An RNA-Cleaving DNAzyme That Requires an Organic Solvent to Function.

Engineering functional nucleic acids that are active under unusual conditions will not only reveal their hidden abilities but also lay the groundwork for pursuing them for unique applications. Although many DNAzymes have been derived to catalyze diverse chemical reactions in aqueous solutions, no prior study has been set up to purposely derive DNAzymes that require an organic solvent to function. Herein, we utilized in vitro selection to isolate RNA-cleaving DNAzymes from a random-sequence DNA pool that were "compelled" to accept 35 % dimethyl sulfoxide (DMSO) as a cosolvent, via counter selection in a purely aqueous solution followed by positive selection in the same solution containing 35 % DMSO. This experiment led to the discovery of a new DNAzyme that requires 35 % DMSO for its catalytic activity and exhibits drastically reduced activity without DMSO. This DNAzyme also requires divalent metal ions for catalysis, and its activity is enhanced by monovalent ions. A minimized, more efficient DNAzyme was also derived. This work demonstrates that highly functional, organic solvent-dependent DNAzymes can be isolated from random-sequence DNA libraries via forced in vitro selection, thus expanding the capability and potential utility of catalytic DNA.

Potential G-quadruplexes within the Promoter Nuclease Hypersensitive Sites of the Heat-Responsive Genes in Rice.

G-quadruplexes (G4s) have been shown to be involved in the regulation of multiple cellular processes. Exploring putative G4-forming sequences (PQSs) in heat-responsive genes of rice and their folding structures under different conditions will help to understand the mechanism in response to heat stress. In this work, we discovered a prevalence of PQSs in nuclease hypersensitive sites within the promoters of heat-responsive genes. Moreover, 50 % of the searched G3 PQSs ((G3+ L1-7 )3+ G3+ ) locate in heat shock transcription factors. Circular dichroism spectroscopy, thermal difference spectroscopy, and UV melting analysis demonstrated the representative PQSs could adopt stable G4s at physiological temperature and potassium concentration. These PQSs were able to stall Klenow fragment (KF) DNA polymerase by the formation of G4s. However, the G4s with Tm values around 50-60 °C could be increasingly unwound by KF with the increase of temperatures from 25 to 50 °C, implying that these G4s could sense the changes in temperature by structural switch. This work offers fresh clues to understanding the potential of G4-involved functions of PQSs and the molecular events in plants in response to heat stress.

Functional Nucleic Acids Under Unusual Conditions.

Functional nucleic acids (FNAs), including naturally occurring ribozymes and riboswitches as well as artificially created DNAzymes and aptamers, have been popular molecular toolboxes for diverse applications. Given the high chemical stability of nucleic acids and their ability to fold into diverse sequence-dependent structures, FNAs are suggested to be highly functional under unusual reaction conditions. This review will examine the progress of research on FNAs under conditions of low pH, high temperature, freezing conditions, and the inclusion of organic solvents and denaturants that are known to disrupt nucleic acid structures. The FNA species to be discussed include ribozymes, riboswitches, G-quadruplex-based peroxidase mimicking DNAzymes, RNA-cleaving DNAzymes, and aptamers. Research within this space has not only revealed the hidden talents of FNAs but has also laid important groundwork for pursuing these intriguing functional macromolecules for unique applications.

Detection of G-Quadruplex Structures Formed by G-Rich Sequences from Rice Genome and Transcriptome Using Combined Probes.

Putative G-quadruplex (G4) forming sequences (PQS) are highly prevalent in the genome and transcriptome of various organisms and are considered as potential regulation elements in many biological processes by forming G4 structures. The formation of G4 structures highly depends on the sequences and the environment. In most cases, it is difficult to predict G4 formation by PQS, especially PQS containing G2 tracts. Therefore, the experimental identification of G4 formation is essential in the study of G4-related biological functions. Herein, we report a rapid and simple method for the detection of G4 structures by using a pair of complementary reporters, hemin and BMSP. This method was applied to detect G4 structures formed by PQS (DNA and RNA) searched in the genome and transcriptome of Oryza sativa. Unlike most of the reported G4 probes that only recognize part of G4 structures, the proposed method based on combined probes positively responded to almost all G4 conformations, including parallel, antiparallel, and mixed/hybrid G4, but did not respond to non-G4 sequences. This method shows potential for high-throughput identification of G4 structures in genome and transcriptome. Furthermore, BMSP was observed to drive some PQS to form more stable G4 structures or induce the G4 formation of some PQS that cannot form G4 in normal physiological conditions, which may provide a powerful molecular tool for gene regulation.

