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PubMed2026

RBFOX3 regulates hippocampal transcriptomic programs to maintain synaptic and ultrastructural integrity.

RBFOX3 is a neuron-specific RNA-binding protein essential for maintaining brain circuit homeostasis and functional connectivity. Genetic disruptions in RBFOX3 are clinically linked to cognitive impairment, epilepsy, and sleep disorders. Although global Rbfox3 knockout (Rbfox3-/-) mouse models have established its necessity in hippocampus-dependent neuronal circuits and behaviors, the underlying hippocampal transcriptomic landscape and synaptic ultrastructure remain poorly understood. To address these gaps, we integrated hippocampal RNA-sequencing from Rbfox3-/- mice with high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation analysis. We identified 3,401 differentially expressed genes in the hippocampus of Rbfox3-/- mice, confirming 1,920 as candidate RBFOX3 targets. Gene Ontology enrichment analysis revealed that these candidate targets converge on pathways governing neuronal morphogenesis, synaptic transmission, dendritic development, and cognition. Furthermore, transmission electron microscopy of the hippocampal dentate gyrus revealed that while the overall presynaptic area remained unaltered, Rbfox3 deletion reduced presynaptic vesicle number, presynaptic mitochondria area, and postsynaptic density thickness. Collectively, our findings demonstrate that RBFOX3 acts as a critical regulator orchestrating the transcriptomic programs required for hippocampal structural and functional maturation, revealing that its loss compromises both the metabolic and structural architecture of the synapse.

Hippocampus↗
PubMed2003

Identification of target genes of the p16INK4A-pRB-E2F pathway.

Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of human cancer. The core members of this pathway include the tumor suppressor protein, pRB, which through binding to a number of cellular proteins, most notably members of the E2F transcription factor family, regulates progression through the cell division cycle. With the aim of identifying transcriptional changes provoked by deregulation of the pRB pathway, we have used cell lines that conditionally express a constitutively active phosphorylation site mutant of pRB (pRBDeltaCDK) or p16INK4A (p16). The expression of pRBDeltaCDK and p16 resulted in significant repression and activation of a large number of genes as measured by high density oligonucleotide array analysis. Transcriptional changes were found in genes that are essential for DNA replication and cell proliferation. In agreement with previous results, we found a high degree of overlap between genes regulated by p16 and pRB. Data we have obtained previously for E2F family members showed that 74 of the genes repressed by pRB and p16 were induced by the E2Fs and 23 genes that were induced by pRB and p16 were repressed by the E2Fs. Thus, we have identified 97 genes as physiological targets of the pRB pathway, and the further characterization of these genes should provide insights into how this pathway controls proliferation. We show that Gibbs sampling detects enrichment of several sequence motifs, including E2F consensus binding sites, in the upstream regions of these genes and use this enrichment in an in silico filtering process to refine microarray derived gene lists.

Amino Acid Motifs↗
PubMed1976

Biochemical actions of follice-stimulating hormone in the sertoli cell of the rat testis.

The sequenc of biochemical events associated with the action of follicle-stimulating hormone (FSH) in the testis has been investigated using a Sertoli cell-enriched testis model system. The Sertoli cell-encriched testis, created by irradiation of male rats in utero, is devoid of germinal elements but contains a normal complement of supportive Sertoli cells. Comparison of the Sertoli cell-enriched testis with normal testis, demonstrates that the two types of testes contain equal numbers of FSH specific receptors, judged by the binding of labeled hormone. In addition, FSH over a concentration range from 6 X 10(-11) to 6 X 10(-9)M will stimulate the production of adenosine 3',5' monophosphate (cAMP) in the Sertoli cell-enriched testis in a manner indistinguishable from that of the normal testis. Incubation of Sertoli cell enriched testis also results in the activation of soluble cAMP-dependent protein kinase. This response to FSH is dependent upon the age of the animal and disappears at about 32 days of age. While sensitivity to the hormone can still be detected in mature Sertoli cell-enriched animals by the addition of the phosphodiesterase inhibitor 1-methyl-3-isobutyl-xanthine, no detectable increase in phosphodiesterase activity is apparent after 30 days of age. Injection of FSH into Sertoli cell-enriched animals results in an increase in total testicular protein synthesis as well as in the production of the Sertoli cell-specific protein, androgen-binding protein within 30 minutes. Furthermore, while hypophysectomy of Sertoli cell-enriched animals result in a decline of the testicular concentration of androgen-binding protein, the injection of FSH will stimulate and maintain the levels of androgen-binding protein in such animals. These results demonstrate that the Sertoli cell-enriched testis is capable of carrying out the sequence of biochemical events previously described for FSH in the normal testis and therefore, suggest that the Sertoli cell is the primary target cell for FSH action.

