Inhaled vitamin A is more effective than intramuscular dosing in mitigating hyperoxia-induced lung injury in a neonatal rat model of bronchopulmonary dysplasia.

Prevention of bronchopulmonary dysplasia (BPD) in premature-birth babies continues to be an unmet medical need. Intramuscular vitamin A is currently employed in preterm neonates to prevent BPD but requires intramuscular injections in fragile neonates. We hypothesized that noninvasive inhaled delivery of vitamin A, targeted to lung, would be a more effective and tolerable strategy. We employed our well-established hyperoxia-injury neonatal rat model, exposing newborn rats to 7 days of constant extreme (95% O2) hyperoxia, comparing vitamin A dosed every 48 h via either aerosol inhalation or intramuscular injection with normoxic untreated healthy animals and vehicle-inhalation hyperoxia groups as positive and negative controls, respectively. Separately, similar vitamin A dosing of normoxia-dwelling animals was performed. Analyses after day 7 included characterization of alveolar histomorphology and protein biomarkers of alveolar maturation [surfactant protein C (SP-C), peroxisome proliferator-activated receptor (PPAR) γ, cholinephosphate cytidylyl transferase, vascular endothelial growth factor and its receptor, FLK-1, and retinoid X receptors (RXR-α, -ß, and -γ], apoptosis (Bcl2 and Bax) key injury repair pathway data including protein markers (ALK-5 and ß-catenin) and neutrophil infiltration, and serum vitamin A levels. Compared with intramuscular dosing, inhaled vitamin A significantly enhanced biomarkers of alveolar maturation, mitigated hyperoxia-induced lung damage, and enhanced surfactant protein levels, suggesting that it may be more efficacious in preventing BPD in extremely premature infants than the traditionally used IM dosing regimen. We speculate lung-targeted inhaled vitamin A may also be an effective therapy against other lung damaging conditions leading to BPD or, more generally, to acute lung injury.

Top-down proteomics on a high-field Fourier transform ion cyclotron resonance mass spectrometer.

Mass spectrometry is the tool of choice for sequencing peptides and determining the sites of posttranslational modifications; however, this bottom-up approach lacks in providing global information about the modification states of proteins including the number and types of isoforms and their stoichiometry. Recently, various techniques and mass spectrometers, such as high-field Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers, have been developed to study intact proteins (top-down proteomics). While the protein molecular mass and the qualitative and quantitative information about protein isoforms can be revealed by FTICR-MS analysis, their primary structure (including the identification of modifications and their exact locations in the amino acid sequence) can directly be determined using the MS/MS capability offered by the FTICR mass spectrometer. The distinct advantage of top-down methods are that modifications can be determined for a specific protein isoform rather than for peptides belonging to one or several isoforms. In this chapter, we describe different top-down proteomic approaches enabled by high-field (7, 9.4, and 12 T) FTICR mass spectrometers, and their applicability to answer biological and biomedical questions. We also describe the use of the free flow electrophoresis (FFE) to separate proteins prior to top-down mass spectrometric characterization.

Free-flow electrophoresis for top-down proteomics by Fourier transform ion cyclotron resonance mass spectrometry.

High-efficiency prefractionation of complex protein mixtures is critical for top-down proteomics, i.e., the analysis of intact proteins by MS. Free-flow electrophoresis (FFE) can be used for IEF to separate proteins within a pH gradient according to their pIs. In an FFE system, this separation is performed entirely in the liquid phase, without the need for particulate chromatographic media, gels, or membranes. Herein, we demonstrated the compatibility of IEF-FFE with ESI-Fourier transform ICR MS (ESI-FTICR-MS) for top-down experiments. We demonstrated that IEF-FFE of intact proteins were highly reproducible between FFE instruments, between laboratories, and between analyses. Applying native (0.2% hydroxypropylmethyl cellulose) IEF-FFE to an enzyme resulted in no decrease in enzyme activity; applying either native or denaturing (8 M urea) IEF-FFE to a four-protein mixture with different pIs resulted in isolation of each protein into separate fractions in a 96-well plate. After desalting, each protein was sequenced by top-down MS/MS. As an application of this technique, chicken erythrocyte histone H2A-IV and its major modified forms were enriched by IEF-FFE. Top-down analysis revealed Lys-5 to be a major acetylation site, in addition to N-terminal acetylation.

Advanced computational techniques for re-sequencing DNA with polymerase signaling assay arrays.

