Dietary nucleic acids (NAs) were important nutrients. However, the digestion of NAs in stomach has not been studied. In this study, the digestion of NAs by enzymes from fish stomach was investigated. The snakehead pepsins (SP) which were the main enzymes in stomach were extracted and purified. The purity of SP was evaluated by SDS-PAGE and HPLC. The snakehead pepsin 2 (SP2) which was the main component in the extracts was used for investigating the protein and NAs digestion activity. SP2 could digest NAs, including λ DNA and salmon sperm DNA. Interestingly, the digestion could be inhibited by treatment of alkaline solution at pH 8.0 and pepstatin A, and the digestion could happen either in the presence or absence of hemoglobin (Hb) and BSA as the protein substrates. Similarly, the stomach enzymes of banded grouper also showed the NAs digestion activity. NAs could be digested by the stomach enzymes of snakehead and banded grouper. It may be helpful for understanding both animal nutrition and NAs metabolic pathway.

In the present study, we demonstrate photoregulation of gene expression in a cell-free translation system from a T7 promoter containing two azobenzene derivatives at specific positions. As photoswitches, we prepared azobenzene-4′-carboxlyic acid (Azo) and 2,6-dimethylazobenzene-4′-carboxylic acid (DM-Azo), which were isomerized from trans to cis upon irradiation with UV light (λ < 370 nm), and 4-methylthioazobenzene-4′-carboxylic acid (S-Azo) and 2,6-dimethyl-4-(methylthio)azobenzene-4′-carobxylic acid (S-DM-Azo), which were cis-isomerized by irradiation with 400 nm visible light. Expression of green fluorescent protein from a promoter modified with S-Azo or S-DM-Azo could be induced by harmless visible light whereas that from a promoter modified with Azo or DM-Azo was induced only by UV light (340–360 nm). Thus, efficient photoregulation of green fluorescent protein production was achieved in a cell-free translation system with visible light without photodamage.Keywords: azobenzene; d-threoninol scaffold; gene expression; photoactivated; transcription; visible light;

•The limit of detection was 19 cells by strip reader and 100 cells by naked eye.•This assay shows good specificity and sensitivity of whole stem cells detection.•The gold-nanoparticle based lateral flow biosensor enables visual detection.Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80 min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells.

An advanced rolling circle amplification (RCA) strategy based on the target-circularization of the targeted double-stranded DNA (dsDNA) was established. Different from the traditional padlock-RCA, in which single-stranded probe DNA was circularized and amplified as signal amplification, our new approach could amplify a double-stranded DNA target. This special circularization of the target was realized by the ligation of target DNA with a biotin-labelled duplex adaptor containing 9 nt sticky ends by complementary base pairing. High specificity was obtained using two primers targeting the target sequence but not the probe itself in traditional padlock-RCA. With the help of streptavidin magnetic beads that immobilized the ligated dsDNA amplicon, the background nucleic acids contributing the most to non-specific amplification were eliminated. Under optimized conditions, less than 60 copies of the target sequence could be detected in the presence of massive background nucleic acids (>1012 copies of unrelated sequences). The sensitivity and specificity can rival canonical PCR. Without thermal cycles, the reduced handling and simpler equipment requirements render this assay a simple and rapid alternative to conventional methods. Based on these advantages, this method is a promising candidate in practical applications such as detecting contaminated food-borne pathogens in comprehensive food samples.

We demonstrated the generality of a strategy for photoswitching the activity of functional oligonucleotides by modulating their topological structure. Our strategy was proved to be versatile because it can be used to photoregulate functional oligonucleotides, e.g., ribozymes and DNAzymes, which have two binding arms and a catalytic loop. Repeated reversible photoregulation of RNA cleavage by a ribozyme or a DNAzyme was achieved by attaching two photoresponsive strands, artificial oligomers involving azobenzene moieties and nucleobases capable of forming a duplex as the supraphotoswitch. Individual strands were attached to the 3′ and 5′ ends of a RNA-cleavage oligonucleotide. Thus, the topological structure of the ribozyme or DNAzyme was constrained, and RNA cleavage was greatly suppressed when the supraphotoswitch duplex formed (OFF state). In contrast, RNA cleavage resumed when the supraphotoswitch duplex dissociated (ON state). Light irradiation was used to repeatedly switch the supraphotoswitch between the ON and OFF states so that RNA cleavage activity could be efficiently photoregulated. Analysis of the regulatory mechanism showed that topological constraints suppressed the RNA cleavage by causing both structural changes at the catalytic site and lower binding affinity between the RNA substrates and the functional oligonucleotides.Keywords: azobenzene; modified nucleic acid; photoswitch; RNA cleavage; topological structure;

