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Gas chromatography-ion mobility spectrum (GC-IMS) is used to analyze and compare the differences in aroma among different tobacco samples. The aroma substances in tobacco samples in Jilin Changchun are the richest, while those in Guangdong Nanxiong are the lowest. The concentrations of aroma substances such as decanal, 1-hydroxy-2-propanone, and 2-methylbutanol were the highest in Guangdong Nanxiong of the three. The concentration of 1-hexanol, cyclohexanone, pentanoic acid, and other aroma substances in Fujian Nanping was high. The concentration of 2-acetylfuran, 2-octanol, isopentanol, 3-methylvaleric acid, phenylacetic acid, and other aroma substances in Changchun area of Jilin Province was low. Through principal component analysis and similarity research, both tobaccos can be distinguished by their production areas and grades from the same.

We report the synthesis and characterization of novel chromophores based on chlorogenic acid. The structure analysis and photophysical properties were studied to investigate the structure of the ne...

Solanesol has antibacterial, anticancer, anti-inflammatory and antiulcer biological activities, and is a key intermediate in the synthesis of coenzyme Q10, vitamin K2, and the anticancer synergist N-solanesyl-N,N'-bis(3,4-dimethoxy-benzyl)ethylenediamine (SDB) and other ubiquinone drugs. Due to its unique chemical structure, the chemical synthesis of solanesol is difficult, so solanesol is currently mainly isolated from solanaceous plants. Tobacco (Nicotiana tabacum) has the highest content of solanesol compared to other solanaceous plants. Currently, the research on the extraction of solanesol from tobacco focuses on its extraction and purification. This article reviews the extraction methods, saponification, separation and purification methods of solanesol, as well as the research progress on tobacco solanesol, in China and abroad in recent years. Studies show that the organic solvent extraction method is time-consuming and has a low extraction rate. Modern technology- assisted extraction methods, such as ultrasound-assisted extraction, microwave-assisted extraction, and supercritical carbon dioxide (CO2) extraction not only can improve the extraction rate but also can effectively shorten the extraction time. The separation and purification of solanesol from the extracting solution are critical steps to meet the purity requirements of synthetic drugs, and are also key in limiting the large-scale industrial production of high-purity solanesol. Molecular Imprinting Technology (MIT) has the advantages of small investment, simple operation and ease of large scale production compared to chromatographic methods. Solanesol separated by MIT can meet the requirements for synthesizing coenzyme Q10. Nowadays, the synthesis of molecularly-imprinted solanesol polymers with better adsorption efficiency is one of the important directions in the future research of solanesol purification.

Nicking endonucleases (NEs) become increasingly attractive for their promising applications in isothermal amplification. Unfortunately, in comparison with their applications, their catalytic mechanism studies have relatively lagged behind due to a paucity of crystal structure information. Nt.BstNBI is one of those widely used NEs. However, many aspects of its catalytic mechanism still remained to be explored. Herein, we employed only rolling circle amplification (RCA) assay as a major analytic tool and succeeded in identifying the potential binding positions and regions of the DNA substrate based on locked nucleic acid modification, DNA duplex length of substrate, and substrate mismatch designs. Based on these data, we, for the first time, revealed that Nt.BstNBI was likely to recognize six adjacent positions of the recognition sequence (G1rt, A2rt, G3rt, A2rb, C3rb, and T4rb) in the major groove and hold three positions of the cleavage sequence (N3ct, N4ct, and N7cb) in the minor groove of DNA duplex for nicking. Moreover, this work also demonstrated the unexpected efficiency of RCA to study the macromolecular interaction for certain kind of nucleases in an easy and high-throughput way.

