Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognition

Thermodynamic and kinetic analyses of biomolecular interactions reveal details of the energetic and dynamic features of molecular recognition processes, and complement structural analyses of the free and complexed conformations. The recent improvements in both isothermal titration calorimetry and surface plasmon resonance sensoring provide powerful tools for analysing biomolecular interactions in thermodynamic and kinetic approaches. The thermodynamic and kinetic parameters obtained for binding between protein and DNA indicate the mechanism of specific DNA recognition, in the high-resolution structures of the protein-DNA complexes. The effects of temperature and ionic strength reflect the conformational changes of the protein and DNA molecules upon complex formation, including important contributions of water and solutes. When combined with mutational studies, the interactions can be reduced to several energetic contributions from individual contacts. These studies should be useful to determine general features of protein functions in genetic regulation.     

Nanogram per milliliter-level immunologic detection of alpha-fetoprotein with integrated rotating-resonance microcantilevers for early-stage diagnosis of heptocellular carcinoma

Nanogram per milliliter-level ultra-low concentration detection of alpha-fetoprotein (AFP), which is an important marker for heptocellular carcinoma, is in favor of early-stage prognosis and disease diagnosis. On-the-spot rapid detection of such antigens as AFP highly requires innovative micro/nano techniques. To meet this requirement, an advanced resonant microcantilever is developed and used for screening the tumor marker at nanogram per milliliter level. The sensing principle of the resonant microcantilever is measuring frequency-shift versus specific-adsorbed mass. With both electromagnetic resonance-exciting and piezoresistive readout elements on-chip integrated, the microcantilever sensor is operated in a rotating resonance mode to improve sensitivity and resolution to specific mass adsorption. Prior to detection of AFP with previously immobilized anti-AFP antibody, the antigen-antibody specific-binding is confirmed with an enzyme linked immunosorbent assay experiment. By implementing the specific reaction in liquid and reading out the sensor signal in lab air environment, the micromechanical sensor has achieved the sensitive scale between 2 and 20 ng/ml. To effectively depress cross-talk signal and improve resolution, the insensitive regions of the cantilever surface are pre-modified with 2-[methoxy (polyethyleneoxy) propyl] trimethoxysilane for nonspecific bio-adsorption minimization. Finally, a better AFP detecting limit than 2 ng/mL is experimentally achieved. The label-free resonant microcantilever sensor is promising in low-cost or even disposable early-stage prognosis and diagnosis of tumors.     

Functionalized Biomicroelectromechanical Systems Sensors for Force Response Study at Local Adhesion Sites of Single Living Cells on Substrates

We present a method of measuring force response of a single living cell, attached to a substrate, in situ, by a functionalized microelectromechanical systems sensor that applies local deformation on the cell. The sensor is a single crystal silicon microcantilever beam with prescribed shape and geometry, and is coated by a thin layer of fibronectin. It is brought in contact with a cell to form adhesion cites, and is then moved by a piezoactuator to deform the cell locally. The force is transmitted from the adhesion site(s) on the cantilever to the sites on the substrate through the cytoskeleton. The interaction force between the cell and the cantilever is measured from the deformation of the cantilever and its spring constant, which can be obtained by several independent means. The force and the cell deformation can be 10 s of nano-Newtons and micrometers, respectively. We demonstrate the method using two families of force sensors with spring constants of 18 and 0.4 nN/m. Several cells, endothelial and fibroblast, are deformed by tens of micrometers until the adhesion sites failed. Their force–deformation response shows strong linearity. Several possible mechanisms are discussed to explain the linear response. © 2003 Biomedical Engineering Society. PAC2003: 8780Fe, 0710Pz, 8716Gj, 8585+j     

Surface plasmon resonance phase-shift interferometry: real-time DNA microarray hybridization analysis

Surface plasmon resonance (SPR) phase-shift interferometry (PSI) is a novel technique which combines SPR and modified Mach-Zehnder PSI to measure the spatial phase variation caused by biomolecular interactions upon a sensing chip. The SPR-PSI imaging system offers high resolution and high-throughout screening capabilities for microarray DNA hybridization without the need for additional labeling, and provides valuable quantitative information. The SPR-PSI imaging system has an enhanced detection limit of 2.5 x 10(-7) refraction index change, a long-term phase stability of pi/100 in 30 min, and a spatial phase resolution of pi/300 with 100 x 100 microm2 detection area. This study successfully demonstrates the label-free observation of 15-mer DNA microarray.     