De novo sequencing and comparative transcriptome analysis of the male and hermaphroditic flowers provide insights into the regulation of flower formation in andromonoecious taihangia rupestris.

BACKGROUND: Taihangia rupestris, an andromonoecious plant species, bears both male and hermaphroditic flowers within the same individual. However, the establishment and development of male and hermaphroditic flowers in andromonoecious Taihangia remain poorly understood, due to the limited genetic and sequence information. To investigate the potential molecular mechanism in the regulation of Taihangia flower formation, we used de novo RNA sequencing to compare the transcriptome profiles of male and hermaphroditic flowers at early and late developmental stages. RESULTS: Four cDNA libraries, including male floral bud, hermaphroditic floral bud, male flower, and hermaphroditic flower, were constructed and sequenced by using the Illumina RNA-Seq method. Totally, 84,596,426 qualified Illumina reads were obtained and then assembled into 59,064 unigenes, of which 24,753 unigenes were annotated in the NCBI non-redundant protein database. In addition, 12,214, 7,153, and 8,115 unigenes were assigned into 53 Gene Ontology (GO) functional groups, 25 Clusters of Orthologous Group (COG) categories, and 126 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. By pairwise comparison of unigene abundance between the samples, we identified 1,668 differential expressed genes (DEGs), including 176 transcription factors (TFs) between the male and hermaphroditic flowers. At the early developmental stage, we found 263 up-regulated genes and 436 down-regulated genes expressed in hermaphroditic floral buds, while 844 up-regulated genes and 314 down-regulated genes were detected in hermaphroditic flowers at the late developmental stage. GO and KEGG enrichment analyses showed that a large number of DEGs were associated with a wide range of functions, including cell cycle, epigenetic processes, flower development, and biosynthesis of unsaturated fatty acid pathway. Finally, real-time quantitative PCR was conducted to validate the DEGs identified in the present study. CONCLUSION: In this study, transcriptome data of this rare andromonoecious Taihangia were reported for the first time. Comparative transcriptome analysis revealed the significant differences in gene expression profiles between male and hermaphroditic flowers at early and late developmental stages. The transcriptome data of Taihangia would be helpful to improve the understanding of the underlying molecular mechanisms in regulation of flower formation and unisexual flower establishment in andromonoecious plants.

Activity Enhancement of G-Quadruplex/Hemin DNAzyme by Flanking d(CCC).

G-quadruplex (G4)/hemin DNAzymes have been extensively applied in bioanalysis and molecular devices. However, their catalytic activity is still much lower than that of proteinous enzymes. The G4/hemin DNAzyme activity is correlated with the G4 conformations and the solution conditions. However, little is known about the effect of the flanking sequences on the activity, though they are important parts of G4s. Here, we report sequences containing d(CCC), flanked on both ends of the G4-core sequences remarkably enhance their DNAzyme activity. By using circular dichroism and UV-visible spectroscopy, the d(CCC) flanking sequences were demonstrated to improve the hemin binding affinity to G4s instead of increasing the parallel G4 formation, which might explain the enhanced DNAzyme activity. Meanwhile, the increased hemin binding ability promoted the degradation of hemin within the DNAzyme by H2O2. Furthermore, the DNAzyme with d(CCC) flanking sequences showed strong tolerance to pH value changes, which makes it more suitable for applications requiring wide pH conditions. The results highlight the influence of the flanking sequences on the DNAzyme activity and provide insightful information for the design of highly active DNAzymes.

Duplex Identification of Staphylococcus aureus by Aptamer and Gold Nanoparticles.

Staphylococcus aureus is the top common pathogen causing infections and food poisoning. Identification of S. aureus is crucial for the disease diagnosis and regulation of food hygiene. Herein, we report an aptamer-AuNPs based method for duplex identification of S. aureus. Using AuNPs as an indicator, SA23, an aptamer against S. aureus, can well identify its target from Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa. Furthermore, we find citrate-coated AuNPs can strongly bind to S. aureus, but not bind to Salmonella enterica and Proteus mirabilis, which leads to different color changes in salt solution. This colorimetric response is capable of distinguishing S. aureus from S. enteritidis and P. mirabilis. Thus, using the aptasensor and AuNPs together, S. aureus can be accurately identified from the common pathogens. This duplex identification system is a promising platform for simple visual identification of S. aureus. Additionally, in the aptasensing process, bacteria are incubated with aptamers and then be removed before the aptamers adding to AuNPs, which may avoid the interactions between bacteria and AuNPs. This strategy can be potentially applied in principle to detect other cells by AuNPs-based aptasensors.

