Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for ...detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).
The success of oligonucleotide ligation assays in probing specific sequences of DNA arises in large part from high enzymatic selectivity against base mismatches at the ligation junction. We describe ...here a study of the effect of mismatches on a new nonenzymatic, reagent-free method for ligation of oligonucleotides. In this approach, two oligonucleotides bound at adjacent sites on a complementary strand undergo autoligation by displacement of a 5′-end iodide with a 3′-phosphorothioate group. The data show that this ligation proceeds somewhat more slowly than ligation by T4 ligase, but with substantial discrimination against single base mismatches both at either side of the junction and a few nucleotides away within one of the oligonucleotide binding sites. Selectivities of >100-fold against a single mismatch are observed in the latter case. Experiments at varied concentrations and temperatures are carried out both with the autoligation of two adjacent linear oligonucleotides and with intramolecular autoligation to yield circular ‘padlock’ DNAs. Application of optimized conditions to discrimination of an H-ras codon 12 point mutation is demonstrated with a single-stranded short DNA target.
A new iodothymidine phosphoramidite enables the placement of a 5′-iodide into oligonucleotides; the iodide is stable to ammonia deprotection and allows nonenzymatic ligations of DNA.
A new ...iodothymidine phosphoramidite (
1) enables the placement of a 5′-iodide into oligonucleotides; the iodide is stable to ammonia deprotection and allows facile nonenzymatic ligations of DNA.
We describe physicochemical and enzymatic properties of 5′ bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is ...examined and no measurable hydrolysis is observed at pH 5–9 after 4 days at 25°C. The abilities of three 3′- and 5′-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (NsiI) cleavage is also attempted at a 5′-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5′-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that Tm values are lowered by a small amount (2°C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5′ bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.
We investigated the recognition of double-stranded DNAs by circular oligonucleotides. Using a circular oligodeoxynucleotide that contains a disulfide crosslink across the center of the circle, a ...complex that consists of a duplex and a displaced single-stranded loop was observed at pH 4.6. However, such displaced complexes were not formed at pH values near neutral. We also developed a method for autoligation of DNAs utilizing a 5 ′-iodide on one strand and a 3′ phosphorothioate on the other. The phosphoramidite derivative of iodothymidine was prepared; this allows 5′ iodide incorporation into DNA during automated DNA synthesis. Several practically useful template-directed ligations including the ligation of single stranded DNAs, cyclization of ssDNAs and the ligation of sticky-end duplexes were achieved using this iodide-phosphorothioate autoligation method. A detailed investigation of physicochemical and enzymatic properties of the 5′-S linkage was carried out. The 5′ -S linkage is found to be stable for extended periods in aqueous buffer and causes only small thermal destabilization of duplexes. The 5′ -S linkage is resistant to some of the exonucleases we investigated, and when placed at the restriction site, strongly inhibits the action of the restriction endonuclease Nsi 1. Surprisingly, the DNAs with 5′-S linkages were found to act as normal templates for DNA and RNA polymerases. The sensitivity of this phosphorothioate - iodide DNA autoligation reaction to single nucleotide mismatches at or near the ligation junction was investigated. Using a pair of probes 7nt and 13nt in length, selectivities of 10–200 fold were seen. Optimized placement of a single-base mismatch was shown to lead to selectivities comparable to those seen with ligase enzymes. This autoligation shows significant promise in solution detection of an H-ras point mutation using fluorescence resonance energy transfer (FRET) autoligation probes. The rate of the autoligation reaction was found to be accelerated by replacing the 3′ phosphorothioate with 3′ phosphoroselenoate. Like the 5′-S linkage, the 5′-Se linkage is found to be stable for extended periods in aqueous buffer and causes only small thermal destabilization of duplexes. The sequence selectivity is about the same as with the sulfur reaction. It was found that autoligation using both 3′ phosphorothioate and 3′ phosphoroselenoate occurs on RNA templates, whereas ligase enzymes are not active on RNAs.
In DNA triple helices, methylation at C-5 of thymine or cytosine is reported to have similar stabilizing effects for both bases. Here we show, however, that methylation of the same positions in RNA ...triplexes has distinctly different effects than in DNA. We have previously described the use of circular triplex-forming RNA oligonucleotides to recognize RNA sequences. Here it is shown that addition of C-5 methyl groups to uracils in these compounds very significantly increases not only affinity but also sequence selectivity in binding a purine-rich RNA target, as measured by thermal denaturation with various target RNAs. Surprisingly, however, addition of C-5 methyl groups to cytosines actually decreases affinity in binding RNA, while the same substitution in DNA is thermally stabilizing. Possible sources of this divergent behavior are discussed. A synthesis of 5-methylcytidine ribonucleoside 2′-
O-silyl-3′-
O-phosphoramidite is also described.
The contrasting effects of addition of methyl groups to the C-5 position of uracil and cytosine on the binding of RNA sequences by triplex-forming circular RNA oligonucleotides are described.