High dielectric constant ceramics fillers are widely used as fillers in polymer matrix to prepare high performance dielectric composites. Homogeneous filler distribution in these composites films is found to be quite difficult to achieve balance between high dielectric constant and high breakdown strength. Herein, multilayer structured composites films containing nickelous hydroxide (Ni(OH)2) as fillers, with different multilayered structures were designed and prepared, including 2–5 layers structure with various filler distribution. The effects of multilayer structure on the dielectric performance are explored by keeping the overall filler content constant. Combined with computer simulation, it is suggested that the variation in filler distribution in these films can effectively redistribute electric field intensity. Meanwhile, the dielectric constant and breakdown strength of the overall composites can be adjusted by changing the volume ratio between high filler content layer and low filler content layer. It is noted that at least 1/3 of the overall film volume should be occupied by a high breakdown strength layer to keep rather high overall breakdown strength. Moreover, high dielectric constant layer should be the outer layers to achieve overall high dielectric constant. Among all the layered structures investigated, the maximum energy density of 6.03 J/cm3 is obtained for film with a 3-layer nonsequential arrangement structure, which is 68% higher than single layer film with identical composition. This study provides some reference value for the preparation of multilayered dielectric polymer composites films for energy storage applications.

Self-powered elastic Conductors based on thermoelectric materials with the ability to harvest energy from the living environment are considered as important for electronic devices under off-grid, maintenance-free, or unfeasible battery replacement circumstances. Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) is perhaps the most well-known organic conductor. However, the application of PEDOT:PSS in flexible devices is limited by its brittleness and various unrecoverable properties under strain. Various polymer blends based on water-soluble polymers and PEDOT:PSS have been prepared. Nevertheless, they fail to illustrate good balance between electrical conductivity and mechanical performance due to various issues, including the phase morphology with PEDOT:PSS as the dispersed phase; thus, the formation of a conductive network between PEDOT:PSS is prohibited. In this study, PEDOT:PSS is incorporated into natural rubber (NR), with NR as the dispersed phase. For 10 wt % PEDOT:PSS-NR composite films doped with dimethyl sulfoxide (DMSO), the conductivity was up to 87 S/cm and the elongation at break was maintained at 490%. More importantly, self-powered temperature- and tensile strain-sensing abilities were also realized. Furthermore, it is also demonstrated that most of the unrecoverable strain and conductivity under cyclic tensile strain could be healed by water and phosphate-buffered saline (PBS) post-treatment. This work provides interesting insights for preparing healed and stretchable self-powered electronic sensors.

Feature recognition is widely used in visual simultaneous localization and mapping and visual odometry system. The most popular feature is the point feature, which includes SIFT feature, SURF feature, ORB feature. But in low textured scenes, RGB-D simultaneous localization and mapping (SLAM) tend to fail due to lack of reliable point features. Line features are as rich as point features in a structured or a low textured environment. However, line feature always contains more texture information for the calculation of pose, making it more complex for a line feature to be extracted, described and parameterized. To overcome the problems. We proposed a robust VO system fusing both points and lines by linearizing points, which not only preserves the information provided by line features, but also speeds up the calculation process. The method in this paper is evaluated on the TUM public real-world RGB-D dataset. The experimental results demonstrate that the algorithm proposed in this paper is more accurate and robust than the pure feature point algorithm in consideration.

Development of CRISPR/Cas-based in vitro diagnostic devices, or CRISPR/Cas-Dx, has become an intensely researched area. Among the different classes of CRISPR/Cas-Dx, the class based on the Cas12a enzyme (i.e., CRISPR/Cas12a-Dx or simply Cas12a-Dx), is predominantly employed for detecting DNA targets. Current research in Cas12a-Dx has focused on appending Cas12a-Dx to preamplification techniques or coupling Cas12a-Dx to different detection modalities, which has inevitably overshadowed the detection performance of Cas12a-Dx and overlooked its intrinsic detection capability without preamplification. We recognize that Cas12a-Dx, which relies on DNA-activated Cas12a to cleave single-stranded DNA, shares significant similarity with other nuclease-based DNA biosensors, whose performances can be influenced by parameters ranging from the reaction buffer to the reaction temperature. We are thus inspired to probe the limits of preamplification-free Cas12a-Dx by exploring and systematically evaluating several potential parameters that may impact its detection sensitivity and time. Using a previously reported fluorescence-based Cas12a-Dx as the test bed, we have identified that the Cas12a enzyme, the reaction buffer, the substrate label, the substrate concentration, and the reaction temperature can be optimized to significantly improve the signal-to-background ratio and the reaction rate of Cas12a-Dx. Based on these findings, we have improved the limit of detection (LOD) of the Cas12a-Dx to 100 fM, while reduced the time-to-positive to