Short DNA sequences, especially those that are repetitive or palindromic, can be used as the seeds for synthesis of long DNA by some DNA polymerases in an unusual manner. Although several elongation mechanisms have been proposed, there is no well-established model that explains highly efficient elongation under isothermal conditions. In the present study, we analyzed the elongation of nonrepetitive sequences with distinct hairpins at each end. These DNAs were elongated efficiently under isothermal conditions by thermophilic Vent (exo–) DNA polymerase, and the products were longer than 10 kb within 10 min of the reaction. A 20-nucleotide DNA with only one hairpin was also elongated. Sequence analysis revealed that the long products are mainly tandem repeats of the short seed sequences. The thermal melting temperatures of the products were much higher than the reaction temperature, indicating that most DNAs form duplexes during the reaction. Accordingly, a terminal hairpin formation and self-priming extension model was proposed in detail, and the efficient elongation was explained. Formation of the hairpin at the 5′ end plays an important role during the elongation.

•The active sites of pepsin towards NA were the same as those for protein digestion.•Asp32, Thr33, Asp215 and Gly217 were the pepsin active sites for NA digestion.•Residues Tyr189 and Val214 were essential for pepsin binding and catalysis of dsDNA digestion.•The Gly34 residue was essential for pepsin binding and catalysis of ssDNA digestion.Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity.

•Obtained unbiased WGA with high efficiency by using type IIS restriction enzymes.•Self-ligation problem was solved by producing more than 256 kinds of sticky ends.•Realized WGA by using adapters with random overhangs and single universal primer.•Amplification efficiency was evaluated by quantitative PCR.Ligation-mediated polymerase chain reaction (LM-PCR) is a whole genome amplification (WGA) method, for which genomic DNA is cleaved into numerous fragments and then all of the fragments are amplified by PCR after attaching a universal end sequence. However, the self-ligation of these fragments could happen and may cause biased amplification and restriction of its application. To decrease the self-ligation probability, here we use type IIS restriction enzymes to digest genomic DNA into fragments with 4–5 nt long overhangs with random sequences. After ligation to an adapter with random end sequences to above fragments, PCR is carried out and almost all present DNA sequences are amplified. In this study, whole genome of Vibrio parahaemolyticus was amplified and the amplification efficiency was evaluated by quantitative PCR. The results suggested that our approach could provide sufficient genomic DNA with good quality to meet requirements of various genetic analyses.

•DNA/chitosan nanocarriers were initially used for astaxanthin loading and delivery.•Astaxanthin-loaded nanosystem can efficiently protect cells from oxidative damage.•Encapsulated-astaxanthin can be quickly absorbed via endocytosis by Caco-2 cells.DNA/chitosan co-assemblies were initially used as nanocarriers for efficient astaxanthin encapsulation and delivery. The obtained astaxanthin-loaded DNA/chitosan (ADC) colloidal system was transparent and homogenous, with astaxanthin content up to 65 μg/ml. Compared to free astaxanthin, ADC nanoparticles with an astaxanthin concentration as low as 3.35 nM still showed a more powerful cytoprotective effect on H2O2-induced oxidative cell damage, and improved cell viability from 49.9% to 61.9%. The ROS scavenging efficiency of ADC nanoparticles was as high as 54.3%, which was 2-fold higher than that of free astaxanthin. Besides this, ADC nanoparticles were easily engulfed by Caco-2 cells in a short time, indicating that the encapsulated astaxanthin could be absorbed through endocytosis by intestinal epithelial cells. The improved antioxidation capability and facilitated cellular uptake enabled the ADC nanoparticles to be good candidates for efficient delivery and absorption of astaxanthin.

A facile and robust RNA preparation protocol was developed by combining rolling circle transcription (RCT) with RNA cleavage by RNase H. Circular DNA with a complementary sequence was used as the template for promoter-free transcription. With the aid of a 2′-O-methylated DNA, the RCT-generated tandem repeats of the desired RNA sequence were disconnected at the exact end-to-end position to harvest the desired RNA oligomers. Compared with the template DNA, more than 4 × 103 times the amount of small RNA products were obtained when modest cleavage was carried out during transcription. Large amounts of RNA oligomers could easily be obtained by simply increasing the reaction volume.