Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells

Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing

We report that combining a DNA analog (2′F-ANA) with rigid RNA analogs [2′F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1–1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells

Eltrombopag markedly decreases levels of ROS in MOLM14 cells.

<p>Flow cytometry analysis of ROS in MOLM14 cells cultured with or without E. A) Representative flow charts show gating strategy (left panel) based on size (Forward Scatter) and granularity (Side Scatter) of the cells, and (central and right panel) Carboxy-H₂DCFDA signal. B) Graphic presentation of ROS levels measured by flow cytometry in cells treated with E (5 μM) or DPI (25 μM), an inhibitor of NADPH oxidase known to decrease an intracellular level of ROS, or untreated (CTR) cells. Results are presented as an average of percent of control (untreated cells) mean fluorescence intensity with SEM. Data represent 4 independent experiments and the differences are statistically significant (*) p = 0.0000018. C) Relative level of ROS in AML cell lines: MOLM14, HL-60, MV4-11 and KG1a (left panel) and primary AML samples (right panel) after exposure to E. Differences between CTR and E treated cells are statistically significant (*) p = 0.0026 for cell lines and p = 0.001 for primary cells. D) Levels of O₂^- in MOLM14 cells exposed to E measures at various time points using Dihydroethidium (DHE) stain. Control represents untreated cells, E—cells exposed to E (5 μM), DPI—cells exposed to DPI (25 μM). Data are presented as an average percent of control calculated based on mean fluorescence from 3 independent experiments and standard error of the mean.</p

Eltrombopag Does Not Significantly Affect Mitochondria Function and ATP Production.

<p>A) Graph shows results of TMRE staining as a measurement of the mitochondria membrane potential in MOLM14 cells after exposure to E at various time points. CCCP, a known mitochondrial membrane potential disrupter was used as a control for the assay. Bars represent percent of control (untreated cells) mean fluorescence intensity (MFI) with SEM. B) Effect of E on mitochondrial membrane potential in AML cell lines (left panel) and primary AML cells (right panel) measured by a TMRE staining. C) Measurements of oxygen consumption rate in control and cells treated with E (5 μM) for 24 hours. The oxygen consumption rates are expressed as nanomoles O₂ consumed/ min/ 10⁶ cells. The differences in maximal and spare respiratory capacity (SRC) between control and E treated cells are statistically significant with p value from 3 independent experiments equal 0.04 and 0.01 respectively. D) Graph represents an ATP production in MOLM14 cells cultured with or without (CTR) E for 20 hours, then exposed either to IAA, AA+O or remained untreated. Dark gray bars (untreated) represent total ATP, black bars (IAA) represent ATP produced by mitochondria and light gray bars (AA+O) represent ATP produced during glycolysis.</p

Modification of intracellular ROS level abrogates eltrombopag’s effect on AML cells.

<p>A) E blocks completely increase of ROS caused by BSO and rescues cells from BSO induced death. Left panel—ROS level in MOLM14 cells untreated (CTR) or pre-treated with BSO for 72 hours and then exposed to E or left untreated. Right panel—proliferation assay—results are presented as an average and SEM of a total number of cells from 3 independent experiments. At day 0 cells cultured in the presence of BSO (50 or 100 μM) for 72hours or in plain medium (CTR) were plated at concentration 5x10^4 per 250 μL of culture medium and exposed to E or left untreated. B) Pre-loading with ferric ammonium citrate (FAC) rescues MOLM14 cells from E cytotoxic effect by inducing ROS level. Levels of H₂O₂ measured using carboxy- H₂DCFDA in MOLM14 cells treated as described above (left panel). Proliferation of MOLM14 cells un-manipulated or pre-loaded with 500 μg/mL of FAC for 24 h and then exposed to E or left untreated (CTR) (right panel).</p

Effectiveness of E cytotoxicity presented as a half maximal growth inhibitory concentration (CC₅₀).

<p>The CC₅₀ was calculated for primary AML samples (top) and AML cell lines (bottom) at days: 4 and 2 respectively, when cultured at 2% serum.</p><p>Effectiveness of E cytotoxicity presented as a half maximal growth inhibitory concentration (CC₅₀).</p

MOLM14 cells incubated with Eltrombopag for 6 hours show higher expression of stress response genes.

<p>Quantitative real time PCR (Q-PCR) measurement of mRNA level expression of five stress-response related genes in untreated cells (CTR), cells treated with rhTPO (100 ng/mL) and E (5 μM) for 6h (top panels) and earlier time point 2–6 h (bottom panel). Expression level of each gene is normalized to β-actin expression and presented as a percent of expression in untreated cells.</p