We have developed a variety of novel DNA biosensors using a new class of oligonucleotide probe, molecular beacon (MB). MB has the fluorescence signal transduction mechanism built within the molecules. It can report the presence of specific nucleic acids with high sensitivity and excellent selectivity. Biotinylated MBs have been designed and synthesized for immobilization onto silica surface through avidin-biotin binding. The effect of the avidin-biotin bridge on the MB hybridization has been studied. Our result shows that using streptavidin has less effect than using avidin in MB hybridization. Two kinds of fiber optical DNA sensors have been prepared and characterized: a fiber optic evanescent wave sensor and a submicrometer optical fiber sensor. The sensors are rapid,stable, highly selective, reproducible and regenerable. They have been applied to detect specific DNA and mRNA sequences and to the study of the DNA hybridization kinetics. Silica nanoparticles have also been used for MB immobilization in order to prepare a large quantity of nanometer sized DNA/RNA biosensors.

In `5'-translational blockage,' significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low-usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream). We tested the hypothesis that ribosomes encountering these low-usage codons prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes, monosomes and ribosomes-free. New hybridization probes, called `molecular beacons,' and standard slot-blots, were used to detect test messages containing either consecutive low-usage AGG (arginine) or synonymous high-usage CGU insertions near the 5' end. The results show an approximately twofold increase in the ratio of free to bound mRNA when the low-usage codons were present compared to high-usage codons. In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low-usage CUA or high-usage CUG (leucine) codons were inserted, or when the arginine codons were inserted near the 3' end. These data indicate that at least some mRNA is released from ribosomes during 5'-translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon.

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a new approach to detect the presence of Salmonella species using these fluorogenic reporter molecules and demonstrated their ability to discriminate between similar E. coli species in real-time PCR assays. A detection limit of 1 CFU per PCR reaction was obtained. The assays were carried out entirely in sealed PCR tubes, enabling fast and direct detection in a semiautomated format.

We report a novel approach to study protein-nucleic acid interactions by using molecular beacons (MBs). Molecular beacons are hairpin-shaped DNA oligonucleotide probes labeled with a fluorophore and a quencher, and can report the presence of target DNA/RNA sequences. MBs can also report the existence of single-stranded DNA binding proteins (SSB) through non-sequence specific binding. The interaction between SSB and MB has resulted in significant fluorescence restoration of the MB. The fluorescence enhancement brought by SSB and by complementary DNA is very comparable. The molar ratio of the binding between SSB and the molecular beacon is 1:1 with a binding constant of 2 X 10⁷ M^-1. Using the MB-SSB binding, we are able to determine SSB at 2 X 10^-10 M with a conventional spectrometer. We have also applied MB DNA probes for the analysis of an enzyme lactic dehydrogenase (LDH), and for the investigation of its binding properties with ssDNA. The biding process between MB and different isoenzymes of LDH has been studied. We also show that there are significant differences in MB binding affinity to different proteins, which will enable selective binding studies of a variety of proteins. This new approach is potentially useful for protein-DNA/RNA interaction studies that require high sensitivity, speed and convenience. The results also open the possibility of using easily obtainable, custom designed, modified DNA molecules for studies of drug interactions and targeting. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has the signal transduction mechanism built within the molecule, and can thus be used for quick protein assay development and for real-time measurements.

Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is a rapidly emerging, universal platform with the ability to provide detailed information regarding genetic variation and the up- and down regulation of their cognate gene products. Herein, we report our progress towards the development of ESI-FTICR mass spectrometry for the characterization of genomic regions which contain both a length and sequence polymorphism (i.e., complex short tandem repeats). Specifically, it is demonstrated for the first time that a high-quality ESI-FTICR mass spectrum of a 82-bp double- stranded PCR product derived from a single, 50 (mu) L PCR reaction with less than 10 X 10^-15 moles injected into the mass spectrometer can be routinely obtained. It is important to note that each measurement, which translates to an accurate genotype, is completed on the timescale of seconds. Progress towards the implementation of flow- injection analysis methodology to increase the throughput is also presented using an alternating injection of a 15-mer and 16-mer oligonucleotide.

