Dependence of melting temperature and mismatch discrimination on oligomer length

<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Average melting temperatures were calculated for 50% g•c DNA duplex oligomers that did not contain any mismatched base pairs as well as oligomers with single g•t or a•c mismatches. Predictions assumed total single strand concentration of 400 nM in 1 M Na buffer

Effects of sodium ions on melting temperatures of DNA–DNA and LNA–DNA duplexes

<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Duplexes were investigated that had a T•A base pair in the X•Y mismatch site. All duplexes had the same base sequence, 5′-ggtcctttcttggtg-3′/3′-ccaggaaagaaccac-5′, where LNA modifications were introduced at various positions (). Solid lines were calculated using a published salt correction ()

Stability and Mismatch Discrimination of Locked Nucleic Acid–DNA Duplexes

Locked nucleic acids (LNA; symbols of bases, +A, +C, +G, and +T) are introduced into chemically synthesized oligonucleotides to increase duplex stability and specificity. To understand these effects, we have determined thermodynamic parameters of consecutive LNA nucleotides. We present guidelines for the design of LNA oligonucleotides and introduce free online software that predicts the stability of any LNA duplex oligomer. Thermodynamic analysis shows that the single strand–duplex transition is characterized by a favorable enthalpic change and by an unfavorable loss of entropy. A single LNA modification confines the local conformation of nucleotides, causing a smaller, less unfavorable entropic loss when the single strand is restricted to the rigid duplex structure. Additional LNAs adjacent to the initial modification appear to enhance stacking and H-bonding interactions because they increase the enthalpic contributions to duplex stabilization. New nearest-neighbor parameters correctly forecast the positive and negative effects of LNAs on mismatch discrimination. Specificity is enhanced in a majority of sequences and is dependent on mismatch type and adjacent base pairs; the largest discriminatory boost occurs for the central +C·C mismatch within the +T+C+C sequence and the +A·G mismatch within the +T+A+G sequence. LNAs do not affect specificity in some sequences and even impair it for many +G·T and +C·A mismatches. The level of mismatch discrimination decreases the most for the central +G·T mismatch within the +G+G+C sequence and the +C·A mismatch within the +G+C+G sequence. We hypothesize that these discrimination changes are not unique features of LNAs but originate from the shift of the duplex conformation from B-form to A-form

Fluorescence emission spectra of target oligomer, 5′-gcgaggpggctt-3′, with single 2-aminopurine (p) reveal the magnitude of stacking interactions

<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Duplexes containing a H-bonded t•p base pair are compared with mismatched g•p base pair duplexes. Both unmodified DNA probes (blue lines) and probes with a LNA triplet at the mismatch site (red lines) were studied. UV melting experiments in 1 M Na buffer showed that LNA triplets increased mismatch discrimination for these sequences

Difference of Δ values between LNA and DNA probes for various mismatches

<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Sequence Set 1 (panel A and B), and Sets 2 and 3 (panel C) are plotted. Positions of LNA residues and set names are shown in Figure 1. A positive difference indicates improved mismatch discrimination for the LNA probe relative to the DNA probe. Dashed lines denote the range (±0.8°C), which is within the experimental error of the measurements

Effect of MDS-MSC on T cell apoptosis.

<p>T cells were incubated for 3 days alone or with MDS-MSC or normal-MSC in the presence of the mitogen PHA.The test was conducted by Annexin-V and PI double staining and analyzed by flow cytometry. Data are expressed as mean±SD of triplicates of 5 separate experiments. Annexin V+ means the cells were PI negative and Annexin V positive. *P≤0.05.</p

Induction of CD4+CD25+Foxp3+Tregs by MDS-MSC is dependent on TGFβ1.