A turn-on fluorescent sensor for zinc and cadmium ions based on perylene tetracarboxylic diimide.

In this paper, we describe a turn-on fluorescent probe for Zn(2+) and Cd(2+) ion detection and discrimination. This probe, N'-bis-(N,N-di-(2-pyridylmethyl)-ethane-1,2-diamine)-perylene-3,4,9,10-tetracarboxylic-diimide (PDI-DIDPA), exhibits very low fluorescence at pH above 6.0 due to the photo-induced electron transfer (PET) process from 2-pyridylmethyl-amine (DPA) to perylene tetracarboxylic diimide (PDI). Zn(2+) and Cd(2+) can chelate PDI-DIDPA to form a stable complex at pH 6.0-7.0 and 9.0 respectively, and inhibit the PET process, which result in the fluorescence recovery of PDI-DIDPA. This fluorescence turn-on behavior allows the detection of Zn(2+) in the range of 0.1-4.0 µM and Cd(2+) in the range of 0.1-5.0 µM. The limit of detection for Zn(2+) and Cd(2+) is as low as 32 nM and 48 nM. The high selectivity, high sensitivity and easy operation make this probe suitable for the rapid detection of Zn(2+) and Cd(2+) respectively. The different response of PDI-DIDPA to Zn(2+) and Cd(2+) at different pH makes it possible to discriminate Zn(2+) and Cd(2+) by simply adjusting the pH of the working solution.

Functional-group specific aptamers indirectly recognizing compounds with alkyl amino group.

Aptamers are usually generated against a specific molecule. Their high selectivity makes them only suitable for studying specific targets. Since it is nearly impossible to generate aptamers for every molecule, it can be of great interest to select aptamers recognizing a common feature of a group of molecules in many applications. In this paper, we describe the selection of aptamers for indirect recognition of alkyl amino groups. Because amino groups are small and positive charged, we introduced a protection group, p-nitrobenzene sulfonyl (p-nosyl) to convert them into a form suitable for aptamer selection. Taking N(ε)-p-nosyl-L-lysine (PSL) as a target, we obtained a group of aptamers using the SELEX technique. Two optimized aptamers, M6b-M14 and M13a exhibit strong affinity to PSL with the K(d) values in the range of 2-5 µM. They also show strong affinity to other compounds containing p-nosyl-protected amino groups except those also possessing an α-carboxyl group. Both aptamers adopt an antiparallel G-quadruplex structure when binding to targets. An aptamer beacon based on M6b-M14 showed good selectivity toward the reaction mixture of p-nosyl-Cl and alkyl amino compounds, and could recognize lysine from amino acid mixtures indirectly, suggesting that aptamers against a common moiety of a certain type of molecules can potentially lead to many new applications. Through this study, we have demonstrated the ability to select aptamers for a specific part of an organic compound, and the chemical conversion approach may prove to be valuable for aptamer selection against molecules that are generally difficult for SELEX.

Specific interactions between adenosine and streptavidin/avidin.

The screening of ligands against proteins plays important role in drug discovery and biological research. Using a dye labelled Streptavidin binding aptamer (SBA) as a competitive reporter probe, we found that adenosine bound to streptavidin specifically. Fluorescence spectral analysis showed that adenosine bound to both avidin and streptavidin with the K(ds) in the range of 0.1-0.2 mM, and these bindings can be blocked by biotin. Although streptavidin and avidin are well-known and widely used in bioanalysis, their biological role is still a riddle so far. Since adenosine is a ubiquitous physiological regulator present in cells, our finding provides new clues for the understanding of the functions of both proteins.

Morphological characteristics of amino acids in wet deposition of Danjiangkou Reservoir in China's South-to-North Water Diversion Project.