Modeling and Optimal Design of High-Sensitivity Piezoresistive Microcantilevers Within Flow Channels for Biosensing Applications

The mechanical design and optimization of piezoresistive cantilevers for biosensing applications is studied via finite element analysis. Models are described for predicting the static behavior of cantilevers with elastic and piezoresistive layers for analyte-receptor binding. The high-sensitivity cantilevers can be used to detect changes in surface stress due to binding and hybridization of biomolecules. The silicon-based cantilevers have thicknesses typically on the order of a few microns and are doped to introduce their piezoresistive characteristics. Parametric modeling based on the finite element method is used to help determine the optimum parameters of cantilever design. Chemo-mechanical binding forces have been analyzed to understand issues of saturation over the cantilever surface. Furthermore, the introduction of stress concentration regions during cantilever fabrication has been discussed which greatly enhances the detection sensitivity through increased surface stress. The spring constant and the resonance frequency change are also analyzed.     

Microcantilever sensing arrays from biodegradable,pH-responsive hydrogels

Biodegradable, pH-responsive hydrogels composed of poly(methacrylic acid) crosslinked with varying molar percentages of polycaprolactone diacrylate were synthesized. The equilibrium swelling properties of these pH-responsive materials were studied. Methods were developed to incorporate these novel hydrogels as sensing components in silicon-based microsensors. Extremely thin layers of hydrogels were prepared by photopolymerization atop silicon microcantilever arrays that served to transduce the pH-responsive volume change of the hydrogel into an optical signal. Organosilane chemistry allowed covalent adhesion of the hydrogel to the silicon beam. As the hydrogel swelled, the stress generated at the surface between the hydrogel and the silicon caused a beam deflection downward. The resulting sensor demonstrated a maximum sensitivity of 1 nm/5.7×10⁻⁵ pH unit. Sensors were tested in protein-rich solutions to mimic biological conditions and found to retain their high sensitivity. The existing theory was evaluated and developed to predict deflection of these composite cantilever beams.     

An integrated reaction-transport model for DNA surface hybridization: implications for DNA microarrays

DNA microarrays have the potential to revolutionize medical diagnostics and development of individualized medical treatments. However, accurate quantification of scantily expressed genes and precise measurement of small differences between different treatments is not currently feasible. A major challenge remains the understanding of physicochemical processes and rate-limiting steps of hybridization of complex mixtures of DNA targets on immobilized DNA probes. To this end, we developed a mathematical model to describe the effects of molecular orientation and transport on the kinetics and efficiency of hybridization. First, we calculated the hybridization rate constant based on the distance between the complementary nucleotides of the target and probe DNA. The surface reaction rate was then integrated with translational and rotational transport of target DNA to the surface to calculate the kinetics of hybridization. Our model predicts that hybridization of short DNA targets is diffusion limited but long targets are kinetically limited. In addition, for DNA targets with wide size distribution, it may be difficult to distinguish between specific binding of long targets from nonspecific binding of short ones. Our model provides novel insight into the process of DNA hybridization and suggests operating conditions to improve the sensitivity and accuracy of microarray experiments.     

