Magnetic labeling, detection, and system integration.

Among the plethora of affinity biosensor systems based on biomolecular recognition and labeling assays, magnetic labeling and detection is emerging as a promising new approach. Magnetic labels can be non-invasively detected by a wide range of methods, are physically and chemically stable, relatively inexpensive to produce, and can be easily made biocompatible. Here we provide an overview of the various approaches developed for magnetic labeling and detection as applied to biosensing. We illustrate the challenges to integrating one such approach into a complete sensing system with a more detailed discussion of the compact Bead Array Sensor System developed at the U.S. Naval Research Laboratory, the first system to use magnetic labels and microchip-based detection.

Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics.

A significant challenge for all biosensor systems is to achieve high assay sensitivity and specificity while minimizing sample preparation requirements, operational complexity, and sample-to-answer time. We have achieved multiplexed, unamplified, femtomolar detection of both DNA and proteins in complex matrices (including whole blood, serum, plasma, and milk) in minutes using as few as two reagents by labeling conventional assay schemes with micrometer-scale magnetic beads, and applying fluidic force discrimination (FFD). In FFD assays, analytes captured onto a microarray surface are labeled with microbeads, and a controlled laminar flow is then used to apply microfluidic forces sufficient to preferentially remove only nonspecifically bound bead labels. The density of beads that remain bound is proportional to the analyte concentration and can be determined with either optical counting or magnetoelectronic detection of the magnetic labels. Combining FFD assays with chip-based magnetoelectronic detection enables a simple, potentially handheld, platform capable of both nucleic acid hybridization assays and immunoassays, including orthogonal detection and identification of bacterial and viral pathogens, and therefore suitable for a wide range of biosensing applications.

The structure of helix III in Xenopus oocyte 5 S rRNA: an RNA stem containing a two-nucleotide bulge.

The solution structure of an oligonucleotide containing the helix III sequence from Xenopus oocyte 5 S rRNA has been determined by NMR spectroscopy. Helix III includes two unpaired adenosine residues, flanked on either side by G:C base-pairs, that are required for binding of ribosomal protein L5. The consensus conformation of helix III in the context provided by this oligonucleotide has the two adenosine residues located in the minor groove and stacked upon the 3' flanking guanosine residue, consistent with biochemical studies of free 5 S rRNA in solution. A distinct break in stacking that occurs between the first adenosine residue of the bulge and the flanking 5' guanosine residue exposes the base of the adenosine residue in the minor groove and the base of the guanosine residue in the major groove. The major groove of the helix is widened at the site of the unpaired nucleotides and the helix is substantially bent; nonetheless, the G:C base-pairs flanking the bulge are intact. The data indicate that there may be conformational heterogeneity centered in the bulge region. The corresponding adenosine residues in the Haloarcula marismortui 50 S ribosomal subunit form a dinucleotide platform, which is quite different from the motif seen in solution. Thus, the conformation of helix III probably changes when 5 S rRNA is incorporated into the ribosome.

The role of serine hydroxymethyltransferase isozymes in one-carbon metabolism in MCF-7 cells as determined by (13)C NMR.

The role of cytosolic and mitochondrial serine hydroxymethyltransferase in supplying one-carbon groups for purine and thymidylate biosynthesis in MCF-7 cells was investigated by observing folate-mediated one-carbon metabolism of l-[3-(13)C]serine, [2-(13)C]glycine, and [(13)C]formate. (13)C NMR was used to follow the incorporation of label into carbons 2 and 8 of purines and the methyl group attached to carbon 5 of thymidylate. The percentage enrichment of the (13)C label in purines was determined from the splitting patterns of the (1)H NMR spectra of C2 and C8 of adenine and C8 of guanine. The results show that formate is the major precursor in the cytosol of the one-carbon group in 10-formyltetrahydrofolate, which is used in purine biosynthesis, and the one-carbon group in 5,10-methylenetetrahydrofolate, which is used in thymidylate biosynthesis. Formate is formed in the mitochondria from carbon 3 of serine. The cleavage of serine to glycine and 5,10-methylenetetrahydrofolate by cytosolic serine hydroxymethyltransferase does not appear to be a major source of one-carbon groups for either purine or thymidylate biosynthesis. Carbon 3 of serine accounts for about 95% of the one-carbon pool, suggesting that other sources of one-carbon groups represent only minor pathways. [2-(13)C]Glycine is not a donor of one-carbons groups, confirming that MCF-7 cells lack a functional glycine cleavage system.

