Cellulose nanocrystals were converted into ratiometric pH-sensing nanoparticles by dual fluorescent labelling employing a facile one-pot procedure. A simple and versatile three-step procedure was also demonstrated extending the number of fluorophores available for grafting. In this method an amine group was introduced via esterification followed by a thiol-ene click reaction.

Adenosine 5-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F 0 F 1 ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.Adenosine 5Ј-triphosphate (ATP) is a highly important biomolecule in living cells: It plays a central role in cell energy metabolism and also serves directly or indirectly in a number of cell signaling processes (1-3). The intracellular concentration of ATP is believed to oscillate in some eukaryotic cells, e.g. in ␤-cells (4) and in cells of the yeast Saccharomyces cerevisiae (5). However, changes in cytoplasmic ATP concentration with high time resolution have so far only been measured in a few circumstances (6, 7), mainly because methods for such continuous measurements are not generally available, or, in the case of NMR, require very high densities of cells or tissue (8).The lack of time-resolved measurements has prohibited the understanding of how the level of ATP and other intracellular metabolites are regulated in the cell and how ATP in turn regulates a number of cellular processes. Most current measurements of ATP in cells use extraction of the cell content and measure the concentration of ATP in the extract by various off-line methods such as HPLC (9), luciferase (10), or other enzyme-based methods (11). A few protein-based sensors exist (6,7,(12)(13)(14), which in principle allow for time-resolved measurements of intracellular ATP or ADP, but some of these methods entail expression of the sensor molecule in situ, which is not always possible. Hence, although it is expected that the intracellular concentration of ATP is anything but stationary, this has not been verified by real-time measurements, except in a few cases where ...

We describe a new type of aptamer-based optical nanosensor which uses the embedding of target responsive oligonucleotides in porous polyacrylamide nanoparticles to eliminate nuclease instability. The latter is a common problem in the use of aptamer sensors in biological environments. These aptamers embedded in nanoparticles (AptaNPs) are proposed as a tool in real-time metabolite measurements in living cells. The AptaNPs comprise 30 nm polyacrylamide nanoparticles, prepared by inverse microemulsion polymerization, which contain water-soluble aptamer switch probes (ASPs) trapped in the porous matrix of the nanoparticles. The matrix acts as a molecular fence allowing rapid diffusion of small metabolites into the particles to interact with the aptamer molecules, but at the same time it retains the larger aptamer molecules inside the nanoparticles providing protection against intracellular degradation. We tested the ability of the AptaNPs to measure the adenine-nucleotide content in yeast cells. Our results successfully demonstrate the potential for monitoring any metabolite of interest in living cells by selecting specific aptamers and embedding them in nanoparticles.

A series of six double-functionalised nucleosides, in which aromatic moieties were inserted into the 5'-(S)-C-position, were synthesised and incorporated into DNA duplexes. The aromatic moieties were thymine-1-yl, phenyl, 1,2,3-triazol-1-yl, 1,2,3-triazol-4-yl, 4-(uracil-5-yl)-1,2,3-triazol-1-yl and 4-phenyl-1,2,3-triazol-1-yl. The DNA duplexes were studied with UV melting curves, CD spectroscopy and molecular modelling. The results showed that the aromatic moieties in some cases interact in the minor groove forming DNA zipper structures. The strongest specific interaction was found between two thymines or between a thymine and a phenyl group in a crossed (-3)-zipper motif (i.e., with two base pairs interspacing the modifications). Modelling revealed that the interaction is aromatic stacking across the minor groove. Also, the extended uracil-triazole moiety demonstrated zipper contacts in the minor groove as well as binding to the floor of the groove.

Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F(0)F(1) ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating the glycolytic oscillation. Measurements of intracellular ATP provide a unique tool to understand the role of these membrane ATPases and how their activities are regulated. We have constructed a new nanobiosensor that can perform time-resolved measurements of intracellular ATP in intact cells. Measurements of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal behaviour of intracellular ATP in yeast strains lacking either of the two membrane bound ATPases have confirmed that F(0)F(1) ATPase and Pma1p contribute significantly to the ATP consumption in the cell and to the regulation of glycolytic oscillation. Furthermore, our measurements also demonstrate that ATPase activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose is used up, the ATP consumption stops immediately. Thus, glucose or some compound derived from glucose must be involved in controlling the activity of these two ATPases.