Identification of whole cell proteins by 2D-PAGE/in-gel digestion combined with mass spectrometry has become routine, but the time required to prepare the gel, perform the separation, and process the gel for mass spectrometry analysis requires several days. To overcome the disadvantages of 2D-PAGE/in-gel digestion, we developed simple separation/in-solution digestion combined with high- resolution matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry for analysis of whole cell proteins. High resolution MALDI TOF mass spectrometry can be used to analyze complex protein mixtures, thus complete protein separation is not required. Microtip column (C8) separation/in-solution digestion minimizes the time and effort required for sample preparation. Solvent-gradient elution from microtip column (C8) is used to fractionate the proteins and the number of samples that must be mass-analyzed is reduced tenfold. Therefore, significantly fewer numbers of mass spectra are used to identify major proteins in the whole cell lysates. The whole procedure from protein extraction to analysis of mass spectral data can be completed in a day and several hundreds proteins can be identified.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a popular analytical tool for the analysis of large biomolecules such as proteins, oligonucleotides and oligosaccharides. This method has proven to be extremely useful for the analysis of water soluble proteins. However, analysis of hydrophobic proteins using MALDI-MS has been more of a challenge. The difficulty arises due to the limited solubility of these compounds in the aqueous solutions used to prepare the MALDI matrix. This study has investigated the effect of surfactants on the MALDI-MS data obtained from hydrophobic peptides and tryptic digests of hydrophobic and hydrophilic proteins. The surfactant sodium dodecylsulfate was found to improve the information content obtained during MALDI-MS analysis of such molecules. Preliminary results which examine the applicability of surfactant-aided MALDI-TOFMS for the characterization of proteolytic digests of proteins will be presented, and the potential use of this analytical strategy in a proteomics-based examination of intrinsic membrane proteins will be discussed.

Infrared multiphoton dissociation (IRMPD) of the hydrogen/deuterium (H/D) exchanged 12+ charge state of gas-phase bovine ubiquitin was performed on a Fourier transform ion cyclotron resonance mass spectrometer. The H/D exchange of the 12+ charge state revealed two distinct isotopic distributions indicating the presence of at least two distinct conformations of the 12+ charge state. Following H/D exchange, IRMPD was used to dissociate the conformations. The fragments clearly showed little or no deuterium scrambling as evidenced by a nonstatistical distribution of deuterium incorporation. Analysis of the deuterium incorporation for the five most abundant fragment ions indicated a slow exchanging region of the fast exchanging conformation that corresponds to a stable (beta) - sheet observed by NMR in alcoholic solutions. The data suggest that protection of the amide hydrogens in the (beta) -sheet may result in the observed slow exchange rate and provides further evidence for the retention of secondary structure in gas phase.

Progress in the development of ion mobility (IM) orthogonal time-of-flight (oTOF) mass spectrometry for rapid analysis of biological samples is presented. The IM-oTOF apparatus described consists of a short drift tube (1 to 15 cm) designed for ion mobility measurement in the low-field limit and a low resolution linear (20 cm) TOF mass spectrometer. Proof of concept is demonstrated by analysis of peptide mixtures generated by proteolytic digestion of proteins.

Nanoelectrospray or nanospray ionization has revolutionized biological mass spectrometry because of its low sample consumption requirements, low flow rates (nL/min), and improved ion transfer efficiency from source to detector versus conventional electrospray ionization. These advantages suggest that nanospray could prove beneficial in the coupling to on-line capillary separations methods, such as capillary electrophoresis and capillary electrochromatography. In order to establish a stable spray, the nanospray emitter must be electrically biased relative to a counter-electrode inlet to the mass spectrometer, this is most easily achieved by coating the nanospray emitter with a conductive metal coating. However, metallized nanospray tips are highly susceptible to deterioration by electrical discharge. Due to this limited lifetime, nanospray emitters are limited for coupling to capillary separations techniques. Here, an alterative coating for nanospray emitters using polyaniline is demonstrated.

Labeling of DNA with fluorescent reporter is generally accomplished by attaching fluorescent dyes to the 5'-end of primers for PCR based applications, including geno-typing and linkage mapping. Requirements of spectral resolution and chemical stability have guided us toward asymmetric fluorescein dye structures whose chemical and physical properties have been fine-tuned through substituent modification. We have developed dyes that are amenable to 5'-prime labeling on solid support as dye-amidites facilitating automated dye-primer synthesis.

We are attempting to design and prepare a new class of dyes for biomedical/biotechnological applications, including detection of oligonucleotides in DNA sequencing and diagnostics. The dyes use through-bond energy transfer to harvest short wavelength irradiation, and fluoresce at much longer wavelengths. Excellent resolution of fluorescent emissions from a 266 nm laser source is observed, with apparent Stokes' shifts of up to 408 nm.

High-density DNA probe arrays offer a massively parallel approach to nucleic acid sequence analysis that will have a major impact on gene-based biomedical research and clinical diagnostics. Light-directed synthesis has enabled the large- scale manufacture of arrays containing hundreds of thousands of oligonucleotide probe sequences on a glass `chip' about 1.6 cm<sup>2</sup> in size. This method is used to produce high- density GeneChip<sup>TM</sup> probe arrays, which are now finding widespread use in the detection and analysis of mutations and polymorphisms (`genotyping'), and in a wide range of gene expression studies. This paper will discuss methods for high-resolution photolithographic array fabrication which integrate solid-phase oligonucleotide synthesis, photochemically removable protecting groups, and lithographic techniques adapted from the microelectronics industry.

