Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Uridine-3H incorporation and RNA concentration were investigated in different parts of the shoot apical meristem ofChenopodium rubrum using autoradiography and cytophotometry. A single inductive cycle was sufficient to bring about postinductive first events in the shoot apex but not for complete flower differentiation. The initial activation of RNA synthesis manifested itself in all zones of the apex. The first increase was more conspicuous in the peripheral than in the central zone. The indications of the first events in the apices after a single inductive cycle disappear prior to morphological reversal to the vegetative state. Induction by three short days led to rapid flower differentiation. The increase in RNA synthesis and concentration was most conspicuous in the central zone in this case. The ratio of RNA synthesis and content between bud and leaf primordia (B/L) also change in relation to photoperiodic induction. In vegetative plants the B/L ratio was low while after induction it increased.

NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 μM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 μM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.

RNA interference (RNAi) is one of the most promising new approaches for disease therapy. The design of a dumbbell-shaped nanocircular RNA allows it to act as a short interfering RNA (siRNA) precursor. To optimize the design, we studied the relationship between the nanostructure and RNAi activity by synthesizing various RNA dumbbells. An RNA dumbbell with a 23-bp stem and 9-nt loops was the most potent. Sequence analysis by mass spectrometry showed that Dicer could edit RNA dumbbells to siRNA species. The reaction offered the slow release of siRNA species, which conferred prolonged RNAi activity. Introduction of DNA into the loop position significantly stabilized the dumbbell in biological fluid without any loss of RNAi activity. In-depth pharmacological evaluation was performed by introducing dumbbells into HeLa cells that stably express the target luciferase gene. The dumbbells provided a rapid silencing effect and retained this effect for a longer time even at a lower concentration than that at which standard siRNA completely lost RNAi activity. We conclude that an RNA dumbbell with DNA loops is the most promising design for in vivo applications for RNA medicine.

Chirality transfer, induction, and circularly polarized luminescence (CPL) using supramolecular hosts, such as macrocycles and cages, have been explored for wide-ranging applications in chiral reco...

Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain.IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

This study was designed to determine the effect of gastrin on colorectal neoplasms in the rat. Multiple colon and rectal cancers were induced in each of 57 rats with methylazoxymethanol. Animals were randomly assigned to groups: (1) antral exclusion, (2) antrectomy, (3) sham, or (4) sham with subsequent pentagastrin injections. Resulting tumors were analyzed for concentration and synthesis of DNA, RNA, and protein. The number of tumors per rat and distribution of tumors within the colons did not vary among groups. Antrectomy did not alter the gastrin level, and tumors developing in these animals did not differ from those of sham controls. Antral exclusion markedly raised serum gastrin levels. Both chronic endogenous hypergastrinemia and administration of exogenous pentagastrin significantly increased tumor synthesis and concentration of DNA, RNA, and protein. We conclude that gastrin exerts a trophic effect on colorectal neoplasms in rats. This biological phenomenon suggests a tumor regulatory role for the hormone.