Which aminoglycoside ring is most important for binding? A hydropathic analysis of gentamicin, paromomycin, and analogues.

The NMR structures of gentamicin and paromomycin in complex with the A-site of Escherichia coli 16S ribosomal RNA were modified with molecular modeling to 12 analogues. The intermolecular interactions between these molecules and RNA were examined using the HINT (Hydropathic INTeractions) computational model to obtain interaction scores that have been shown previously to be related to free energy. The calculations correlated well with experimental binding data, and the interaction scores were used to analyze the specific structural features of each aminoglycoside that contribute to the overall binding with the 16S rRNA. Our calculations indicate that, while ring I binds to the main binding pocket of the rRNA A-site, ring IV of paromomycin-based aminoglycosides contributes significantly to the overall binding.

Investigation of the properties of an ion-etched plane laminar holographic grating.

We have measured the efficiency over the range 125-225 A of a bare ion-etched plane laminar holographic grating made of fused silica and with 1000 grooves/mm. The measured efficiency of each order oscillates with wavelength because of constructive and destructive interference between radiation diffracted from the lands and the grooves. We measured the grating groove profile with an atomic force microscope, and the resulting groove depth of 434 ? 6 A agrees well with the values determined independently from the oscillatory behavior of the efficiency measurements. Grating efficiency in the +1 order peaked at values of 0.027%, 0.011%, and 0.005% at wavelengths of 191, 157, and 132 A, respectively; and the derived groove efficiencies are 27%, 25%, and 27%. The irregular shape at the land-groove edges dominates the large grating roughness of 23-45-A rms, but even regions far from the edges have a roughness of 10-18-A rms. The average groove profile was used to model the grating efficiency, and the resulting wavelengths predicted for different order maxima and minima agree well with measured wavelengths, although the calculated efficiencies are greater than the measured results by 10-20%.

On the Z-E photoisomerization of chiral 2-pentenoate esters: stationary irradiations, laser-flash photolysis studies, and theoretical calculations.

Chiral pentenoates 1-3 in both Z and E isomeric forms underwent stationary irradiations in several solvents and in the presence of different photosensitizers. The photostationary-state ratio has been determined for each Z/E couple showing a predominance of the thermodynamically more stable isomer for 1 and 3. Moreover, transient species were generated by pulsed laser excitation and detected by their characteristic ultraviolet absorptions, being the first time that enoate-originated triplets are detected. Stern-Volmer quenching studies afforded a quantitative measure for the efficiency of the photosensitization processes induced by benzophenone or acetophenone and allowed the determination of the corresponding quenching rate constants. Density functional calculations permitted the determination of the geometries and the energies of the diastereomeric excited states. Two diastereomeric orthogonal and two diastereomeric planar structures result as a consequence of the presence of a chiral substituent. The orthogonal triplets are the energy minima in all cases, whereas the planar triplets are the transition states linking these orthogonal structures, the corresponding energy barriers being 8-10 kcal mol(-1) for enoates 1-3. The computed S(0) to T(1) excitation energies show a trend which is consistent with the quenching rate constants. On the other hand, the triplet lifetimes determined for 1 and 2 are unusually long (1-20 micros) if compared with the data already described for several enones, in the range of nanoseconds. This fact has been rationalized from calculations of spin-orbit coupling at several points of the T(1) potential energy surface. This coupling is maximum for structures with a torsional angle close to 45 degrees, which are 4-5 kcal mol(-1) above the minima of T(1). Calculations done on the hypothetical aldehyde 4 and methyl vinyl ketone show much lower energy barriers, thus accounting for the shorter lifetimes reported for enone triplets.

Comparison of the crystal and solution structures of two RNA oligonucleotides.