Age Factors↗
PubMed2005

AMOD: a morpholino oligonucleotide selection tool.

AMOD is a web-based program that aids in the functional evaluation of nucleotide sequences through sequence characterization and antisense morpholino oligonucleotide (target site) selection. Submitted sequences are analyzed by translation initiation site prediction algorithms and sequence-to-sequence comparisons; results are used to characterize sequence features required for morpholino design. Within a defined subsequence, base composition and homodimerization values are computed for all putative morpholino oligonucleotides. Using these properties, morpholino candidates are selected and compared with genomic and transcriptome databases with the goal to identify target-specific enriched morpholinos. AMOD has been used at the University of Minnesota to design approximately 200 morpholinos for a functional genomics screen in zebrafish. The AMOD web server and a tutorial are freely available to both academic and commercial users at http://www.secretomes.umn.edu/AMOD/.

DNA Primers↗
PubMed2026

Matrine Alleviates Sepsis-Induced Acute Lung Injury by Reinforcing NQO1/SLC7A11/GPX4-Associated Anti-Ferroptotic Defenses and Attenuating NF-κB-Driven Inflammation.

BACKGROUND: Sepsis triggers dysregulated systemic inflammation and multiple-organ dysfunction, with the lungs being particularly susceptible to injury. Sepsis-induced acute respiratory distress syndrome (ARDS) is mainly driven by TLR4/NF-κB-mediated hyperinflammation and alveolar macrophage activation. Matrine, a bioactive alkaloid derived from Sophora flavescens, has been reported to modulate redox homeostasis and ferroptosis-associated lipid peroxidation. However, the target-specific mechanisms underlying its effects on ferroptosis and inflammatory signaling in sepsis-induced acute lung injury (SALI) remain incompletely understood. PURPOSE: This study aimed to evaluate the therapeutic effects of matrine in a cecal ligation and puncture (CLP)-induced SALI model and to determine whether its protective effects involve reinforcement of NQO1/SLC7A11/GPX4-associated anti-ferroptotic defenses and suppression of NF-κB-driven inflammation. METHODS: We analyzed the single-cell RNA-sequencing (scRNA-seq) dataset GSE273924 to characterize CD45-enriched pulmonary immune-cell subsets in sham mice and mice with intratracheal Escherichia coli-induced pneumonia. Network pharmacology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to predict Kushen (KS)-related targets and pathways associated with SALI. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) of GSE245013 were used to identify candidate targets. Matrine-NQO1 binding and intracellular target engagement were evaluated using molecular docking, molecular dynamics simulations, surface plasmon resonance (SPR), and the cellular thermal shift assay (CETSA). The therapeutic effects of matrine were assessed in mice with CLP-induced SALI and in lipopolysaccharide (LPS)-stimulated MH-S cells. Lung histopathology, inflammatory cytokine production, target protein expression, ferroptosis-associated indicators, and NF-κB activation were evaluated using molecular, biochemical, and histological assays. The functional contribution of NQO1 was further examined using the NQO1 inhibitor ES936. RESULTS: scRNA-seq analysis of GSE273924 revealed substantial remodeling of the CD45-enriched pulmonary immune-cell landscape in mice with intratracheal E. coli-induced pneumonia, including macrophage transcriptional programs associated with ferroptosis and inflammatory signaling. Integrated network pharmacology and bioinformatics analyses prioritized NQO1 as a candidate target of matrine and identified NF-κB signaling as a potentially relevant pathway. Molecular docking, molecular dynamics simulations, SPR, and CETSA supported matrine-NQO1 binding and intracellular target engagement. Functionally, matrine improved survival, attenuated lung injury, reinforced NQO1/SLC7A11/GPX4-associated anti-ferroptotic defenses, and suppressed NF-κB activation in CLP mice. Similar protective effects were observed in LPS-stimulated MH-S cells. ES936 partially attenuated the matrine-mediated improvements in cell viability, redox homeostasis, ferroptosis-associated indicators, and NF-κB p65 phosphorylation, supporting a functional contribution of NQO1 to the protective effects of matrine. CONCLUSION: Matrine alleviates SALI by reinforcing NQO1/SLC7A11/GPX4-associated anti-ferroptotic defenses and attenuating NF-κB-driven inflammation.

Animals↗
PubMed2005

Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.

Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n = 10) of 2-22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2-7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coefficients (R(2)) of 0.995 and 0.989, respectively. Absolute analyte specificity for each peptide was demonstrated using MRM-triggered MS/MS scans. Reliable detection of L-selectin (measured at 0.67 microg/ml) indicates that proteins down to the microg/ml level can be quantitated in plasma with minimal sample preparation, yielding a dynamic range of 4.5 orders of magnitude in a single experiment. Peptide MRM measurements in plasma digests thus provide a rapid and specific assay platform for biomarker validation, one that can be extended to lower abundance proteins by enrichment of specific target peptides (stable isotope standards and capture by anti-peptide antibodies (SISCAPA)).

Amino Acid Sequence↗
PubMed1996

Allelic imbalance at NME1 in microdissected primary and metastatic human colorectal carcinomas is frequent but not associated with metastasis to lymph nodes or liver.

Allelic imbalance at the NME locus on chromosome 17q21 was analyzed in colorectal cancer patients using a highly polymorphic microsatellite repeat sequence within NME1 itself. Duplicate samples of carcinoma and adjacent normal tissue was obtained by microdissection from 6 to 7-microns paraffin sections of 94 primary carcinomas (treatment years 1979-1993) and available lymph node and liver secondaries. In 55 patients informative (heterozygous) at this locus, allelic imbalance was examined in primary and secondary carcinomas. Microsatellite instability prevented assessment of allelic balance in two cases, and there was no evidence of homozygous loss at NME1 in any case analyzed. Allelic imbalance at the NME locus in carcinomas was frequent (27/53; 51%), and concordant results were obtained between primary carcinoma and secondary deposits in 30 of 33 (91%) cases. Three discordant cases showed allelic imbalance in secondary deposits but not the primary lesion. Although frequent, allelic imbalance at NME1 had no relationship to Dukes' stage at presentation or with subsequent hepatic metastasis, nor with the primary carcinoma site (proximal versus distal), tumor size, or mitotic or apoptotic index. Moreover, neither disease-free nor overall survival differed between patients with carcinomas showing NME1 allelic imbalance and patients with carcinomas that did not. Our results show that although allelic imbalance is frequent at the NME locus in primary and secondary colorectal carcinomas, there is no evidence to link this with clinical or pathological features or with metastatic potential. Microsatellite PCR and microdissection of enriched populations of carcinoma cells allowed uniformly successful analysis of samples from archival formalin-fixed paraffin-embedded tissue up to 15 years old and clear assessment of allelic imbalance in tumor specimens. Target sequences (e.g., microsatellites and minisatellites) up to approximately 200-250 bp may be reliably analyzed for allelic balance, suggesting that this method is of general utility in the genetic analysis of primary and metastatic neoplasia.

Adenocarcinoma↗
PubMed2001

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins.

BACKGROUND: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor kappa B (NF-kappa B) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. RESULTS: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-kappa B p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-kappa B p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:10(8) and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. CONCLUSIONS: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Blotting, Western↗
PubMed1992

Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs.

A vast amount of data suggests that homologous recombination in mammalian cells is relatively rare as compared to random integration, imposing the need for sophisticated selection protocols to enrich for cells in which homologous recombination has occurred. We here show that one of the key factors in efficient homologous recombination is the use of isogenic DNA to prepare the targeting vectors. Homologous recombination at the retinoblastoma susceptibility gene (Rb) in embryonic stem cells derived from mouse strain 129 was 20-fold more efficient with a 129-derived targeting construct than with a BALB/c-derived construct. The two constructs were identical, except for a number of base sequence divergences between 129 and BALB/c DNA, including base-pair substitutions, small deletions/insertions, and a polymorphic CA repeat. Transfection with an isogenic DNA construct, containing 17 kilobases of homology, yielded a targeting frequency of 78% (of a total of 20,000 drug-resistant colonies), without the use of an enrichment protocol for homologous recombination. This result indicates that, also in mammalian cells, homologous recombination rather than random integration can be the predominant event.

Animals↗
PubMed2001

Geographic distribution of Desulfitobacterium frappieri PCP-1 and Desulfitobacterium spp. in soils from the province of Quebec, Canada.