<p>Supplementary materials and methods. Suppl Table 1. MMS combination PF50 cell line assays. Concentration-dependent potentiation of MMS (PF50 ratios). Suppl Table 2. Permeability of AZD2461 and olaparib were assessed. Suppl Table 3. Activity of olaparib and AZD2461 alone or in combination with the P-gp inhibitor verapamil in the colorectal cancer cell line HCT-15, which expresses high levels of P-gp. Suppl Fig. 1. Immunofluorescence for poly(ADP-ribose) in human A549 cells. Suppl Fig. 2. Analysis of both in vivo pharmacokinetics (PK) and pharmacodynamics (PD) in tumors taken from SW620 (human colon carcinoma) xenograft-bearing mice. Suppl Fig. 3. Colony formation (clonogenic) assays in the intrinsically high P-gp-expressing MRE11-deficient HCT-15 human colorectal cell line. Suppl. Fig. 4. Combination efficacy studies. Suppl Fig. 5. Relative body weight changes in mice following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide. Suppl. Fig. 6. A serial dilution of RNA concentration was used to establish a standard curve for determining the reaction efficiency of PARP1, PARP2, PARP3, and housekeeper gene (PPIA: cyclophilin A) TaqMan RT-PCR assays in both rat and mouse species; Determination of optimal reference gene for rat-mouse data normalization. Suppl Fig. 7.A. A serial dilution of reference RNA (Qiagen) concentration was used to establish a standard curve for determining the reaction efficiency of PARP3 and housekeeper genes (PPIA: cyclophilin and YWHAZ: tyrosine 3-monooxygenase/tryptohpan 5-monooxygenase activation protein, zeta polypeptide) SybrGreen RT-PCR assays in both rat and human species; Determination of optimal reference gene for rat-human data normalization. Suppl Fig. 8. Relative body weight changes in rat following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide; Relative body weight changes in rat following treatment with 20 mg/kg AZD2461 and olaparib in combination with temozolomide.</p>

Elucidation of inhibitory effects of bioactive anthraquinones towards formation of DNA advanced glycation end products (DNA-AGEs)

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

Dipyridamole is an effective inhibitor of cardiovirus growth in cell culture. The effects of dipyridamole on mengovirus replication in vivo and in vitro were examined in the hope the drug could be used as an experimental analog of the poliovirus inhibitor guanidine. Guanidine selectively inhibits poliovirus RNA synthesis but not RNA translation, and as such, has been a valuable research tool. Although guanidine does not inhibit cardiovirus infection, a compound with similar discriminatory characteristics would be experimentally useful for parallel work with these viruses. We found that mengovirus plaque formation in HeLa or L cells was inhibited nearly 100% by the presence of 80 muM dipyridamole. The inhibitory effect was reversible and targeted an early step in the replication cycle. Studies with luciferase-expressing mengovirus replicons showed that viral protein synthesis was unaffected by dipyridamole, and rather, RNA synthesis was the step targeted by the drug. This assessment was confirmed by direct analyses of viral translation and RNA synthesis activities in a Krebs-2-derived in vitro system that supported complete, infectious cardiovirus replication. In Krebs extracts, dipyridamole specifically inhibited viral RNA synthesis to more than 95%, with no concomitant effect on viral protein translation or polyprotein processing. The observed inhibition reversibly affected an early step in both minus-strand and plus-strand RNA synthesis, although inhibition of plus-strand synthesis was more profound than that of minus-strand synthesis. We conclude that dipyridamole is a potent experimental tool that readily distinguishes between cardiovirus translation and RNA replication functions.

BackgroundTranslational capacity (i.e. ribosomal mass) is a key determinant of protein synthesis and has been associated with skeletal muscle hypertrophy. The role of translational capacity in muscle atrophy and regrowth from disuse is largely unknown. Therefore, we investigated the effect of muscle disuse and reloading on translational capacity in middle‐aged men (Study 1) and in rats (Study 2).MethodsIn Study 1, 28 male participants (age 50.03 ± 3.54 years) underwent 2 weeks of knee immobilization followed by 2 weeks of ambulatory recovery and a further 2 weeks of resistance training. Muscle biopsies were obtained for measurement of total RNA and pre‐ribosomal (r)RNA expression, and vastus lateralis cross‐sectional area (CSA) was determined via peripheral quantitative computed tomography. In Study 2, male rats underwent hindlimb suspension (HS) for either 24 h (HS 24 h, n = 4) or 7 days (HS 7d, n = 5), HS for 7 days followed by 7 days of reloading (Rel, n = 5) or remained as ambulatory weight bearing (WB, n = 5) controls. Rats received deuterium oxide throughout the study to determine RNA synthesis and degradation, and mTORC1 signalling pathway was assessed.ResultsTwo weeks of immobilization reduced total RNA concentration (20%) and CSA (4%) in men (both P ≤ 0.05). Ambulatory recovery restored total RNA concentration to baseline levels and partially restored muscle CSA. Total RNA concentration and 47S pre‐rRNA expression increased above basal levels after resistance training (P ≤ 0.05). In rats, RNA synthesis was 30% lower while degradation was ~400% higher in HS 7d in soleus and plantaris muscles compared with WB (P ≤ 0.05). mTORC1 signalling was lower in HS compared with WB as was 47S pre‐rRNA (P ≤ 0.05). With reloading, the aforementioned parameters were restored to WB levels while RNA degradation was suppressed (P ≤ 0.05).ConclusionsChanges in RNA concentration following muscle disuse and reloading were associated with changes in ribosome biogenesis and degradation, indicating that both processes are important determinants of translational capacity. The pre‐clinical data help explain the reduced translational capacity after muscle immobilization in humans and demonstrate that ribosome biogenesis and degradation might be valuable therapeutic targets to maintain muscle mass during disuse.