Transcription factor (TF)-based metabolite detection mainly depends on TF-regulated gene expression in cells. From TF activation to gene transcription and translation, the signal travels a relatively long way before it is received. Here, we propose a TF-splinting duplex DNA nanoswitch to detect metabolites. We show its feasibility using tryptophan repressor (TrpR) to detect l-tryptophan as a model. The assay has been optimized and characterized after obtaining a proof of concept, and the detection of l-tryptophan in complex biological samples is feasible. Unlike an equivalent gene expression approach, the whole process is a single-step, enzyme-free, and signal-on method. It can be completed within 20 min. This proposed TF-splinting duplex has the potential to be applied to the quick and convenient detection of other metabolites or even TFs.

Oligonucleotides were screened for strongly silver-stained repetitive sequences. An 'AG'-clustered purine sequence showed strong staining, and the staining density can be compromised by disrupting the continuity of the 'AG'-clustered sequence. The staining-favored sequence was then employed in rolling circle amplification (RCA) for its product detection. A tube-staining method was developed for convenient and visual RCA assay. Moreover, by introducing GalR into RCA, d-galactose was detected by RCA tube-staining with naked eyes without any equipment. About 10 mM d-galactose can be easily identified, and the detection of d-galactose was specific in comparison with that of several other monosaccharides.

Inspired by the hierarchically dendritic structure of plant root and its extraordinary ability of grabbing soil, polyurethane (PU) fibers with such bio-mimic structure were fabricated by non-solvent induced precipitation under turbulent force to grab multi-walled carbon nanotubes (CNTs) in this work, forming interpenetrating segregated network (IPSN) with CNTs coated on hierarchically dendritic polyurethane (PU) fibers (HDPUFs) and filled into voids between entangled HDPUFs. Prepared CNT/PU composites illustrate an ultrahigh maximum CNT loading of 80 wt% with good flexibility, while only 10% could be achieved for common segregated structure. Meanwhile, a low percolation threshold of 0.25 wt% and a maximum conductivity of 25 S/cm is achieved. Benefiting from significantly reduced insulating polymer coating on CNTs, the electrical conductivity of CNT/HDPUF composites at low CNT loading (≤7 wt%) is orders of magnitude higher than that of CNT/PU composites containing randomly dispersed CNTs with similar filler content. Simultaneously, the specific electromagnetic interference (EMI) shielding efficiency (SE) of CNT/HDPUF composite reaches up to 10526.1 dB cm2/g thanks to their high electrical conductivity and low density, which has been rarely reported previously for CNT-based composites. This work provides a simple and low-cost method to fabricate thin, lightweight, and flexible composite with high filler content, high electrical conductivity and outstanding electromagnetic shielding performance.

Film capacitors can be used as energy storage for fast storage and release energy, but the low energy density limits their applications. In our previous study, we have demonstrated that a small amount of 2D titanium dioxide (TiO2) nanobelts in PVDF could significantly enhance the energy density of these composite films. In order to increase the dielectric constant and energy density of these composites, multi-walled carbon nanotubes (MWCNTs) are introduced into these 2D TiO2 nanobelts obtained during electrospinning, and parallelly oriented along these 2D nanobelts. The shell formed by insulating TiO2 can avoid the formation of conductive network and realize high polarization efficiency. It’s shown that when the ratio of MWCNTs and TiO2 in the composite filler is 1:30, the breakdown voltage of the filler can achieve 478 kV/mm, the polarizability is 8.34 μC/cm2, and the energy density is increased from 2.87 J/cm3 to 17.97 J/cm3 comparing with neat PVDF.