Re-sequencing, the identification of the specific variants in a sequence of interest compared with a known genomic sequence, is a ubiquitous task in today's biology. Universal arrays, which interrogate all possible oligonucleotides of a certain length in a target sequence, have been suggested for computationally determining a polynucleotide sequence from its oligonucleotide content. We present here new methods that use such arrays for re-sequencing. Our methods are applied to data obtained by the polymerase signaling assay, which arrays single-based primer extension reactions for either universal or partial arrays of pentanucleotides. The computational analysis uses the spectrum alignment algorithm, which is refined and enhanced here in order to overcome noise incurred by the use of such short primers. We present accurate re-sequencing results for both synthetic and amplified DNA molecules.

beta-Adrenergic receptor polymorphisms and responses during titration of metoprolol controlled release/extended release in heart failure.

OBJECTIVE: beta-Blockers require careful initiation and titration when used in patients with heart failure. Some patients tolerate beta-blocker therapy initiation without difficulty, whereas in other patients this period presents clinical challenges. We tested the hypothesis that polymorphisms at codons 389 (Arg389Gly) and 49 (Ser49Gly) of the beta(1)-adrenergic receptor would be associated with differences in initial tolerability of beta-blocker therapy in patients with heart failure. We also tested whether polymorphisms in the beta(2)-adrenergic receptor, G-protein alpha s subunit (G(s)alpha), and cytochrome P450 (CYP) 2D6 genes or S-metoprolol plasma concentrations were associated with beta-blocker tolerability. METHODS: Sixty-one beta-blocker-naive patients with systolic heart failure were prospectively enrolled. Patients began taking 12.5 to 25 mg metoprolol controlled release/extended release with titration every 2 weeks (as tolerated) to 200 mg/d or the maximum tolerated dose over a period of 8 to 10 weeks. Decompensation was the composite of death, heart failure hospitalization, increase in other heart failure medications, or need to discontinue metoprolol. End points were assessed during the titration period. RESULTS: The overall rate of decompensation was not different between the codon 49 or 389 genotypes. However, a significantly greater percentage of patients with the Gly389 variant required increases in heart failure medications as compared with Arg389 homozygotes (48% versus 14%, respectively; P = .006). Similarly, patients with the Ser49 homozygous genotype were significantly more likely to require increases in concomitant heart failure therapy as compared with Gly49 carriers (41% versus 11%, respectively; P = .03). Neither CYP2D6 genotypes nor metoprolol pharmacokinetics differed between patients with and those without a decompensation event. There was no association between the beta(2)-adrenergic receptor or G(s)alpha polymorphisms with decompensated heart failure. CONCLUSIONS: Patients with the Gly389 variant and Ser49Ser genotype were significantly more likely to require increases in heart failure medications during beta-blocker titration and thus may require more frequent follow-up during titration.

Auto-validation of fluorescent primer extension genotyping assay using signal clustering and neural networks.

BACKGROUND: SNP genotyping typically incorporates a review step to ensure that the genotype calls for a particular SNP are correct. For high-throughput genotyping, such as that provided by the GenomeLab SNPstream instrument from Beckman Coulter, Inc., the manual review used for low-volume genotyping becomes a major bottleneck. The work reported here describes the application of a neural network to automate the review of results. RESULTS: We describe an approach to reviewing the quality of primer extension 2-color fluorescent reactions by clustering optical signals obtained from multiple samples and a single reaction set-up. The method evaluates the quality of the signal clusters from the genotyping results. We developed 64 scores to measure the geometry and position of the signal clusters. The expected signal distribution was represented by a distribution of a 64-component parametric vector obtained by training the two-layer neural network onto a set of 10,968 manually reviewed 2D plots containing the signal clusters. CONCLUSION: The neural network approach described in this paper may be used with results from the GenomeLab SNPstream instrument for high-throughput SNP genotyping. The overall correlation with manual revision was 0.844. The approach can be applied to a quality review of results from other high-throughput fluorescent-based biochemical assays in a high-throughput mode.

Pharmacokinetics and CYP2D6 genotypes do not predict metoprolol adverse events or efficacy in hypertension.