Samples of RNA, isolated from uteri of ovariectomized adult rats treated with estrogen, have been analyzed on sucrose gradients. Treatment with estrogen either for 20 min or 2 hr increased the specific activity of all classes of uterine RNA, but produced no significant alteration in the distribution of radioactivity in the gradients, when animals received [(3)H]uridine intraperitoneally 15 min before they were killed. After labeling periods of 30 min, 1 hr, or 2 hr, however, the RNAs isolated from animals treated with estrogen had a smaller percentage of rapidly sedimenting (faster than 28S) species of RNA than did RNA from animals not treated with the hormone. The decreased percentage of high molecular weight RNA correlated with increases in both the specific activity of 28S and 18S RNA and the concentration of RNA in the whole organ. The labeled RNA of high molecular weight was also demonstrated, by the use of actinomycin D in vivo, to have a more rapid turnover rate in the estrogen-stimulated uterus. Our results indicate that estrogen increases not only the rate of synthesis of ribosomal RNA in the uterus of the ovariectomized adult rat, but also the rate or efficiency of processing of precursor RNA species of high molecular weight.

A practical solid-phase synthesis of deoxynucleic guanidine (DNG), a positively charged DNA backbone analogue, is reported. The nucleoside coupling step in the solid-phase synthesis of DNG involves the attack of a terminal 3'-amine upon an electronically activated 5'-carbodiimide to create a protected guanidinium internucleoside linkage. The activated carbodiimide is synthesized in situ by the mercury(II) abstraction of sulfur from an unsymmetrically substituted thiourea in which one substituent is an electron-withdrawing protecting group and the other is the 5'-nucleoside monomer. This produces, in addition to the carbodiimide, a mercury sulfide precipitate which accumulates in the pores of the solid support, restricting solvent and reagent access and reducing the coupling yields with each successive cycle. This obstacle is overcome by a simple washing step involving a thiophenol solution which readily removes the mercury salt. The addition of this step to the cycle enables DNG oligomers to be synthesized using standard macroporous SPS supports. Coupling yields of 98% were estimated from the HPLC analysis of the product mixtures. An octameric thymidyl oligomer (II) was synthesized and the fidelity of binding to octameric adenyl DNA oligomers containing cytidyl mismatches was determined. Binding was studied by thermal denaturation (Tm), Job plots, and circular dichroism spectrophotometry. The DNG oligomer (II) formed a 2:1 complex with octameric adenyl DNA (III) with a melting temperature of 63 °C. Each cytidyl mismatch induced a penalty of 4 to 5 °C in the observed melting temperatures. DNA sequences with four or more mismatches showed no base pairing in the presence of II. No association was observed between II and octameric cytidyl DNA. These observations demonstrate that DNG oligomers of moderate length are able to discriminate between complementary and mismatched DNA oligomers.

Analyzing the stability of gene expression using a simple reaction-diffusion model in an early Drosophila embryo

Rethinking hepatitis C viral kinetics: Insights into host-virus interactions in ‘difficult-to-treat’ groups and implications for novel treatment approaches