Three 5-modified 2'-deoxyuridine nucleosides were synthesized and incorporated into oligonucleotides and compared with the previously published 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine monomer W. The introduction of an aminomethyl group on the phenyl group led to monomer X, which was found to thermally stabilize a 9-mer DNA:RNA duplex, presumably through the partial neutralization of the negative charge of the backbone. By also taking advantage of the stacking interactions in the major groove of two or more of the monomer X, an extremely high thermal stability was obtained. A regioisomer of the phenyltriazole substituent, that is the 5-(4-phenyl-1,2,3-triazol-1-yl)-2'-deoxyuridine monomer Y, was found to destabilize the DNA:RNA duplex significantly, but stacking in the major groove compensated for this when two to four monomers were incorporated consecutively. Finally, the 5-phenyl-2'-deoxyuridine monomer Z was incorporated for comparison, and it was found to give a more neutral influence on duplex stability indicating less efficient stacking interactions. The duplexes were investigated by CD spectroscopy and MD simulations.

[a] ATP (adenosine-5'-triphosphate) is central to cellular metabolism as a multifunctional intermediate in cellular processes. Many cellular reactions depend on the hydrolysis of ATP, for example, ion transport across membranes, cell motility, and biosynthetic reactions.[1] In cell-signaling cascades, protein kinases transfer a phosphate group from ATP to key regulatory proteins that serve in the control of cell metabolism, growth, and differentiation.[2] Thus, assays that monitor ATP concentration in both bioassays and in cellular environments have wide applications in biochemical and biomedical research. Despite the important role played by ATP in biological systems, only a handful of sensors that can monitor ATP in real-time exist and several of them have limitations in intracellular usage.In most ATP-utilizing metabolic reactions, ATP is converted to ADP (adenosine-5'-diphosphate) and only to a lesser extent to AMP (adenosine-5'-monophosphate). ADP is recycled to ATP through phosphorylation reactions. Therefore, the challenge in developing a specific ATP sensor is to produce one that can differentiate between ATP and ADP. However, only a few such sensors have been reported. [3][4][5][6][7] Furthermore, most of the available sensors suffer from the fact that they have high affinity for ATP and therefore will only have a limited use in many bioassays and in cellular environments where the ATP concentration is in the millimolar range. Recently, four kinds of biosensor were reported for real-time monitoring of ATP : 1) A sandwich stacking of pyrene-adenine-pyrene was designed to measure ATP concentrations in HeLa cells, [3] 2) a protein-based biosensor for ADP was developed by engineering the bacterial actin ParM to measure ATPase and kinase activity, [4] 3) two genetically encoded biosensors have been reported: i: the e-subunit of F0F1-ATP synthase was sandwiched between two fluorescent proteins to develop a FRET-type indicator of ATP [5] and ii: the bacterial regulatory protein, GlnK1 was combined with GFP to measure ATP:ADP ratios, [6] 4) enzyme activity-coupled sensors in the form of a luciferase-carbon nanotube ATP sensor. [7] An alternative approach to designing an ATP sensor is to develop a functional aptamer sensor for simple and accurate real-time ATP detection in bioassays and in cellular environments. Aptamers are single-stranded nucleic acids with specific affinity for their targets. In vitro selection can provide an aptamer for almost any kind of target with preselected affinity that thus may be considered an attractive diagnostic and sensing molecule. Moreover, once an aptamer has been selected, it can be chemically synthesized and used directly as a probeunlike genetically encoded sensors, which often require molecular cloning and cellular expression procedures.In developing a nanobiosensor specific for ATP we selected a new DNA aptamer sequence that preferentially binds ATP. A selection method based on Flu-Mag SELEX [8] was designed to obtain ATP-specific enrichment through a total of 14 cycle...

A series of double-headed nucleosides were synthesized using the Sonogashira cross-coupling reaction. In the reactions, additional nucleobases (thymine, cytosine, adenine, or guanine) were attached to the 5-position of 2'-deoxyuridine or 2'-deoxycytidine through a propyne linker. The modified nucleosides were incorporated into oligonucleotides, and these were combined in different duplexes that were analyzed by thermal denaturation studies. All of the monomers were well tolerated in the DNA duplexes and induced only small changes in the thermal stability. Consecutive incorporations of the monomers led to increases in duplex stability owing to increased stacking interactions. The modified nucleotide monomers maintained the Watson-Crick base pair fidelity. Stable duplexes were observed with heavily modified oligonucleotides featuring 14 consecutive incorporations of different double-headed nucleotide monomers. Thus, modified duplexes with an array of nucleobases on the exterior of the duplex were designed. Molecular dynamics simulations demonstrated that the additional nucleobases could expose their Watson-Crick and/or Hoogsteen faces for recognition in the major groove. This presentation of nucleobases may find applications in providing molecular information without unwinding the duplex.