Various literatures have reported that incorporating functional fillers into thermoplastic vulcanizates (TPVs) is an ideal method to prepare functional TPVs with balanced properties. Nevertheless, it is quite difficult to achieve that for thermoplastic silicone vulcanizate (TPSiV) due to their rather high silicon rubber (SiR) content as well as delicate balance between processing ability and various physical properties. Herein, a novel method involves using CNT/TPU phase as polymer matrix to disperse TPSiV particles through solution processing is proposed. Compared with randomly dispersed CNTs and traditional segregated CNTs network with TPSiV particles, composites fabricated by such method illustrate much better balance between various properties, including: low percolation threshold (Pc), comprehensive mechanical performance, better electromagnetic interference (EMI) shielding ability, etc. CNT/TPU anchored TPSiV double-percolated composite illustrates comparable Pc (0.42 wt%) to traditional segregated TPSiV/CNT composite (0.38 wt%), while the TPSiV composite with randomly dispersed CNT shows apparently high Pc (1.37 wt%). More importantly, both tensile strength and elongation at break are improved for these compoistes with double-percolation structure. It is even better than neat TPSiV composites at the same CNT loading. The prepared two types of segregated composites demonstrate similar electrical conductivity at high filler loading (≥1 wt%), enabling them with similar EMI performance, but the one with CNT/TPU as adhesive phase is much more stable after 100 times bending, illustrating negligible attenuation. This study provides a novel approach for incorporating functional fillers into TPSiV matrix for balanced properties.

Various fillers and methods have been utilized to enhance the thermal conductivity (TC) of polymer-based electronic packaging materials, however, the enhancement was limited by disconnected pathway for phonon transferring. Herein, a novel fibrillated hybrid skeleton (f-AO@BNNS skeleton) was fabricated by dispersing boron nitride nanosheet (BNNS) into alumina (AO) sol, electrospinning and sintering, where long-range phonon transferring highway was successfully constructed by both macroscopically interconnected fiber and microscopically oriented and overlapped BNNSs confined in nanofiber. Besides, crystal AO and atomic-level bonding were acquired on BNNS-AO interface by sintering, largely decreasing interface thermal resistance inside skeletons. Prepared polybenzoxazine/f-AO@BNNS composites exhibited the highest TC of 3.24 Wm−1 K−1 at a skeleton loading of 6.9 vol%. The higher TC was achieved at lower filler content when compared with other reported skeletons, demonstrating the superiority of f-AO@BNNS skeleton. This work provides a new strategy of preparing fibrillated hybrid skeleton for high thermally conductive composites.

Two-dimensional (2D) nanomaterials have been widely used as advanced functional materials due to their special nanostructures and various functionalities. Researchers working on 2D nanomaterials are facing several problems, the difficulties in synthesis, scarcity of species, largely unclear formation mechanism and non-universal preparation method. Herein, this paper reports a new method based on electrospinning to fabricate 2D inorganic materials (titanium dioxide) with thickness around 20 nm and width/thickness ratio of >50. These 2D nanobelts TiO2 could enhance the dielectric performance of PVDF based composites much more significantly, and the energy density of 12.45 J/cm3 could be easily achieved with a small volume fraction (3 vol%) of the nanobelts. Such method has many advantages such as safe production, low production cost, and continuous production. More importantly, this approach is potential to become a universal method to fabricate a range of 2D inorganic nanomaterials in the future due to its universality.