OBJECTIVE: Beta-Blocker use can be associated with adverse effects that may have an impact on adherence or harm patients. The commonly prescribed beta-blocker metoprolol is metabolized by the polymorphic cytochrome P450 (CYP) 2D6 enzyme, resulting in widely variable drug exposure. We investigated whether metoprolol plasma concentrations, CYP2D6 polymorphisms, or genotype-derived phenotype was associated with adverse effects or efficacy in patients with hypertension. METHODS: Fifty hypertensive patients received metoprolol by use of a dose-titration algorithm until target blood pressure was reached, intolerable side effects occurred, or maximal daily dose was achieved. CYP2D6 genotype was determined by methods based on polymerase chain reaction-restriction fragment length polymorphism and included 19 allelic variants. Patients were assigned to standard phenotype groups on the basis of genotype. Patients were also assigned activity scores based on functional activity of the alleles. General and dose-limiting adverse events and blood pressure responses were analyzed in relation to metoprolol steady-state pharmacokinetic profile and CYP2D6 genotype-derived phenotype. RESULTS: Poor metabolizers had a significantly longer elimination half-life, higher S-metoprolol area under the plasma concentration-time curve (AUC), and lower oral clearance (P < or = .007 for all parameters). There was a 29.6-fold variability in AUC among extensive metabolizers, which was largely explained by CYP2D6 activity scores (P = .032 for ordered differences in AUC by activity score among extensive metabolizers). Overall general and dose-limiting adverse event rates were 46% and 14%, respectively. General adverse event rates did not differ by AUC quartile (66.7% [95% confidence interval (CI), 35.4%-88.7%] and 41.7% [95% CI, 16.5%-71.4%] in the lowest and highest quartiles, respectively; P = .09 among all quartiles). Dose-limiting adverse event rates were also not different by AUC quartile (16.7% [95% CI, 2.9%-49.1%] and 8.3% [95% CI, 0.4%-40.2%] in the lowest and highest quartiles; P = .35 among all quartiles). Furthermore, adverse event rates did not differ by activity scores or between extensive, intermediate, or poor metabolizers. Antihypertensive response rate and blood pressure changes also were not influenced by differences in plasma concentrations or CYP2D6 genotypes. CONCLUSIONS: As expected, CYP2D6 genotype-phenotype correlates with differences in metoprolol pharmacokinetics. However, there was no association between variable pharmacokinetics or CYP2D6 genotype and beta-blocker-induced adverse effects or efficacy.

SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery.

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orrhid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-IT tag array single base extension genotyping technology The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHTArray Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1,000,000 genotyping assays with >96% of samples giving genotypes with >99% accuracy

Minimizing preanalytical variation of plasma samples by proper blood collection and handling.

Blood samples collected for proteome studies are subject to a variety of preanalytical instability, among which intrinsic proteolysis activities cause a broad spectrum of protein and peptide degradation. This chapter describes two MALDI MS-based methods for plasma peptidomic analyses; a direct MALDI-TOF MS and an LC MALDI-TOF MS. Using these methods, we compared peptides and their time-dependent changes in traditional serum, four plasma samples with different anticoagulants and additives: EDTA-based, citrate-based, or heparin-based, and EDTA-based with protease inhibitors. For minimizing plasma sample instability and preanalytical variation, we suggest using an optimized blood collection device, minimizing the dwell time during blood collection and handling, controlling centrifugation and handling at room temperature, and saving plasma samples for use at most one freeze/thaw cycle. We have optimized our protocol to achieve reproducibility in peptidomic analyses of plasma samples using MALDI-TOF MS by minimizing preanalytical and analytical variability.

Investigation of peptide biomarker stability in plasma samples using time-course MS analysis.

Peptide biomarkers in plasma or serum are subject to proteolytic degradation caused by intrinsic peptidase activities, resulting in a potential barrier in translating a discovered biomarker into clinical application. This chapter describes a method using time-course MALDI-TOF MS analysis to investigate the stability of a plasma peptide biomarker under a variety of preanalytical situations. A synthesized peptide with the same primary sequence as a potential endogenous biomarker is spiked into a blood sample, and the sample is incubated over time at r.t. (25 ± 1°C) or other preanalytical situations. At a specific period of incubation time, the sample is quenched with the addition of acid with or without an internal control peptide. The spiked peptides in the sample are extracted with one of three procedures for highly soluble, moderately soluble, or essentially insoluble peptides. The peptide samples are then analyzed using MALDI-TOF MS. The abundance changes of the peptide biomarker are monitored by time-course changes of the mass spectra. These changes over-time are measured and fitted to a first-order degradation reaction so that stability of the peptide biomarker (half-life) can be calculated. Kinetics analysis of both parent and shorter (daughter) peptides are also possible by fitting to a sequential multiple-step reaction (SMSR) model. This optimized method facilitates evaluation of biomarker stability, and helps to define sample handling and analytical processing steps that contribute to instability of measured peptide biomarker(s).