Until recently, there were no examples of RNAs whose structures had been determined by both NMR and x-ray crystallography, and thus there was no experimental basis for assessing the accuracy of RNA solution structures. A comparison of the solution and the crystal structures of two RNAs is presented, which demonstrates that NMR can produce solution structures that resemble crystal structures and thus validates the application to RNA of a methodology developed initially for the determination of protein conformations. Models for RNA solution structures are appreciably affected by the parameters used for their refinement that describe intramolecular interactions. For the RNAs of interest here, the more realistic those parameters, the greater the similarity between solution structures and crystal structures.

Thin-film interference effects on the efficiency of a normal-incidence grating in the 100-350-A wavelength region.

Thin-film interference effects were observed in the normal-incidence efficiency of a 2400-groove/mm replica grating. The efficiency was measured in the 100-350-A wavelength range and had an oscillatory behavior that resulted from the presence of a thin SiO(2) coating. The thicknesses of the SiO(2) and the underlying oxidized aluminum layers were inferred from computer modeling of the zero-order efficiency. The efficiencies in the diffracted orders were calculated with the modified integral approach and accounting for the multilayer coating and the groove profile derived from atomic force microscopy. The calculated and measured efficiencies were in good agreement.

Normal-incidence efficiencies of 4800-grooves/mm-ruled replica gratings with multilayer and gold coatings in the 125-325-A wavelength region.

The normal-incidence efficiencies of two 4800-grooves/mm ruled replica gratings, one with a dual-bandpass molybdenum/silicon multilayer coating and the other with a gold coating, were measured by use of synchrotron radiation in the 125-325-A wavelength region. The peak reflectance of the multilayer coating was 22% in the first Bragg order near 235 A and 28% in the second Bragg order near 126 A. The peak efficiency of the multilayer grating was 2.6% in the first diffraction order near 225 A and 0.3% in the second diffraction order near 125 A. The efficiencies of the multilayer grating were much higher than the corresponding efficiencies of the gold grating. The characterization of the surfaces of the gratings by atomic force microscopy indicated rms microroughness values in the 5-18-microm(-1) frequency range of 12-20 A for the multilayer grating and 22-32 A for the gold grating. Both gratings had bumpy surface features larger than the nominal groove height. The rather large surface roughness and groove irregularities had a detrimental effect on the grating efficiencies.

Efficiency calibration of the first multilayer-coated holographic ion-etched flight grating for a sounding rocket high-resolution spectrometer.

We have fabricated the four flight gratings for a sounding rocket high-resolution spectrometer using a holographic ion-etching technique. The gratings are spherical (4000-mm radius of curvature), large (160 mm x 90 mm), and have a laminar groove profile of high density (3600 grooves/mm). They have been coated with a high-reflectance multilayer of Mo/Si. Using an atomic force microscope, we examined the surface characteristics of the first grating before and after multilayer coating. The average roughness is approximately 3 A rms after coating. Using synchrotron radiation, we completed an efficiency calibration map over the wavelength range 225-245 A. At an angle of incidence of 5 degrees and a wavelength of 234 A, the average efficiency in the first inside order is 10.4 +/- 0.5%, and the derived groove efficiency is 34.8 +/- 1.6%. These values exceed all previously published results for a high-density grating.

Predicting suicidality.

In a sample of 102 college students, prior suicidality was associated with self-reports of depressive tendencies, but not with manic or obsessive-compulsive tendencies, religiosity or irrational thinking.

N 2-methylguanosine is iso-energetic with guanosine in RNA duplexes and GNRA tetraloops.

Modified nucleotides are resource-intensive alternatives to the four nucleotides that constitute the bulk of natural RNAs. Yet, even in cases where modifications are highly conserved, their functions are difficult to identify. One possible function might be to modulate the stability of RNA structures. To investigate this possibility for N 2-methylguanosine (m2G), which is present in a wide variety of RNAs, we have determined the thermodynamic consequences of substituting m2G for G in G-C Watson-Crick pairs and G@U wobble pairs within RNA duplexes. The m2G substitution is iso-energetic with G in all cases, except for aninternal m2G@U pair, where it has a modest (0.3 kcal/mol) stabilizing effect. We have also examined theconsequences of replacing G by m2G, and A by N 6, N 6-dimethyladenosine (m26A) in the helix 45 tetraloop of 16S rRNA, which would otherwise be a standard GNRA tetraloop. This loop is a conserved, hypermethylated region of the ribosome where methylation appears to modulate activity. m26A substitution destabilizes the tetraloop, presumably because it prevents the formation of the G@A sheared pair it would otherwise contain. m2G substitution has no effect on tetraloop stability. Together, these results suggest that m2G is equally stable as either the s-cis or s-trans rotamer. The lack of a significant effect on secondary structural stability in these systems suggests that m2G is introduced into naturally occurring RNAs for reasons other than modulation of duplex stability.