High-purity solanesol can be used for pharmaceutical applications, but the current method for purifying solanesol has high cost and difficult continuous operation, and the use of molecular imprinting to purify natural products is a hot research topic of current research. Solanesol molecularly imprinted polymers were synthesized via emulsion polymerization for the first time. The morphology of the SSO-MIPs was observed with a scanning electron microscope, and the effects of the synthesis time, initiator dosage, functional monomer dosage, and cross-linking agent dosage on the adsorption effects under high-temperature and rapid synthesis conditions were discussed. The results demonstrate that the optimum synthesis conditions were a ratio of the template molecules to the functional monomers to the cross-linking agents of 1:8:30 (mol:mol:mol), 10 mg of the initiator, and a synthesis temperature of 70 &deg, C. The imprinting factor of SSO-MIPs synthesized under the optimized process was found to reach 2.51, and the SSO-MIPs synthesized by this method exhibited a good adsorption effect, emitted less pollution during the synthesis process, and are convenient for demulsification. This research reports a reliable method for the synthesis of solanesol molecularly imprinted polymers.

Rolling circle amplification (RCA) has been considered as a powerful tool for nucleic acids detection. Here, a novel repressor-RCA-based method for L-tryptophan (L-Trp) detection was developed. This method utilizes the specific interaction between the RCA circular template and the Trp repressor protein (TrpR) involved in trp operon of Escherichia coli (E. coli). In the absence of L-Trp, the TrpR protein could not bind to the RCA template, and the RCA process can be continued. When L-Trp is present, the activated TrpR will bind to the operon sequence on the RCA template and inhibit the RCA reaction. Thus, the concentration of L-Trp is correlated directly with the fluorescent RCA signals. We succeeded in detecting L-Trp in a single step in simple homogeneous reaction system. The detection limit was estimated to be 0.77 μM (S/N=3) with good linearity. The method can unambiguously distinguish L-Trp from other 19 standard amino acids and L-Trp analogs. This strategy is also promising for detecting many small molecules such as other amino acids and carbohydrates.

Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

To further understand the ligation mechanism, effects of 2'-O-methyl nucleotide (2'-OMeN) on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5'- and 3'-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ± 4.0%, respectively. Substitutions at both 5'- and 3'-ends decreased the ligation efficiency by 76.6% ± 1.3%. Corresponding kinetic parameters, Vmax, Km, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2'-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5'- and 3'-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3'-end impairs the ligation efficiency more profoundly than that at 5'-end. Different concentrations of Mg(2+), Ca(2+), K(+), Na(+), and ATP were also demonstrated to affect the T4 DNA ligase activity. These results enriched our knowledge about the effects of 2'-OMeN substitutions on the T4 DNA ligase.

Induction of endonucleolytic DNA cleavage is an essential event that links the initiating stimuli to the final effects of cells. The cleavage efficiency and thus the final yield could be affected by many factors, including structures of DNA substrates, composite structures of enzymes–substrates or enzymes–nucleic analogs and so on. However, it is not clear whether a nucleotide derivative-substituted in DNA substrates can influence the efficiency of enzymatic cleavage. To investigate the effect of sugar pucker conformation on DNA–protein interactions, we used 2′-O-methyl modified nucleotides (OMeN) to modify DNA substrates of isocaudemers BamHI and BglII in this study, and used FRET assay as an efficient method for analysis of enzyme cleavage. Experimental results demonstrated that OMeN-substituted recognition sequences influenced the cleavage rates significantly in a position-dependent manner. OMeN substitutions can reduce the cleavage as expected. Surprisingly, OMeN substitutions can also enhance the cleavage rates. The kinetics parameters of V max and K m have been obtained by fitting the Michaelis-Menten kinetic equation. These 2'-OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in synthetic biology.

We have developed a rapid and high-throughput assay based on rolling circle amplification, to distinguish individual strand cleavage of DNA duplexes by restriction endonucleases. As an illustration, we analyzed nicking activity of Nb.BbvCI and uneven cleavage of LNA modified DNA by EcoRI. This assay has potential for analyzing protein-DNA interactions.