The Reversed Enzyme Activity DNA Interrogation Test (READIT^TM) System provides a new, rapid method for genotype determination. The system can be used for the analysis of single nucleotide polymorphisms as well as insertions, deletions and chromosomal rearrangements. The READIT^TM System is based upon driving various enzymatic reactions in the reverse direction from the direction they are normally considered. The method is based on a pyrophosphorylation reaction that is the reverse of DNA polymerization. Interrogation probes annealed to DNA fragments with which they have a 3- mismatch are not a substrate for the reaction and do not generate significant signals compared with probes that match the interrogation sequence perfectly. By interrogation of specific DNA sequences with carefully designed probes, mutant genes can be identified with speed and precision.

The Human Genome Project has led to the discovery of hundreds of thousands of single nucleotide polymorphisms (SNPs). SNPs can act as genetic markers to create high- density maps of the human genome for large-scale genetic analysis for evaluating links between genetic mutations and human diseases and for performing association studies. To create those maps, assays capable of detecting many different SNPs must be developed rapidly, as additional SNPs are discovered. When both the design of and the technology used in the assays can be partially or fully automated, the development process and the time to results can be accomplished quickly and efficiently. Invader^TM technology offers a highly sensitive signal amplification system that detects and quantifies mutations and SNPs from unamplified human genomic DNA in two sequential steps.

Traditional anion exchange purification of nucleic acids requires the elution of the DNA or RNA in a salt solution, necessitating the precipitation or desalting of the nucleic acid prior to many molecular biology applications. A pH dependent anion exchange purification method is described which allows the purification of nucleic acids at one pH, followed by the elution of the nucleic acid in a low salt buffer at a second, higher pH. The benefits of this method include the avoidance of alcohol washes and the drying steps required for alcohol removal, as well as the benefits of anion exchange purification without the need for desalting of the purified DNA or RNA.

Microarray technology makes it possible to monitor expression levels of thousands of genes simultaneously during single or multiple experiments. Routinely, in order to analyze gene expressions level quantitatively, two fluorescent-labeled RNAs are hybridized to an array of cDNA probes on a glass slide. Ratios of gene expression levels arising from two co-hybridized samples are obtained through image segmentation and signal detection methods. During the past three years, we have developed a gene expression analysis system in which ratio statistics have been applied to expression analysis, and a ratio confidence interval has been established to identify ratio outliers.

A cDNA microarray is a complex biochemical-optical system whose purpose is the simultaneous measurement of gene expression for thousands of genes. This paper describes a general statistical environment for finding associations among gene expression patterns, and between genes and external conditions, via the coefficient of determination. This coefficient measures the degree to which the transcriptional levels of an observed gene set can be used to improve the prediction of the transcriptional state of a target gene relative to the best possible prediction in the absence of observations. Various aspects of the method are discussed: prediction quantification, deign of predictors given small numbers of replicated microarrays, and constrained prediction using ternary perceptrons. A main focus is the supporting software and its facilities for data analysis and visualization.

Choline oxidase converts one molecule of choline to generate two molecules of H₂O₂. In a 1:1 reaction stoichiometry, horseradish peroxidase-catalyzed oxidation of the nonfluorescent and colorless H₂O₂ indicator, the Amplex Red reagent, by H₂O₂ produces the highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nm. Using the Amplex Red reagent as an H₂O₂ indicator, assays of phospholipase D, phosphatidylcholine-specific phospholipase C, acetylcholine, acetylcholinesterase, and sphingomyelinase have been developed in choline oxidase- and HRP-coupled reactions. These assays have been developed in a microplate with a one-step procedure.

We have developed novel microarray technology for performing large numbers (up to 10⁶) of chemical and cell-based assays in parallel. This technology is particularly relevant to the high-throughput screening methods used by the pharmaceutical industry to identify potential drug candidates. In this paper we provide an overview of the system and its enabling technologies, including an economical manufacturing process for creating these microarrays, a fluorescence imaging system for detecting `hits', a fluid delivery system for loading arrays, and a method for mixing reagents.

In many medical, biological, chemical, and environmental applications it is desirable not only to monitor one specific chemical or biological species, but several simultaneously. Thus, we have focused our efforts on development of a detection system for multi-analyte sensor arrays that is able to monitor the changes in fluorophore lifetimes (via the detection of phase shifts) corresponding to the presence of many analytes of interest in near-real time. We describe a phase-sensitive electronic detection system employing a multianode photomultiplier tube. This system utilizes the frequency-domain method of time-resolved spectroscopy and is also suitable for lifetime-based imaging.