The presence of indigenous Desulfitobacterium species in 44 soil samples taken from various sites in the southern part of the province of Quebec (Canada) and four from locations outside Quebec was investigated. Twenty-four of these soils were sampled from contaminated industrial sites. Indigenous Desulfitobacterium bacteria from soil samples were enriched by cultivation in anaerobic soil slurry culture. Total DNA was then extracted from these slurries and polymerase chain reaction (PCR) amplifications were performed with primers targeting 16S ribosomal RNA gene sequences of Desulfitobacterium spp. and of Desulfitobacterium frappieri PCP-1. A positive PCR signal was obtained in 31 soil slurry cultures. Resolution of single-strand DNAs of some of the PCR products by a single-strand conformational polymorphism protocol suggests that more than one species of Desulfitobacterium were present in the corresponding slurry cultures. These results suggest that Desulfitobacterium are ubiquitous in soils in the province of Quebec, especially in soils from the St. Lawrence valley and the southern part of the province.

Journal Article↗
PubMed2026

Balancing under constraint: Structural insights into norovirus evolution and antigenic innovation.

Norovirus is the leading cause of acute viral gastroenteritis worldwide. While genomic studies have revealed its diversity and evolutionary patterns, the structural mechanisms driving viral adaptation remain poorly understood. Here, we establish a comprehensive structural database of norovirus VP1 P-domains across nine genogroups (GI-GIX) through large-scale AlphaFold2 predictions. By integrating phylogenetic analysis of VP1 sequences and structures, we demonstrate that sequence and structural evolution show overall concordance under purifying selection, yet significant local discrepancies reveal distinct patterns of convergent evolution shaped by structural constraints and functional divergence. Focusing on the predominant GII.4 genotype, we found that compared to near-full-genome and nucleotide trees, only the VP1 amino acid tree reliably clustered GII.4 variants in chronological order as monophyletic groups. We further identify a hierarchical evolutionary strategy: positive selection may drive structural hypervariability in major antigenic epitopes D and C for immune escape, with epitope D exhibiting pronounced structural flexibility that complicates its structural characterization, whereas coevolutionary analysis uncovers a broad network of compensatory interactions spanning multiple epitopes, with striking enrichment in epitope A. These epitopes exhibited a pattern of "sequence plasticity with structural conservation", maintained by coevolutionary constraints that preserve conformational integrity. Together, these findings suggest that norovirus vaccine strategies targeting the structurally conserved conformations of epitopes A and G could overcome the limitations of traditional strain-specific approaches, offering a pathway toward broad protection against evolving viral diversity.

Norovirus↗
PubMed2003

Real-time multiplex SYBR green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes.

A multiplex SYBR Green I-based PCR assay has been developed for simultaneous detection of Salmonella serovars and Listeria monocytogenes using a LightCycler. Primers were designed to amplify an 85-bp sequence from the gene encoding a fimbrinlike protein (fimI) of Salmonella Enteritidis and a 98-bp sequence from the hemolysin gene (hly) of L. monocytogenes. These primers allowed the amplification of PCR products having distinct melting temperature values, resulting in the formation of two distinct peaks representing the two targets. Background signals, resulting from primer-dimer formation in the late cycles of PCR, are eliminated through the acquisition of data at a high temperature (>75 degrees C), but several degrees lower than required for detection of the specific PCR products. A rapid and simple method for the extraction of bacterial genomic DNA from liquid culture, coupled with duplex PCR using LightCycler SYBR Green-based PCR assays, detected the presence of 2.5 cells and 1 cell of Salmonella serovars and L. monocytogenes, respectively, within an hour. Following overnight enrichment, target DNA was present in sufficient quantities in 1 microl of culture to enable direct detection with the LightCycler.

Animals↗
PubMed1999

Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells.

Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors.

Animals↗
PubMed1998

DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs.

The repeated modification of a genomic locus is a technically demanding but powerful strategy to analyze the function of a particular gene product or the role of cis-regulatory DNA elements in mammalian cells. The initial step is "tagging" a site with a selectable marker which is done by homologous recombination (HR) to modify a known locus or by random integration to study cis-regulatory elements at a reproducibly accessible genomic location. The tag is then used to target the construct of choice during an exchange step. Presented here is a novel technique in which the exchange is independent of HR and does not introduce vector sequences. It relies on our previous studies on the replacement of DNA cassettes by FLP-recombinase, whereby some common limitations can be overcome. To this end, the tag, a hygtk positive/negative selection marker, is integrated into the genome of embryonic stem (ES) cells. This marker is flanked by a wild-type Flp-recognition target (FRT) site on one end and by a modified heterospecific FRT site on the other. Successful Flp-mediated replacement of the hygtk cassette is enriched by ganciclovir (GANC) selection for cells that lack the encoded fusion protein. Thereby, the hygtk gene can be exchanged for virtually any sequence in a single efficient step without the need of introducing a positive selectable marker. The system can hence be used to analyze the function of either a gene product or regulatory sequences in ES cells or the transgenic mice derived thereof.