Small molecule metabolites and their allosterically regulated repressors play an important role in many gene expression and metabolic disorder processes. These natural sensors, though valuable as good logic switches, have rarely been employed without transcription machinery in cells. Here, two pairs of repressors, which function in opposite ways, were cloned, purified and used to control DNA replication in rolling circle amplification (RCA) in vitro. By using metabolites and repressors as inputs, RCA signals as outputs, four basic logic modules were constructed successfully. To achieve various logic computations based on these basic modules, we designed series and parallel strategies of circular templates, which can further assemble these repressor modules in an RCA platform to realize twelve two-input Boolean logic gates and a three-input logic gate. The RCA-output and RCA-assembled platform was proved to be easy and flexible for complex logic processes and might have application potential in molecular computing and synthetic biology.

We used a novel asymmetric cleavage analysis method based on rolling circle amplification (RCA) to determine the effects of LNA modification of substrate on the two subunits of R.BbvCI cleavage. We designed two sets of cleavage circular substrates by using two different ligation strategies and analyzed the single strand cleavage efficiency affected by different modification positions both from the cleaved strands and the uncleaved strands. Results showed that the effects of LNA on cleavage rates of modified strands and unmodified strands were both site-dependent. The Nb.BbvCI and Nt.BbvCI were affected by LNA modification in different way. Most of the modification positions showed strong inhibition of both of these two nickases cleavage. However, the modification in T3 position of bottom strand hardly affected both of the two nickases activities. The results suggested an intimated interaction between the two subunits of R.BbvCI and the T3 position in bottom strand might be a less tight position which was hard to be disturbed.

Dumbbell probe (DP) attracts increasing interests in rolling circle amplification (RCA). A universal DP production method through cleavage-ligation of hairpin was proposed and optimized. The production is characterized by restriction endonuclease (RE)-induced cleavage ends ligation. It has the advantage of phosphorylation-free, splint-free and purification-free. To optimize designing, we found that the position of RE cleavage sequence in the stem and the primer position in the loop affected the formation and amplification of DP obviously. Both sticky and blunt ends cleaved by RE produce DP efficiently. Moreover, we introduced this DP into circle to circle (C2C) RCA based on the same cleavage-ligation principle and acquired high sensitivity. By combining a two-ligation design and the C2C strategy, specificity for detecting let-7 family members was increased extremely. Furthermore, coreaction of different steps facilitated convenient formation and amplification process of DP.

Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2′-OMeRNA modification, which might be due to the size of the C2′-modified moieties. 2′-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction. Schematic of RCA affected by nucleotide analogs. (A) The structure comparison of DNA and nucleotide analogs used. (B) Analogs were substituted in circular template for RCA.

Rolling circle amplification (RCA) is a robust bioanalytical method invented based on the principle of viral DNA reproduction. Since RCA offers many technical advantages of convenient operation, cost-effective experimental setup, rapid result delivery, etc., it has been widely used for analysis and detection of DNAs, RNAs, and proteins. It was recently shown that the RCA-based methods are suitable for analysis of micro RNAs (miRNAs). To optimize the RCA-based miRNA analysis, we evaluated ligation efficiencies of T4 RNA ligase 2 for different DNA/RNA heteroduplexes using a molecular beacon-based assay. Then, we designed padlock probes, each being specific to particular miRNA, and examined their ability to differentiate between members of the let-7 family of miRNAs having high sequence homology. It was found that each padlock probe could accurately and unambiguously identify the target miRNA at concentrations as low as 2.5 fmol. Results from this study are important for the development of RCA-based diagnostics of miRNA biomarkers in clinical and environmental specimens.