The structure of a methylated tetraloop in 16S ribosomal RNA.

BACKGROUND: Ribosomal RNAs contain many modified nucleotides. The functions of these nucleotides are poorly understood and few of them are strongly conserved. The final stem loop in 16S-like rRNAs is an exception in both regards. In both prokaryotes and eukaryotes, the tetranucleotide loop that caps the 3'-terminal stem contains two N6, N6-dimethyladenosine residues. The sequence and pattern of methylation are conserved within the loop, and there is evidence that these methylated nucleotides play an important role in subunit association and the initiation of protein synthesis. Because of the integral role that helix 45 plays in ribosome function, it is important to know what consequences these methylated nucleotides have on its structure. RESULTS: We have solved the solution structure of a 14-nucleotide analog of the terminal stem loop of bacterial 16S rRNA, which contains N2-methylguanosine as well as two N6,N6-dimethyladenosines. CONCLUSIONS: The methylation of the 16S rRNA stem loop completely alters its conformation, which would otherwise be a GNRA tetraloop. It is likely that the conformation of this loop is crucial for its function, having implications for its interaction with ribosomal subunits and its role in the initiation of protein synthesis.

Measurement of diffusion constants for nucleic acids by NMR.

Pulsed field-gradient NMR experiments can be used to measure the diffusion constants of nucleic acids. The diffusion constants measured in this way for double-helical DNAs of defined length agree well both with theory and with measurements done using other techniques. When applied to RNAs, this experiment easily distinguishes duplex RNAs from RNA hairpins and thus it can solve one of the perennial problems faced by RNA spectroscopists, i.e. assessing whether their samples are monomeric or not.

Efficiency of a multilayer-coated, ion-etched laminar holographic grating in the 14.5 16.0-nm wavelength region.

The efficiency of an ion-etched laminar holographic grating was measured at near-normal incidence in the 14.5-16.0-nm wavelength range. The grating had an electron-beam-evaporated Mo/Si multilayer coating matched to the grating groove depth. The efficiency peaked at 16.3% in the first inside order at 15.12 nm and 15.0% in the first outside order at 14.94 nm. These are believed to be the highest efficiencies obtained to date from a multilayer-coated laminar grating at near-normal incidence in the EUV (lambda<30.0nm) . Zero and even orders were almost completely suppressed. The grating groove efficiency in the first order approached the theoretical limit of 40.5%.

Grazing-incidence efficiencies in the 28-42-A wavelength region of replicas of the Skylab 3600-line/mm concave grating with multilayer and gold coatings.

The efficiencies of replicas of the Skylab 3600-line/mm concave grating with multilayer and gold coatings were measured by using synchrotron radiation at an angle of incidence of 79 degrees and in the 28-42-A wavelength range. The blaze angle of the grating facets that faced the incident radiation was 3.1 degrees , and the average angle of the opposite facets was 6 degrees . For the gold grating, the -1 outside order had the highest efficiency of any diffracted order (excluding the zero order) over the entire wavelength range. Calculations of the grating efficiency indicated that the high efficiency in the -1 order resulted from the rather small angle (6 degrees ) of the facets opposite the incident radiation. For the multilayer grating, the efficiency in the on-blaze +2 inside order was enhanced in the 30-34-A wavelength region as a result of the high reflectance of the multilayer coating. The maximum efficiency in the +2 order occurred at the wavelength (32 A) corresponding to the peak of the reflectance of the multilayer coating on the facets facing the incident radiation. These results further demonstrate that a multilayer coating can be used to enhance the efficiency, in a selected wavelength range and in the on-blaze order, of a grating operating at a small grazing angle (11 degrees ).