Accuracy of an array comparative genomic hybridization (CGH) technique in detecting DNA copy number aberrations: comparison with conventional CGH and loss of heterozygosity analysis in prostate cancer

Although genomic DNA microarray (array comparative genomic hybridization [CGH]) technique is a rapid and powerful diagnostic tool for the comprehensive analysis of detailed chromosomal alterations of DNA copy numbers, its accuracy has not been well demonstrated. To clarify the accuracy of this technique, we applied array CGH spotted with 283 specific genes to 11 clinical prostate cancers, and the results were compared with comparative genomic hybridization (conventional CGH) and loss of heterozygosity (LOH) analysis using microsatellite DNA markers. The overall rate of correspondence between array CGH and conventional CGH with respect to the loss of DNA sequences was 94.5%. When the results of both CGH techniques were compared with those of LOH analysis, the correspondence rate of array CGH was significantly higher than that of conventional CGH (93.4% vs. 72.2%, P<0.05). In conclusion, the accuracy of array CGH was higher than that of conventional CGH in detecting losses of the DNA sequences. Array CGH is shown to be a promising tool for screening to identify unknown genes involved in tumorigenesis in prostate cancer.     

The peptide nucleic acids: a new way for chromosomal investigation on isolated cells?

The development of nucleic acid analogues has become an important feature due to the potential use of this new biomolecular tool in genetic diagnostics and investigations. Among all the synthetic oligonucleotides designed, the peptide nucleic acids (PNA) constitute a remarkable class of nucleic acid mimics, with important physico-chemical properties which have been exploited to develop a wide range of powerful biomolecular tools, including molecular probes, biosensors and anti-gene agents. New applications of PNA involve their use as hybridization probes, and consequently the PNA technology is now developing within the field of in situ hybridization techniques. Recent studies have reported the successful use of centromeric PNA probes on human lymphocytes, sperm as well as on isolated oocytes and blastomeres. Muticolour PNA protocols have been described for the specific identification of several human chromosomes. These data show that PNA could become a powerful complement to fluorescence in situ hybridization (FISH) for in situ chromosomal investigation, especially on isolated cells. The present paper gives an overview of the properties of PNA and the assays exploiting PNA technology in molecular genetics and cytogenetics.     

Bioenzymatic detection of troponin C using micro-opto-electro-mechanical systems

Diagnosis and monitoring of critical diseases such as acute myocardial infarction (AMI) require a quantitative analysis of biological molecules. A high-throughput identification of these biological molecules can be generated by using micro-electro-mechanical systems (MEMS) structures like simple cantilever beams, which respond to the intermolecular forces resulting from binding these molecules. Biochemical markers like troponin C are considered the primary markers for myocardial injury and have generated considerable interest. A 26-residue lytic membrane protein of bee venom melittin (ME) is chosen to interact with rabbit skeletal muscle troponin C (TnC) on the surface of the cantilever beams. An optical beam deflection method is employed to identify the enzymatic reaction on the surface of the cantilever due to these proteins. Identification of these proteins is also done using fluorescence spectroscopy (FS) to compliment the optical monitored deflection method. A second set of proteins like horse radish peroxide (HRP) and hydrogen peroxide (H2O2) are applied to atomic force microscopy (AFM) cantilever beams to study their behavior under the enzymatic reactions of proteins. Identification of these proteins is done using Fourier transform infrared spectroscopy (FTIR). An analytical model of the cantilever beam is developed, and its mode shapes are studied by employing orthogonal polynomials in the classic Rayleigh-Ritz method. The surface stress caused by the enzymatic reaction of the proteins that leads to pure bending on the top surface of the cantilever is evaluated. The information provided by the experimental and analytical modeling reported in this work will be useful in the development of a portable biosensor for the detection of AMI.     

A two-colour technique for chromosome in situ hybridization in tissue sections.

By extending non-isotopic in situ hybridization of DNA probes (targeted to metaphase chromosomes or interphase nuclei) to hybridization using tissue sections, additional topological information on the DNA structure and specific alterations can be obtained. We have established a method for the application of two different, chromosome-specific probes labelled with two colour dyes allowing simultaneous detection of two-colour signals. This method was tested in and is applicable to tissue sections of various origins. To demonstrate its sensitivity, prostate carcinomas (either as cryosections or as sections from paraffin blocks) were investigated for the presence or absence of chromosomes 1 and Y. The technique presented here, comparable to immunohistochemical staining, is particularly useful for routine application in diagnostic laboratories and testing of fresh or archival material.     