Application of free flow electrophoresis to the analysis of the urine proteome.

Urine is a complex fluid, which is thought to contain valuable diagnostic information regarding general health. In particular, there is great diagnostic potential in the peptide and/or protein content of urine, but the information is present in low abundance. Most traditional proteomic techniques lack sufficient sensitivity/dynamic range, especially for dilute and/or complex samples. However, orthogonal separation methods can be applied prior to protein/peptide analysis to increase the success rate of urine proteomic studies and access this potentially valuable information. In this chapter, we describe isoelectric focusing (IEF) of intact urine proteins, via free flow electrophoresis (FFE), prior to typical peptide-based mass spectrometry analysis, facilitating the deep analysis of urine protein detection and identification, for biomarker discovery. Our work demonstrates that such an approach can be used as a preprocessing step and can be integrated into a workflow for the successful identification of protein components (biomarkers) from urine.

Thrombin induces broad spectrum proteolysis in human serum samples.

BACKGROUND: During clotting, a thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of a thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially a thrombin. METHODS: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal a thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by a thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion. RESULTS: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by a thrombin confirms these observations. CONCLUSIONS: The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum.

Resolution of adiponectin oligomers in human plasma using free flow electrophoresis.

Adiponectin is an important adipocytokine hormone which circulates in blood as homo-oligomers (trimer, hexamer and high molecular weight (HMW) forms) as well as a truncated form corresponding to the globular domain. Free flow electrophoresis (FFE) used in zone electrophoresis mode revealed the presence of isoforms within these oligomeric forms in plasma. HMW adiponectin oligomer showed two isoforms which carry different charge density at pH 4.7, only one of which is susceptible to dissociation by SDS. The adiponectin hexamer was shown to consist of a doublet and also shown to have at least two isoforms. A truncated form of adiponectin was identified as the main constituent of adiponectin in plasma and appeared to circulate bound to a basic protein, potentially one of the chemokines reported to bind to the globular domain. Analysis of the monomer composition of the oligomers revealed differences in monomeric isoforms used to build up the oligomers.

Intrinsic peptidase activity causes a sequential multi-step reaction (SMSR) in digestion of human plasma peptides.

Human plasma and serum samples, including protein and peptide biomarkers, are subjected to preanalytical variations and instability caused by intrinsic proteases. In this study, we directly investigated the stability of peptide biomarkers by spiking an isotopically labeled peptide into human plasma and serum samples and then monitoring its time-dependent change. Fibrinogen peptide A (FPA) was used as a model substrate, and its degradation in a conventional serum and plasma either with citrate, heparin, or EDTA as the anticoagulant, or EDTA plus protease inhibitors (inhibited plasma), was measured using time-course MALDI-TOF MS analysis. The FPA and other peptides tested in this study vary in these samples. However, the peptides are most stable in the inhibited plasma followed by, in general order, EDTA plasma, citrate plasma, heparin plasma and serum, demonstrating the benefit of plasma versus serum, and protease inhibitors for biomarker stabilization. Kinetic analysis indicates that intrinsic peptidases cause an observed first-order Sequential Multiple-Step Reaction (SMSR) in digestion of the peptide. Modeling analysis of the SMSR demonstrates that step reactions differ in their kinetic rate constants, suggesting a significant contribution of the truncated end residue on the substrate specificity of the intrinsic peptidase(s). Our observations further show that synthetic peptides introduced into plasma as internal controls can also be degraded, and thus, their (in)stability as a preanalytical variable should not be overlooked.

Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma.

Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750-3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI-MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolytic activities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome.

Region-specific detection of neuroblastoma loss of heterozygosity at multiple loci simultaneously using a SNP-based tag-array platform.