<p>Western blot confirmed efficient knockdown of TGF-β1. (B) CD4+CD25-T cells were cultured with TGF-β1 knockdown MDS-MSC or normal-MSC for 5 days, and CD4+ T cells were collected. The expression of CD25 and Foxp3 on CD4+ T cells was analyzed by FACS. Results are expressed as mean±SD of triplicates of 5 separate experiments. *P≤0.05. (C) CD4+CD25-T cells were cultured with mutant siRNA transfected MDS-MSC or normal-MSC for 5 days, and CD4+ T cells were collected. The expression of CD25 and Foxp3 on CD4+ T cells was analyzed by FACS. Results are expressed as mean±SD of triplicates of 6 separate experiments. *P≤0.05. (D) anti-rhTGF-β1 mAb was added at the beginning of coculture of CD4+CD25-T cells and untransfected MDS-MSC or normal-MSC for 5 days, and CD4+ T cells were collected. The expression of CD25 and Foxp3 on CD4+ T cells was analyzed by FACS. Results are expressed as mean±SD of triplicates of 6 separate experiments. *P≤0.05.</p

MDS-MSC inhibit T-lymphocyte proliferation.

<p>Irradiated (15 Gy) MDS-MSC or normal-MSC were cultured for 5 days with CD2+ T-lymphocyte in the presence of PHA, then assessed by [3H]-thymidine incorporation. Data are expressed as mean±SD of triplicates of 5 separate experiments. *P≤0.05.</p

MDS-MSC induce CD4+CD25+Foxp3+Tregs.

<p>(A) CD4+CD25-T cells were cultured with MDS-MSC or normal-MSC for 5 days, and CD4+ T cells were collected. The expression of CD25 and Foxp3 on CD4+ T cells was analyzed by FACS. Results are expressed as mean±SD of triplicates of 4 separate experiments. *P≤0.05. (B) CD4+T cells were cocultured with MDS-MSC generated CD4+CD25+Foxp3+Tregs or normal-MSC generated CD4+CD25+Foxp3+Tregs in the presence of PHA, and the T-lymphocyte proliferation was measured on day 5 by [3H]-thymidine incorporation. Results are expressed as mean±SD of triplicates of 4 separate experiments. *P≤0.05. (C) MDS-MSC generated CD4+CD25+Foxp3+Tregs or normal-MSC generated CD4+CD25+Foxp3+Tregs inhibited the response of allogeneic T-lymphocyte in a dose-dependent manner. Responder CD2+ T-lymphocyte were stimulated with PHA for 5 days with or without graded dosed of MDS-MSC generated CD4+CD25+Foxp3+Tregs or normal-MSC generated CD4+CD25+Foxp3+Tregs. Results are expressed as mean±SD of triplicates of 4 separate experiments. *p≤0.05. (D) CD4+CD25-T cells were cultured with high-risk MDS-MSC or low-risk MDS-MSC for 5 days, and CD4+ T cells were collected. The expression of CD25 and Foxp3 on CD4+ T cells was analyzed by FACS. Results are expressed as mean±SD of triplicates of 4 separate experiments. *P≤0.05.</p

Highly Functional Bioinspired Fe/N/C Oxygen Reduction Reaction Catalysts: Structure-Regulating Oxygen Sorption

Tuna is one of the most rapid and distant swimmers. Its unique gill structure with the porous lamellae promotes fast oxygen exchange that guarantees tuna’s high metabolic and athletic demands. Inspired by this specific structure, we designed and fabricated microporous graphene nanoplatelets (GNPs)-based Fe/N/C electrocatalysts for oxygen reduction reaction (ORR). Careful control of GNP structure leads to the increment of microporosity, which influences the O₂ adsorption positively and desorption oppositely, resulting in enhanced O₂ diffusion, while experiencing reduced ORR kinetics. Working in the cathode of proton-exchange membrane fuel cells, the GNP catalysts require a compromise between adsorption/desorption for effective O₂ exchange, and as a result, appropriate microporosity is needed. In this work, the highest power density, 521 mW·cm^–2, at zero back pressure is achieved