A high accuracy cantilever array sensor for early liver cancer diagnosis

Early liver cancer diagnosis has clinical significance in treating cancer. Joint detection of multiple biomarkers has been considered as an effective and reliable method for early cancer diagnosis. In this work, a novel biosensor based on microcantilever array was batch-fabricated for multiple liver cancer biomarkers detection with high sensitivity, high accuracy, high throughput, and high specification. A micro-cavity was designed in the free end of the cantilever for local reaction between antibody and antigen, which can dramatically reduce the effect of adsorption-induced stiffness coefficient k variation. Furthermore,the pillar arrays in the micro-cavity were designed for increasing detection upper limit. A linear relationship between the relative frequency shift and the antigen concentration was observed for three liver cancer biomarkers, alpha-fetoprotein (AFP), γ-glutamyltranspeptidase II (GGT-2), and hepatocyte growth factor (HGF). Slight cross-reaction response to different antigens ensures high specificity of the sensor. These features will promote clinical application of the cantilever sensors in early cancer diagnosis.     

Murine cytomegalovirus: detection of latent infection by nucleic acid hybridization technique.

The technique of nucleic acid hybridization was used to detect the presence of murine cytomegalovirus (MCMV)-specific deoxyribonucleic acid (DNA) in cell cultures and salivary gland tissues. The presence of approximately 4.5 and 0.2 genome equivalents per cell of MCMV-specific DNA was identified in cultures of salivary (ISG2) and prostate gland (IP) cells, respectively. These cells, derived from animals with experimentally induced latent infections, were negative for virus-specific antigens by immunofluorescence and on electron microscopy revealed no visible evidence of the presence of herpesviruses. A cell line derived from the salivary gland of an uninoculated animal (NSG2) was also found to possess MCMV-specific DNA (0.2 genome equivalents per cell). For this reason, salivary gland tissues from uninoculated animals supplied as "specific pathogen-free" mice by three commercial sources were tested upon arrival for the presence of MCMC-specific DNA. MCMV-specific DNA was detectable in pooled salivary gland extracts from uninoculated animals derived from two commercial sources. All of these animals were seronegative and virus negative by conventional infectivity assays.     

Early reduction in the aneuploidy at chromosomes 7 and 8 are significantly correlated with clinical effect in high-dose rate brachytherapy with external beam radiotherapy in localized prostate cancer.

Although reduction in the serum prostate specific antigen (PSA) correlates with clinical outcome for high dose rate Iridium-192 (HDR Ir-192) brachytherapy, it takes a long latency period. We investigated numerical chromosome changes of prostatic cancer during the pre- and post-treatment periods of HDR Ir-192 brachytherapy (and external beam radiotherapy), using fluorescence in situ hybridization (FISH) to clear the effect of treatment in early phase. Transitional changes in the frequency of aneuploidy for chromosomes 7, 8, 10, 12, 16, X, and Y in prostate cancer during the pre- and post-treatment periods were observed. Gains of chromosomes 7, 8 and 12 were noted in the pre-treatment samples (4 out of 12 cases in chromosomes 7 and 8; 1 out of 12 cases in chromosome 12), while a notable reduction in the number of cells with extra copies of these chromosomes was observed in post-treatment specimens. This change appears earlier than the reduction in the value of prostate specific antigen (PSA) and strongly reflects the effect of HDR brachytherapy with external beam radiotherapy in localized prostate cancer. Decrease in the number of cells with high ploidies of chromosomes 7, 8 and 12 at 12 weeks after treatment may predict clinical effects of radiation therapy, which may explain the radiation dependency of localized prostate cancer cells.     

In situ hybridization for cytomegalovirus DNA in AIDS patients.

