senescence. Telomere shortening is caused partly by the inability of semiconservative DNA replication to copy a linear strand of DNA to its very end. Post-replicative processing of telomeric ends, producing single-stranded G-rich 3' overhangs, has also been suggested to contribute to telomere shortening. This suggestion implies that a positive correlation should exist between the length of 3' overhangs and the rate of telomere shortening. We confirmed shortening of overhangs as human lung (MRC5) and foreskin (BJ) fibroblasts approach senescence by measuring overhang length using in-gel hybridization. However, a large study of fibroblast strains from 21 donors maintained under conditions which lead to two orders of magnitude of variation in telomere shortening rate failed to show any correlation between telomere overhang length and shortening rate, suggesting that overhang length is neither a cause nor a correlate of telomere shortening.     

GW body disassembly triggered by siRNAs independently of their silencing activity

GW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies.     

Synthesis of 2-5As possessing base-modified adenosines and their activities to human recombinant RNase L.

The unique 2',5'-oligoadenylate (2-5A) acts as a potent inhibitor of translation in vertebrate cells through the activation of a constituent latent 2-5A-dependent endoribonuclease (RNase L). This 2-5A system plays a major role in the interferon natural defense mechanism against viral infection. We report the syntheses of base-modified adenosine-substituted 2-5A derivatives, their interaction with recombinant human RNase L and their biological stability.     

Synthesis of 4'-ethynyl-purine nucleosides possessing anti-HIV activity.

Searching for more effective anti-HIV agents, we have prepared 4'-ethynyl-purine nucleosides. They were derived in several steps from 4-C-triethylsilylethynyl ribose, which was used as an intermediate in the synthesis of pyrimidine nucleosides. The adenine derivative exhibited significant anti-HIV activity and favorable cytotoxicity profile in vitro.     

Synthesis and base-pairing properties of the nuclease-resistant alpha-anomeric dodecaribonucleotide alpha-[r(UCUUAACCCACA)]

The non natural oligoribonucleotide alpha-[r(UCUUAACCCACA)] consisting exclusively of alpha-anomeric ribonucleoside units was synthesized according to the phosphoramidite methodology and the solid support technology. For this purpose, the base-protected alpha-ribonucleosides were synthesized and converted into their O-methylphosphoramidites. Assembling was carried out on a DNA synthesizer with an average efficiency of 97% per step. Base composition of this nuclease-resistant alpha-RNA strand was ascertained after chemical and enzymatic hydrolysis and HPLC analysis of the hydrolysate. Whereas no spectroscopic evidence of base pairing was found above 0 degrees C between alpha-[r(UCUUAACCCACA)] and beta-[d(TGTGGGTTAAGA)], a clear UV absorbance transition (Tm 25.5 degrees C) was observed during the hybridization of the same alpha-RNA strand with beta-[d(AGAATTGGGTGT)]. In this latter case, the mixing curve titration suggests formation at low temperature of a triplex involving two alpha-RNA and one beta-DNA strands. Moreover, this alpha-decaribonucleotide complementary in parallel orientation of the splice receptor of HIV-1 tat mRNA was found to inhibit (10 microM less than ED50 less than 20 microM), with apparent lack of sequence specificity, the de novo HIV-1 infection in cultured cells.     

Wetting of nonconserved residue‐backbones: A feature indicative of aggregation associated regions of proteins

Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using “Dehydrons.” “Dehydrons” are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein–protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non‐conserved dehydron‐residues) from other interaction interfaces (conserved dehydron‐residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation. Proteins 2016; 84:254–266. © 2015 Wiley Periodicals, Inc.