Amino acids are an important constituent in organic nitrogen deposition, and changes in the content of their components have a direct impact on the nitrogen input to the ecosystem. From December 2018 to November 2019, 176 precipitation samples were collected at Danjiangkou Reservoir, the source of the middle line of the South-to-North Water Diversion Project, and the variation characteristics of dissolved free amino acids (DFAA) and dissolved combined amino acids (DCAA) were analyzed. The volume-weighted value concentration ranges of DFAA and DCAA were 0.159-1.136 µmol/L and 1.603-7.044 µmol/L, respectively, and amino acids were dominated by DCAA in wet deposition. Our results showed that glutamic acid (Glu), glycine (Gly), and aspartic acid (Asp) were the dominant amino acids in both DFAA and DCAA. The concentration of DFAA was highest in winter, while the concentration of DCAA was in autumn. Dissolved total amino acids (DTAA) were insignificantly correlated with DFAA, whereas they were linearly correlated with DCAA, indicating a significant influence of agricultural activities on DTAA. The analysis of the backward trajectory of air masses showed that amino acids were mainly influenced by proximity inputs around the reservoir. The bioavailability of organic matter was higher in the southeastern of the reservoir than in the northwestern. The wet deposition flux of TDN was 14.096 kg N/ha/year, and the potential ecological impact on water bodies cannot be ignored. This study was conducted to clarify the variation characteristics of amino acids fractions in wet deposition and to provide parameters for regional assessment of amino acids wet deposition. The ecological impact of nitrogen wet deposition on water bodies will be explored to provide a basis for nitrogen pollution control and water quality protection in the middle line of the South-to-North Water Diversion Project.

Characterization of G-quadruplex/hemin peroxidase: substrate specificity and inactivation kinetics.

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.

G-quadruplex DNA aptamers generated for systemin.

Ligands specific to bioactive molecules play important roles in biomedical researches and applications, such as biological assay, diagnosis and therapy. Systemin is a peptide hormone firstly identified in plant. In this paper we report the selection of a group of DNA aptamers that can specifically bind to systemin. Through comparing the predicted secondary structures of all the aptamers, a hairpin structure with G-rich loop was determined to be the binding motif of these aptamers. The G-rich loop region of this binding motif was further characterized to fold into an antiparallel G-quadruplex by truncation-mutation assay and CD spectrum. The apparent equilibrium dissociation constant (K(d)) of one strong binding sequence (S-5-1) was measured to be 0.5 µM. The specificity assay shows that S-5-1 strongly bind to whole systemin, weakly bind to truncated or mutated systemin and does not bind to the scrambled peptide with the same amino acid composition as systemin. The high affinity and specificity make S-5-1 hold potentials to serve as a molecular ligand applied in detection, separation and functional investigation of systemin in plants.

A pH sensitive ratiometric fluorophore and its application for monitoring the intracellular and extracellular pHs simultaneously.

Intracellular and extracellular pH plays a key role in many cell biological processes. Probes for simultaneously monitoring the pH change inside and outside living cells are rarely available. In this paper, we describe a new ratiometric pH fluorophore that was synthesized by condensation of 4-bromine-1,8-naphthalimides and 3-amino-1,2,4-triazole. In the range of pH 5-8, the only N-H in the heterocycle-fused aromatic ring system of this fluorophore undergoes a reversible deprotonation-protonation process, which results in a large red shift of the absorption and emission spectra. In aqueous solution, this fluorophore exhibits good pH selectivity, high photostability, high tolerance to ionic strength, and high fluorescence quantum yield in both the acid and base forms. A long chain derivative of this fluorophore (HNNA) was designed for cellular pH sensing. HNNA was found to locate on the membrane structure of the cells, and was successfully used for mapping the pH change in both the extracellular microenvironment and the inner cells by confocal imaging.

Rational design of Hg2+ controlled streptavidin-binding aptamer.

Through substituting two canonical base pairs of a streptavidin-binding aptamer with T-T mismatched base pairs, a new aptamer was constructed. Its binding ability could be controlled by Hg(2+) through the formation of T-Hg-T metal-base pairs.

General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

G-quadruplexes (G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs) are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes) have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

Selective isolation of G-quadruplexes by affinity chromatography.

G-quadruplex (G4) is a characteristic secondary structure of nucleic acids containing repetitive tandem guanines. G4-forming sequences are found prevalent in the human genome by bioinformatics analysis. Accumulating evidence has suggested that G4s are involved in many biological processes. Selective isolation of G4s would be an effective tool in the study of G4s. In this paper, we prepared four affinity matrixes using hemin or a perylene derivative (N,N'-Bis-(2-(amino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide, Pery01) as ligand, and investigated the retention behaviors of different G4s on these matrixes. Our experimental results suggest that the π-π stacking interaction between ligand and G-tetrad plays a key role in the selective isolation of G4s, whereas the electrostatic interaction between DNA and matrix causes the nonspecific binding. One matrix prepared by immobilizing Pery01 on polyglycidylmethacrylate (PGMA) beads through an aminocaproic acid spacer exhibits good selectivity for parallel structure G4s and has been successfully used to directly isolate a spiked parallel G4 from plasma.