Sequence Context in DNA i-Motifs Can Nurture Very Stable and Persistent Kinetic Traps.

I-motifs are non-canonical DNA structures with recognized biological significance and a proven utility in material engineering. Consequently, understanding and control of i-motif properties is essential to sustain progress across both disciplines. In this work, we systematically investigate how proximity to the most common form of DNA, a double-stranded duplex, influences the thermodynamic and kinetic properties of adjacent i-motifs.

Zero-Background Small-Molecule Sensors for Near-IR Fluorescent Imaging of Biomacromolecular Targets in Cells.

In this study, we report a general approach to the design of a new generation of small-molecule sensors that produce a zero background but are brightly fluorescent in the near-IR spectral range upon selective interaction with a biomolecular target. We developed a fluorescence turn-on/-off mechanism based on the aggregation/deaggregation of phthalocyanine chromophores. As a proof of concept, we designed, prepared, and characterized sensors for in-cell visualization of epidermal growth factor receptor (EGFR) tyrosine kinase.

Activatable G-quadruplex based catalases for signal transduction in biosensing.

Discovery of oxidative catalysis with G-quadruplex•hemin constructs prompted a range of exciting developments in the field of biosensor design. Thus, G-quadruplex based DNAzymes with peroxidase activity found a niche as signal transduction modules in a wide range of analytical applications. The ability of nucleic acid scaffolds to recognise a variety of practically meaningful markers and to translate the recognition events into conformational changes powers numerous sensor design possibilities.

Stoichiometric approach to quantitative analysis of biomolecules: the case of nucleic acids.

Majority of protocols for quantitative analysis of biomarkers (including nucleic acids) require calibrations and target standards. In this work, we developed a principle for quantitative analysis that eliminates the need for a standard of a target molecule. The approach is based on stoichiometric reporting.

Rational design of guiding elements to control folding topology in i-motifs with multiple quadruplexes.

Nucleic acids are versatile scaffolds that accommodate a wide range of precisely defined operational characteristics. Rational design of sensing, molecular computing, nanotechnology, and other nucleic acid devices requires precise control over folding conformations in these macromolecules. Here, we report a new approach that empowers well-defined conformational transitions in DNA molecular devices.

Rational Design of Memory-Based Sensors: the Case of Molecular Calorimeters.

Thermodynamic characterization is crucial for understanding molecular interactions. However, methodologies for measuring heat changes in small open systems are extremely limited. We document a new approach for designing molecular sensors, that function as calorimeters: sensors based on memory.

Design of Turn-On Near-Infrared Fluorescent Probes for Highly Sensitive and Selective Monitoring of Biopolymers.

Simple, sensitive, and selective detection of specific biopolymers is critical in a broad range of biomedical and technological areas. We present a design of turn-on near-infrared (NIR) fluorescent probes with intrinsically high signal-to-background ratio. The fluorescent signal generation mechanism is based on the aggregation/de-aggregation of phthalocyanine chromophores controlled by selective binding of small-molecule "anchor" groups to a specific binding site of a target biopolymer.

Multidimensional Tunability of Nucleic Acids Enables Sensing over Unknown Backgrounds.

A longstanding challenge in quantitative analysis is the relationship between a sensor's dynamic range and a background: the response range must align with the target's background value. If this condition is not met, a reliable measurement is impossible. The requirement is especially critical for sensing systems displaying sharp responses.

Quantitation without Calibration: Response Profile as an Indicator of Target Amount.

Quantitative assessment of biomarkers is essential in numerous contexts from decision-making in clinical situations to food quality monitoring to interpretation of life-science research findings. However, appropriate quantitation techniques are not as widely addressed as detection methods. One of the major challenges in biomarker's quantitation is the need to have a calibration for correlating a measured signal to a target amount.

Rational Control of Folding Cooperativity in DNA Quadruplexes.

Availability of basic tools for engineering molecular systems with precisely defined properties is crucial toward progress in development of new responsive materials. Among such materials are systems capable of generating an ultrasensitive response (i.e.

A dual input DNA-based molecular switch.

We have designed and characterized a DNA-based molecular switch which processes two physiologically relevant inputs: pH (i.e. alkalinisation) and enzymatic activity, and generates a chemical output (in situ synthesized oligonucleotide).

Rational design of highly responsive pH sensors based on DNA i-motif.

Availability of strategies for molecular biosensing over a finely adjustable dynamic range is essential for understanding and controlling vital biological processes. Herein we report design principles of highly responsive pH sensors based on a DNA i-motif where both response sensitivity and transition midpoint can be tuned with high precision over the physiologically relevant pH interval. The tuning is accomplished via rational manipulations of an i-motif structure as well as incorporation of allosteric control elements.

Parallel affinity-based isolation of leukocyte subsets using microfluidics: application for stroke diagnosis.

We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection.

Design and evaluation of an i-motif-based allosteric control mechanism in DNA-hairpin molecular devices.

Molecular devices designed to assess and manipulate biologically relevant conditions with required accuracy and precision play an essential role in life sciences research. Incorporating allosteric regulation mechanism is an attractive strategy toward more efficient artificial sensing and switching systems. Herein, we report on a new principle of regulating switching parameters of a DNA-based molecular device based on allosteric interaction between spatially separated hairpin stem and a tetraplexed fragment (i.

Hydrodynamic shearing of DNA in a polymeric microfluidic device.