The smooth and natural interaction between human and lower limb exoskeleton is important. However, one of the challenges is that obtaining the joint rotation angles in time and accurately is ...difficult. In this paper, we propose the stacked convolutional and long–short term memory networks (Conv-LSTM) to estimate the hip, knee, and ankle joint angles from sEMG signals in locomotion modes including walk, run, stair descent, stair ascent, stand-to-sit, sit-to-stand, and jump. The joint angles are calculated from the kinematic models using the Euler angle signals measured by IMUs. The sEMG and joint angles are segmented according to the gait cycles measured by footswitch signals. Time–frequency analysis of sEMG signals is carried out using continuous wavelet transform. The Conv-LSTM model can extract the spatiotemporal information from the input to establish the mapping from sEMG sequences to multi-joint angle sequences. The evaluation effects of coefficient of determination (R2), root mean squared error, and Dynamic Time Warping on estimation performance are compared. The time domain (TD) features of sEMG perform better on joint angle estimation than the frequency domain and time–frequency domain features (p<0.05). The Conv-LSTM model with TD features as input outperforms the BP and state-of-the-art machine learning algorithms (kernel ridge, random forest, and support vector regression) on multi-joint angle estimation (R2: 0.9334, 0.9110, 0.9236, 0.9238, 0.8999, 0.9430, 0.9351, p<0.05). The estimation results are simulated in V-REP for exoskeleton control.
•Lower limb joint angle estimation of daily movements is realized using sEMG signals.•A method is proposed to calculate joint angles using Euler angles measured by IMU.•Time–frequency analysis of multi-channel sEMG signals is performed in the gait cycles.•TD features outperform FD or TFD features in lower limb multi-joint angle estimation.•Conv-LSTM extracts spatiotemporal information and obtain high estimation performance.
Although previous studies have provided some exploratory evidence of the gender‐taste association, empirical evidence on the causal relationships and downstream consequences of such an association in ...the consumer behavior domain is still lacking. To address this gap, the present research empirically documents the association between gender and taste, and explores the spillover effect of brand gender on consumer preference for sweet food. Across four studies, we demonstrate that feminine and sweet are cognitively associated (Study 1), and accordingly, products launched by a feminine brand are perceived to be sweeter than those launched by a masculine brand (Study 2). Furthermore, a feminine (vs. masculine) brand leads to a higher preference for its sweet (vs. unsweet) products, which is mediated by the perceived congruence between brand gender and product taste (Study 3). Finally, we identify brand type as a moderator, showing that the congruence effect between brand gender and product taste holds for traditional brands but is attenuated for innovative brands (Study 4). Our findings advance the understanding of brand gender association and its impact on consumer behavior and offer meaningful implications for market positioning and communication.
Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) play critical roles in human diseases. We aimed to clarify the role of lncRNA X-inactive specific transcript (XIST)/miR-149-3p/forkhead box P3 ...(FOXP3) axis in ovarian cancer (OC) cell growth. XIST, miR-149-3p and FOXP3 expression in OC tissues and cell lines was assessed, and the predictive role of XIST in prognosis of OC patients was analyzed. The OC cell lines were screened and accordingly treated with silenced/overexpressed XIST plasmid or miR-149-3p mimic/inhibitor, and then the proliferation, invasion, migration, colony formation ability, apoptosis, and cell cycle distribution of OC cells were measured. Effect of altered XIST and miR-149-3p on tumor growth in vivo was observed. Online website prediction and dual luciferase reporter gene were implemented to detect the targeting relationship of lncRNA XIST, miR-149-3p, and FOXP3. XIST and FOXP3 were upregulated, whereas miR-149-3p was downregulated in OC tissues and cells. High XIST expression indicated a poor prognosis of OC. Inhibition of XIST or elevation of miR-149-3p repressed proliferation, invasion, migration, and colony formation ability, and promoted apoptosis and cell cycle arrest of HO-8910 cells. In SKOV3 cells upon treatment of overexpressed XIST or reduction of miR-149-3p, there exhibited an opposite tendency. Based on online website prediction, dual luciferase reporter gene, and RNA pull-down assays, we found that there was a negative relationship between XIST and miR-149-3p, and miR-149-3p downregulated FOXP3 expression. This study highlights that knockdown of XIST elevates miR-149-3p expression to suppress malignant behaviors of OC cells, thereby inhibiting OC development.
This paper investigates the problem of stability, H∞ performance analysis, and H∞ control for continuous-time Markov jump nonlinear systems, where the nonlinear subsystems are approximated by the ...piecewise-affine technique. The proposed Markov jump piecewise-affine systems contain different modes and regions, both of which are determined by Markov chains and piecewise-affine partitions, respectively. A new admissible adjacent region switching paths (AARSPs) algorithm is proposed for the first time in the continuous-time domain to decrease the conservatism of the complete adjacent region switching paths (CARSPs) algorithm. This new algorithm optimizes the path selection conditions of the next instantaneous time region switching in the CARSPs algorithm, and effectively reduces the computational complexity and the conservatism of the CARSPs algorithm. Furthermore, a state-feedback piecewise-linear controller is designed by means of the ellipsoidal outer approximation estimation method, such that the corresponding closed-loop system is stochastically stable and has a guaranteed H∞ performance index. Finally, the effectiveness and practicability of both the AARSPs algorithm and the piecewise-linear control strategy are fully demonstrated via two illustrative examples including a class of tunnel diode circuit systems.
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