To understand the protein transduction domain (PTD)-mediated protein transduction behavior and to explore its potential in delivering biopharmaceutic drugs, we prepared four TAT-EGFP conjugates: TAT(+)-EGFP, TAT(-)-EGFP, EGFP-TAT(+) and EGFP-TAT(-), where TAT(+) and TAT(-) represent the original and the reversed TAT sequence, respectively. These four TAT-EGFP conjugates were incubated with HeLa and PC12 cells for in vitro study as well as injected intraperitoneally to mice for in vivo study. Flow cytometric results showed that four TAT-EGFP conjugates were able to traverse HeLa and PC12 cells with almost equal transduction efficiency. The in vivo study showed that the TAT-EGFP conjugates could be delivered into different organs of mice with different transduction capabilities. Bioinformatic analyses and CD spectroscopic data revealed that the TAT peptide has no defined secondary structure, and conjugating the TAT peptide to the EGFP cargo protein would not alter the native structure and the function of the EGFP protein. These results conclude that the sequence orientation, the spatial structure, and the relative location of the TAT peptide have much less effect on the TAT-mediated protein transduction. Thus, the TAT-fused conjugates could be constructed in more convenient and flexible formats for a wide range of biopharmaceutical applications.

To further understand the ligation mechanism, effects of 2'-O-methyl nucleotide (2'-OMeN) on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5'- and 3'-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ± 4.0%, respectively. Substitutions at both 5'- and 3'-ends decreased the ligation efficiency by 76.6% ± 1.3%. Corresponding kinetic parameters, Vmax, Km, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2'-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5'- and 3'-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3'-end impairs the ligation efficiency more profoundly than that at 5'-end. Different concentrations of Mg(2+), Ca(2+), K(+), Na(+), and ATP were also demonstrated to affect the T4 DNA ligase activity. These results enriched our knowledge about the effects of 2'-OMeN substitutions on the T4 DNA ligase.

DNA polymerase I (PolI) digested by protease produces a small fragment (SF) containing 5'-3' exonuclease activity. The 5'-3' exonuclease activity of polI cleaves the downstream RNA primer strands during DNA replication in vivo. Previous in vitro studies suggested its capability of cleaving duplex from 5' terminal and a flap-structure-specific endonuclease activity. From the crystal structures of other nucleases and biochemical data, a two-metal-ion mechanism has been proposed but has not been determined. In this study, we cloned, expressed, and purified the SF protein, and established a novel fluorescence resonance energy transfer (FRET) assay to analyze the catalytic activity of the SF protein. The effects of several metal ions on its catalytic capability were analyzed using this FRET assay. Results showed that Mg2+, Mn2+, and Zn2+ were able to activate the cleavage of SF, while Ca2+, Ni2 +, and Co2+ were not suitable for SF catalysis. The effects of K+, Na+, and dNTP were also determined.

The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5'-3' polymerase activity can extend the 5' overhanging sticky end to the blunt end, and 3'-5' exonuclease activity can cleave the 3' overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3'-5' exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5' overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.

To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2′-O-methyl nucleotide (2′-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2′-O-methyl nucleotide has a C3′-endo sugar pucker conformation different from the C2′-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2′-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2′-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2′-fluoro nucleic acid (2′-FNA) and locked nucleic acid (LNA) having similar C3′-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2′-endo to C3′-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2′-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.

DNA polymerization products by Klenow fragment (KF) are blunt-ended. In the present study, we found that the Klenow fragment mutants with partial deletions of thumb subdomain were unable to extend primers to the 5' terminal of templates, thus creating 5' overhanging sticky ends 2 nt long. We termed this phenomenon as PmTP (premature termination of polymerization). The KF mutants produced homogenous sticky-ended products only under mild reaction conditions, whereas under vigorous reaction conditions, the sticky ends were prone to be blunt-ended. It was also identified that deletions of more than four residues of KF thumb subdomain could induce PmTP, and tworesidue deletion of KF thumb subdomain only induced PmTP in a lower-concentration situation. Structure modelling analysis suggested that shortening or destruction of alpha helix H1 at the tip of the thumb subdomain was crucial to PmTP, while the conserved residues in front of alpha helix was less important. PmTP might be caused by the reduced DNAbinding affinity of the mutants. The sticky ends made by PmTP have potential applications in gene splicing and molecular cloning techniques.