With the advent of next-generation sequencing (NGS) systems and the associated high throughput they afford, the input to these machines requires manageable lengths of fragments (~1000 bp) produced from chromosomal DNAs. Therefore, it is critical to develop devices that can shear DNA in a controlled fashion. We report a polymer-based microfluidic device that establishes an efficient and inexpensive platform with performance comparable to a commercially available bench-top system.

Near-IR single fluorophore quenching system based on phthalocyanine (Pc) aggregation and its application for monitoring inhibitor/activator action on a therapeutic target: L1-EN.

Controlled H-aggregation of single Pc-labeled oligonucleotides is utilized as a fluorescence quenching system to discern changes in enzyme activity for the discovery of inhibitors for Long Interspersed Element 1 endonuclease (L1-EN), which is involved in genome instability and implicated in many different diseases.

Mono-amine functionalized phthalocyanines: microwave-assisted solid-phase synthesis and bioconjugation strategies.

Phthalocyanines (Pcs) are excellent candidates for use as fluors for near-infrared (near-IR) fluorescent tagging of biomolecules for a wide variety of bioanalytical applications. Monofunctionalized Pcs, having two different types of peripheral substitutents, one for covalent conjugation of the Pc to biomolecules and others to improve the solubility of the macrocycle, are ideally suited for the desired applications. To date, difficulties faced during the purification of monofunctionalized Pcs limited their usage in various types of applications.

Phthalocyanine dimerization-based molecular beacons using near-IR fluorescence.

Herein we demonstrate the use of a novel dimerization-based molecular beacon (MB) probe consisting of two metallo-phthalocyanine (Pc) fluorophores that use near-IR fluorescence, appropriate for highly specific and sensitive in vivo and/or in vitro DNA/RNA detection. Pc's possess a propensity to form nonfluorescent H-dimers that is utilized as the molecular "off" switch in the closed MB conformation. The "on" switch, which is generated when the solution target binds to the loop of the MB forming the open form, also provides two fluorophores for transduction resulting in a doubling of the extinction coefficient and improving the resulting fluorescence yield compared to a classical single-fluorophore/quencher MB system.

Solid-phase synthesis of asymmetrically substituted "AB3-type" phthalocyanines.

Synthesis of phthalocyanines with asymmetrical substitution on the periphery is often difficult due the problems in purification of the phthalocyanine mixtures obtained. Using a poly(ethylene glycol) (PEG)-based support with a Wang-type linker, we have developed the synthesis of monohydroxylated, oligoethylene glycol substituted phthalocyanines utilizing an amidine-base-promoted phthalonitrile tetramerization reaction. The use of a hydrophilic support allows symmetrical phthalocyanine product formed in solution to be readily and completely removed by washing while leaving the "AB3" product on the support.

Mitochondria targeting by guanidine- and biguanidine-porphyrin photosensitizers.

We report the syntheses of three new amphiphilic porphyrin derivatives, containing a guanidine, a biguanidine, or an MLS peptide, that were designed to target the cell mitochondria. The guanidine- and biguanidine-porphyrins are poorly soluble in water, forming J-type aggregates in aqueous solutions. On the other hand, the porphyrin-MLS peptide conjugate bearing a low molecular weight PEG spacer is highly water-soluble and does not aggregate in aqueous media.

Metallo-phthalocyanine near-IR fluorophores: oligonucleotide conjugates and their applications in PCR assays.

Water soluble, metallo-pthalocyanine (MPc) near-IR fluorophores were designed, synthesized, and evaluated as highly stable and sensitive reporters for fluorescence assays. Their conjugation to oligonucleotides was achieved via succinimidyl ester-amino coupling chemistry with the conditions for conjugation extensively examined and optimized. In addition, various conjugate purification and isolation techniques were evaluated as well.

Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates.

Two Zn-Pc-peptide conjugates bearing either a short linker or a long PEG-linker between the macrocycle and a bifunctional peptide containing the nucleoplasmin and HIV-1 Tat 48-60 sequences have been synthesized in order to increase the Pc cell-targeting ability and to evaluate the effect of the linker. The presence of the peptide chain increased the water solubility of the Pc macrocycle and, consequently, its fluorescence in aqueous solutions. The highest fluorescence quantum yields were observed at low pH (5.

Modern Immunotherapy in Clinical Medicine: Present and Future.

In this manuscript an attempt is made to review the most important information pertinent to the main immunotropic preparations used now in different fields of clinical medicine. Numerous studies have confirmed high clinico-immunological efficiency of natural and synthetic thymus preparations: tactivin, thymalin, thymoptin, thymogen, thymomodulin, thymovocal, thymostimulin, thymopentin TP-5 and others. First of all, imidazole compounds, such as levamisol (decaris), dibazol, metromidazol are referred as synthetic preparations with positive action on T cell immunity.

Current Views on the Role of Neutrophil Granulocyte System.

At present there is no doubt concerning the capability of neutrophil granulocytes for cooperative interaction with other immunocompetent cells and substances of different nature, which as a whole provides a wide variety of forms of interaction and makes it possible for neutrophils to occupy key positions in the regulation of the functions of other cells both by direct contact and by secretion of regulatory mediators - neutrophilokines. Neutrophils realize their functional capabilities on a background of stimulating actions in the form of intensification of migrational, adhesive capability, readjustment of metabolism for activation of phagocytic and secretory function. It is also known that many cytokines support the viability of neutrophils, preventing their apoptosis, progressing proportional to the degree of severity of the purulent-septic process.