Many cancers are characterized by chromosomal aberrations that may be predictive of disease outcome. Human neuroblastomas are characterized by somatically acquired copy number changes, including loss of heterozygosity (LOH) at multiple chromosomal loci, and these aberrations are strongly associated with clinical phenotype including patient outcome. We developed a method to assess region-specific LOH by genotyping multiple SNPs simultaneously in DNA from tumor tissues. We identified informative SNPs at an average 293-kb density across nine regions of recurrent LOH in human neuroblastomas. We also identified SNPs in two copy number neutral regions, as well as two regions of copy number gain. SNPs were PCR-amplified in 12-plex reactions and used in solution-phase single-nucleotide extension incorporating tagged dideoxynucleotides. Each extension primer had 5' complementarity to one of 2000 oligonucleotides on a commercially available tag-array platform allowing for solid-phase sorting and identification of individual SNPs. This approach allowed for simultaneous detection of multiple regions of LOH in six human neuroblastoma-derived cell lines, and, more importantly, 14 human neuroblastoma primary tumors. Concordance with conventional genotyping was nearly absolute. Detection of LOH in this assay may not require comparison to matched normal DNAs because of the redundancy of informative SNPs in each region. The customized tag-array system for LOH detection described here is rapid, results in parallel assessment of multiple genomic alterations, and may speed identification of and/or assaying prognostically relevant DNA copy number alterations in many human cancers.

HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples.

There is a substantial list of pre-analytical variables that can alter the analysis of blood-derived samples. We have undertaken studies on some of these issues including choice of sample type, stability during storage, use of protease inhibitors, and clinical standardization. As there is a wide range of sample variables and a broad spectrum of analytical techniques in the HUPO PPP effort, it is not possible to define a single list of pre-analytical standards for samples or their processing. We present here a compendium of observations, drawing on actual results and sound clinical theories and practices. Based on our data, we find that (1) platelet-depleted plasma is preferable to serum for certain peptidomic studies; (2) samples should be aliquoted and stored preferably in liquid nitrogen; (3) the addition of protease inhibitors is recommended, but should be incorporated early and used judiciously, as some form non specific protein adducts and others interfere with peptide studies. Further, (4) the diligent tracking of pre-analytical variables and (5) the use of reference materials for quality control and quality assurance, are recommended. These findings help provide guidance on sample handling issues, with the overall suggestion being to be conscious of all possible pre-analytical variables as a prerequisite of any proteomic study.

High-density single-nucleotide polymorphism maps of the human genome.

Here we report a large, extensively characterized set of single-nucleotide polymorphisms (SNPs) covering the human genome. We determined the allele frequencies of 55,018 SNPs in African Americans, Asians (Japanese-Chinese), and European Americans as part of The SNP Consortium's Allele Frequency Project. A subset of 8333 SNPs was also characterized in Koreans. Because these SNPs were ascertained in the same way, the data set is particularly useful for modeling. Our results document that much genetic variation is shared among populations. For autosomes, some 44% of these SNPs have a minor allele frequency > or =10% in each population, and the average allele frequency differences between populations with different continental origins are less than 19%. However, the several percentage point allele frequency differences among the closely related Korean, Japanese, and Chinese populations suggest caution in using mixtures of well-established populations for case-control genetic studies of complex traits. We estimate that approximately 7% of these SNPs are private SNPs with minor allele frequencies <1%. A useful set of characterized SNPs with large allele frequency differences between populations (>60%) can be used for admixture studies. High-density maps of high-quality, characterized SNPs produced by this project are freely available.

The thermodynamics of template-directed DNA synthesis: base insertion and extension enthalpies.

We used stopped-flow calorimetry to measure the overall enthalpy change associated with template-directed nucleotide insertion and DNA extension. Specifically, we used families of hairpin self-priming templates in conjunction with an exonuclease-free DNA polymerase to study primer extension by one or more dA or dT residues. Our results reveal exothermic heats between -9.8 and -16.0 kcal/bp for template-directed enzymatic polymerization. These extension enthalpies depend on the identity of the inserting base, the primer terminus, and/or the preceding base. Despite the complexity of the overall process, the sign, magnitude, and sequence dependence of these insertion and extension enthalpies are consistent with nearest-neighbor data derived from DNA melting studies. We recognize that the overall process studied here involves contributions from a multitude of events, including dNTP to dNMP hydrolysis, phosphodiester bond formation, and enzyme conformational changes. It is therefore noteworthy that the overall enthalpic driving force per base pair is of a magnitude similar to that expected for addition of one base pair or base stack per insertion event, rather than that associated with the rupture and/or formation of covalent bonds, as occurs during this catalytic process. Our data suggest a constant sequence-independent background of compensating enthalpic contributions to the overall process of DNA synthesis, with discrimination expressed by differences in noncovalent interactions at the template-primer level. Such enthalpic discrimination underscores a model in which complex biological events are regulated by relatively modest energy balances involving weak interactions, thereby allowing subtle mechanisms of regulation.