Infection by cytomegalovirus (CMV) is a frequent cause of morbidity and mortality in patients with acquired immune deficiency syndrome (AIDS). The authors studied the distribution of CMV in 4 patients with AIDS using a commercially available, biotin-labeled CMV DNA probe for in situ hybridization and immunohistochemical staining for the detection of CMV antigen in formalin-fixed paraffin-embedded tissues. The sensitivity and specificity of the hybridization procedure was demonstrated by appropriate controls. The immunohistochemical test for the detection of CMV antigen in routine histologic sections was less sensitive than the in situ hybridization method. CMV DNA was detected not only in cytomegalic inclusion cells, but also in nuclei and cytoplasm of histologically normal-appearing cells such as endothelial cells, pneumocytes, hepatocytes, biliary epithelium, gastrointestinal epithelium, Langerhans islet cells, acinar and duct epithelium of pancreas, adrenal cortical and medullary cells, and prostate epithelium. In addition, CMV DNA, but not CMV antigen, was found in polymorphonuclear leukocytes. These cells may serve as intermediate host or reservoir of CMV and may transmit posttransfusion CMV infection. In situ hybridization on routine histologic sections with a biotinylated CMV DNA probe is a rapid, sensitive, and specific method for diagnostic and experimental pathology.     

iFRET: an improved fluorescence system for DNA-melting analysis

Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.     

"Prostatic" kallikreins, sex hormones and insulin-like growth factors: complex of male and female regulatory elements in health and carcinogenesis

Publications (up to the year 1997) on the interactions of "prostatic" kallikreins (prostatic specific antigen-PSA, etc.), sex hormones, insulin-like growth factors (IGF) and proteins binding them (IGFBP) in physiological processes (ageing, menstrual cycle, pregnancy) and oncogenesis (prostatic and mammary cancer) are reviewed. The concentrations of PSA, IGF, and IGFBP in organs and liquid media of men and women are presented. A concept of similarity in the mechanisms of interactions of sex hormones (dihydrotestosterone in men and progesterone in women), PSA, IGFBP, and IGF during activation of anabolic and proliferative processes in health and carcinogenesis is presented as a scheme. The diagnostic and prognostic value of PSA as a cancer marker should not be confined to male tumors (prostatic cancer and benign prostatic hyperplasia). Our data permits us to regard PSA as an oncofetal marker for men and women, indicating normal and neoplastic proliferative processes in the prostatic, mammary, salivary, and other glands and in the lungs and endometrium. Diagnostic and prognostic significance of PSA in breast cancer is shown. The traditional name "PSA" does not reflect its physiological and pathogenetic role as a member of the kallikrein family with chymotrypsin-like activity. PSA is not absolutely specific towards the producer organ and sex. Its relative specificity for the prostate is undoubted, because the content of PSA in prostatic tissue and seminal plasma is 10(6)-10(8) times higher than in the serum and other organs of men and women. Therefore, although the terms "prostatic, "specific", and "antigen" now became trivial and it is difficult to refuse from them, they can be used only in quotation marks.     

Engineering Issues in the Fabrication of a Hybrid Nano-Propeller System Powered by F 1 -ATPase

A hybrid nanoscale device consisting of nanofabricated structures integrated with a biomolecular motor was constructed on a 25-mm diameter round glass cover slip. Glass substrates were patterned using electron beam lithography and reactive ion etching to define elevated nickel anchor points with a minimum feature size of 60 nm in diameter. Subsequent additions of histidine-tagged F₁-ATPase biomolecular motor resulted in selective attachment of the biomotors to the nanofabricated substrates. Electron beam lithography also was employed to fabricate 1 m long nickel propellers, which subsequently were released through wet etching, collected, and concentrated in buffer solution. Biotin-Cys-6x-His peptides were attached to nickel propellers for interfacing with a biotinylated biomolecular motor through a streptavidin linker. Successful assembly of the hybrid system was demonstrated with observation of rotating nickel propellers. This engineered hybrid nanomechanical system demonstrates the potential application of a new class of devices with enhanced functionality in biological environments.