Animals↗
PubMed1997

Interacting RNA species identified by combinatorial selection.

RNA molecules were selected from a random sequence library for their ability to bind to an RNA stem-loop target. Oligonucleotides with extensive Watson-Crick complementarity to the RNA ligand were selected against by inclusion of a blocking oligodeoxynucleotide in the binding phase of the selection protocol. After 18 generations of SELEX (systematic evolution of ligands by exponential enrichment) a single RNA family was predominant in the binding population. The winning aptamer RNA bound the target RNA with an apparent Kd = 70 nM. Structural mapping and Fe(II)-EDTA protection indicated that the target RNA interacted with small unpaired loops in the aptamer structure.

Base Composition↗
PubMed2004

A conformation-constrained peptide library based on insect defensin A.

Here, we reported a conformation-constrained peptide library, that was constructed based on the scaffold of a 29 amino acids peptide derived from insect defensin A. The peptide scaffold was designed utilizing the InsightII molecular modeling software and then displayed on M13 filamentous bacteriophage by fusion with coat protein III. The library was constructed by randomization of seven positions located within the two loops of the peptide scaffold generating approximately 8.3 x 10(8) transformants. Sequences from 14 randomly selected phage clones indicated that the distribution of nucleotides and amino acids paralleled with the expected frequency. Screening against the target proteins: tumor necrosis factor alpha, TNF receptor 1, TNF receptor 2 and monoclonal antibody against BMP-2 showed significant enrichment in all cases. The results presented here show that the reconstructed insect defensin A domain will be a promising non-antibody protein scaffold for the presentation of a phage-displayed constrained peptide library.

Amino Acid Sequence↗
PubMed2001

Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches.

High density oligonucleotide arrays have been used extensively for expression studies of eukaryotic organisms. We have designed a prokaryotic high density oligonucleotide array using the complete Escherichia coli genome sequence to monitor expression levels of all genes and intergenic regions in the genome. Because previously described methods for preparing labeled target nucleic acids are not useful for prokaryotic cell analysis using such arrays, a mRNA enrichment and direct labeling protocol was developed together with a cDNA synthesis protocol. The reproducibility of each labeling method was determined using high density oligonucleotide probe arrays as a read-out methodology and the expression results from direct labeling were compared to the expression results from the cDNA synthesis. About 50% of all annotated E.coli open reading frames are observed to be transcribed, as measured by both protocols, when the cells were grown in rich LB medium. Each labeling method individually showed a high degree of concordance in replica experiments (95 and 99%, respectively), but when each sample preparation method was compared to the other, approximately 32% of the genes observed to be expressed were discordant. However, both labeling methods can detect the same relative gene expression changes when RNA from IPTG-induced cells was labeled and compared to RNA from uninduced E.coli cells.

Adenosine Triphosphate↗
PubMed2007

A mathematical analysis of SELEX.

Systematic evolution of ligands by exponential enrichment (SELEX) is a procedure by which a mixture of nucleic acids that vary in sequence can be separated into pure components with the goal of isolating those with specific biochemical activities. The basic idea is to combine the mixture with a specific target molecule and then separate the target-NA complex from the resulting reaction. The target-NA complex is then separated by mechanical means (for example by filtration), the NA is then eluted from the complex, amplified by polymerase chain reaction (PCR) and the process repeated. After several rounds, one should be left with a pool of [NA] that consists mostly of the species in the original pool that best binds to the target. In Irvine et al. [Irvine, D., Tuerk, C., Gold, L., 1991. SELEXION, systematic evolution of nucleic acids by exponential enrichment with integrated optimization by non-linear analysis. J. Mol. Biol. 222, 739-761] a mathematical analysis of this process was given. In this paper we revisit Irvine et al. [Ibid]. By rewriting the equations for the SELEX process, we considerably reduce the labor of computing the round to round distribution of nucleic acid fractions. We also establish necessary and sufficient conditions for the SELEX process to converge to a pool consisting solely of the best binding nucleic acid to a fixed target in a manner that maximizes the percentage of bound target. The assumption is that there is a single nucleic acid binding site on the target that permits occupation by not more than one nucleic acid. We analyze the case for which there is no background loss (no support losses and no free [NA] left on the support). We then examine the case in which such there are such losses. The significance of the analysis is that it suggests an experimental approach for the SELEX process as defined in Irvine et al. [Ibid] to converge to a pool consisting of a single best binding nucleic acid without recourse to any a priori information about the nature of the binding constants or the distribution of the individual nucleic acid fragments.

Ligands↗