A new digital imaging spectrophotometer and a series of colorimetric solid phase arrays have been developed to screen bacterial libraries expressing mutagenized enzymes undergoing directed evolution. This high-throughput solid- phase array system (known as `Kcat Technology') can detect less than a 20% difference in enzyme rates within microcolonies grown at a nearly confluent density of 500 colonies per cm² on an assay disk. Each microcolony is analyzed simultaneously at single-pixel resolution (1.5 megapixels; 75 micron/pixel), requiring less than 100 nanoliters of substrate per measurement, a 1000-fold reduction over conventional liquid phase assays. Here we report the successful identification of variants of Agrobacterium (beta) -glucosidase--a glycosidase with broad substrate specificity that favors cleavage of glucosides over galactosides--by simultaneously assaying two different substrates tagged with spectrally distinct chromogenic reporters.

Modern chemical synthesis and screening technologies have the ability to create large numbers of lead components but still do not answer questions of efficacy, dosing, toxicity and optimal patient population. SurroMed was founded to develop discovery technologies for new biological markers that will answer these questions. Biological markers will be derived from the results of many different assays; cell surface, serum factors and others, many performed using whole blood and other fluids and tissues. We report on the design of a Microvolume Laser Scanning Cytometer (MLSC) and disposable capillary arrays to be used in biological marker discovery. The MLSC machines are used primarily for cell surface assays, though they are suitable for other fluorescence assays as well. Each capillary requires a very small sample volume per assay, less than twenty micro- liters, and so allows hundreds of assays to be performed on a single ten milliliter blood draw. The new MLSC is capable of optimally detecting four fluorescence colors at different scan rates. HeNe excitation and red emission permits the use of whole blood, so that no lysing or cell separation is required. The MLSC instrument and disposable capillary arrays are in routine use for biological marker discovery at SurroMed.

The spectrophotometer we have developed enables us to measure absorbance in a simple way without recovering and contaminating the analyte. The absorbance of the analyte can be measured with the sample solution being retained in the tip of the pipette. The traditional cuvette is totally unnecessary. Both the tip and pipette used in this spectrophotometer are commercially available. We have measured the absorbance of analytes containing DNA dissolved in water and compared them with results obtained by the conventional spectrophotometer. Both absorbance values sufficiently agree in repeated tests. The paper will present the features of our instrument and detailed results.

Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

The results of studying the process of glucose' thermal decomposition in consequence of the sterilization in the autoclave and the shelf life are considered. The research of the process was carried out for 5% glucose solution for injections before and after the sterilization in the autoclave by saturated steam at 110 degree(s)C for 30 minutes by the study of UV absorption spectra, granulometric analysis in the interval 0.2 - 25.0 mkm and pH measurements. The spectra of the glucose solutions before the sterilization were found to have no absorption bands in the range from 200 nm to 400 nm. After the sterilization two absorption bands with maximal at 229 nm and 284 nm respectively appeared. After three-month storage only one absorption band with maximum at 284 nm remains. In the report the observed spectral effects are explained.

A spectrophotometric method optical density ratio for simultaneous determination of four derivatives as for deoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin and methemoglobin in a blood sample is described. The normalized averaged spectrum of derivatives hemoglobin in the spectral range 14200 - 21000 cm^-1 is reduced.

We describe two optical methods for in vitro drug trials on living cells and cell cultures. Response of each single target cell to drug impact is measured as the change of cell functional and structural state by photothermal imaging and the changes of cell viability is controlled with Laser Viability Test. Both photothermal methods are sensitive to specific cell reaction but although to reaction of general origin. High sensitivity of the methods allows to use minor amounts of drug and cell materials. Pharmaceutical applications are illustrated with the results of in vitro experiments being obtained for human living blood cells.

Compounds that interact with the cytoskeleton affect mobility and division, making them useful for treatment of certain types of cancer. Actin binding drugs such as the phallotoxins (small, bicyclic peptides) bind to and stabilize actin polymers (F-actin) without binding to actin monomers (G-actin). It has been shown that the intensity of fluorescently labeled phallotoxins such as fluorescein- phalloidin and rhodamine-phalloidin increases upon bind F- actin. We used LJL BioSystems' new FLARe^TM technology to measure excited state lifetime changes of fluorescein- phalloidin and rhodamine-phalloidin upon binding to F- actin.

With a large part of the Human Genome Project behind us and several smaller genomes already finished, we are facing new challenges. Until now most of the collective effort has been focused on sequencing genomes, but the activities to interpret and use this information are now rapidly accelerating. Technology developed for the long read lengths typically required for sequencing projects are not necessarily well suited for the emerging needs of applied genomics where identification of known genes or the study of Single Nucleotide Polymorphism, can be achieved with only a few bases of sequence information. Several new methods such as Taqman, Molecular Beacons, Invader Probes, Mini sequencing (single base extension) etc are being developed to meet these new needs but very few of these has the ability to read more than one base. This paper describes Pyrosequencing, a new way of sequencing DNA based on Sequencing By Synthesis and with the ability to sequence short to medium length stretches of DNA with high accuracy.