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PubMed1998

Design of a combinatorial oligonucleotide library containing all possible hexamer palindromes: PCR synthesis and application for identifying restriction cleavage sites.

An algorithm for designing a combinatorial library comprehensively representing all hexamer palindrome sequences at uniquely defined sites is described. The expected size for such a library of 64 possible hexamer palindromes is 384 bases, which is reduced to 266 bases spread over 8 oligonucleotides through a linear overlap of rationally selected hexamer palindromes. The single stranded oligonucleotides of the designed sets were chemically synthesized and converted into corresponding duplex dimers using PCR primer-dimer method. The utility of these duplex oligomers for identifying cleavage sites of restriction enzymes recognizing hexamer palindromes has been demonstrated using some representative enzymes. The library is also useful for screening restriction enzymes with tetramer cleavage sites and identifying the "star" sites of restriction enzymes. The sets of oligonucleotides with high information content, though designed for direct and unambiguous characterization of cleavage sites of isolated restriction enzymes, have potential applications as templates for characterizing sequence selective binding and interaction of small molecules nucleic acid.

Algorithms↗
PubMed2003

Chemoenzymatic route to macrocyclic hybrid peptide/polyketide-like molecules.

Hybrid peptide-polyketides are a class of medically and biologically important natural products characterized by stereochemical and functional diversity. In their biosynthesis, hybrids are often macrocyclized to achieve rigid structures that populate bioactive conformations. We herein present a chemoenzymatic strategy to access the stereochemical and functional diversity found in macrocyclic hybrid natural products in a manner amenable to efficient library synthesis. Our method makes use of small building blocks in the form of Fmoc-protected epsilon-amino acids containing embedded polyketide functionality. The building block approach allows for combinatorial synthesis of linear molecules that can be activated as soluble thioesters or tethered to a solid-phase resin. We demonstrate that these linear molecules are substrates for macrocyclization by a tolerant catalyst, TycC TE, derived from a nonribosomal peptide synthetase. The method should allow for access to diverse structures with hybrid peptide-polyketide character that can be screened for improved or novel activities.

Amides↗
PubMed1984

Sequence repetition and genomic distribution of small polydisperse circular DNA purified from HeLa cells.

Covalently closed circular DNA molecules (cccDNA) from the human HeLa cell line were purified (96% pure by weight) by use of ATP-dependent deoxyribonuclease, and cloned into the HindIII site of phage lambda vector Charon 7. From the cccDNA library thus obtained, nine recombinants carrying mitochondrial DNA and 36 recombinants carrying small polydisperse circular (spc) DNA were picked at random for subsequent tests. The inserted fragments of spcDNA ranged in size from 0.6 to 7.6 kb with a mean length of 1.9 kb, a value which is the same as the average length of spcDNA. Analysis of the cloned spcDNA fragments revealed that (a) all the spcDNA clones investigated shared homologies with chromosomal DNA sequences, (b) all but one cloned DNA contained repetitive sequences, (c) the sequence organization could be roughly classified according to the reiteration frequency as greater than 10(5) (Alu family class), 10(4) to 10(5) (KpnI family class), 10(3) to 10(4) (mitochondrial DNA class) and less than 10(3) times per haploid genome, and (d) most of the repetitive sequences were dispersed in the genome, although some appeared clustered.

Cloning, Molecular↗
PubMed2004

Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor alpha.

The cyclic peptide AF17121 (Ac-VDECWRIIASHTWFCAEE) that inhibits interleukin 5 (IL-5) function and IL-5 receptor alpha-chain (IL-5Ralpha) binding has been derived from recombinant random peptide library screening and follow-up synthetic variation. To better understand the structural basis of its antagonist activity, AF17121 and a series of analogs of the parent peptide were prepared by solid phase peptide synthesis. Sequence variation was focused on the charged residues Asp(2), Glu(3), Arg(6), Glu(17), and Glu(18). Two of those residues, Glu(3) and Arg(6), form an EXXR motif that was found to be common among library-derived IL-5 antagonists. The E and R in the EXXR motif have a proximity similar to charged residues in a previously identified receptor alpha binding region, the beta-strand between the C- and D-helices of human IL-5. Optical biosensor interaction kinetics and cell proliferation assays were used to evaluate the antagonist activities of the purified synthetic peptides, by measuring competition with the highly active single chain IL-5. Analogs in which acidic residues (Asp(2), Glu(3), Glu(17), and Glu(18)) were replaced individually by Ala retained substantial competition activity, with multiple replacements in these residues leading to fractional loss of potency at most. In contrast, R6A analogs had strongly reduced competition activity. The results reveal that the arginine residue is crucial for the IL-5Ralpha binding of AF17121, while the acidic residues are not essential though likely complex-stabilizing particularly in the Asp(2)-Glu(3) region. By CD, AF17121 exhibited mostly disordered structure with evidence for a small beta-sheet content, and replacement of the arginine had no influence on the observed secondary structure of the peptides. The dominance of Arg(6) in AF17121 activity corresponds to previous findings of dominance of the positive charge balance in the antiparallel beta-sheet of IL-5 composed of (88)EERRR(92) in one strand of the CD turn region of IL-5 and with Arg(32) in the neighboring beta-strand. These results argue that AF17121 and related library-derived peptides function by mimicking the CD turn receptor alpha recognition epitope in IL-5 and open the way to small molecule antagonist design.

Amino Acid Sequence↗
PubMedDate not supplied

Using high-throughput screening data to discriminate compounds with single-target effects from those with side effects.

The most desirable compound leads from high-throughput assays are those with novel biological activities resulting from their action on a single biological target. Valuable resources can be wasted on compound leads with significant 'side effects' on additional biological targets; therefore, technical refinements to identify compounds that primarily have effects resulting from a single target are needed. This study explores the use of multiple assays of a chemical library and a statistic based on entropy to identify lead compound classes that have patterns of assay activity resulting primarily from small molecule action on a single target. This statistic, called the coincidence score, discriminates with 88% accuracy compound classes known to act primarily on a single target from compound classes with significant side effects on nonhomologous targets. Furthermore, a significant number of the compound classes predicted to have primarily single-target effects contain known bioactive compounds. We also show that a compound's known biological target or mechanism of action can often be suggested by its pattern of activities in multiple assays.

Drug-Related Side Effects and Adverse Reactions↗
PubMed2002

A small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells.

Purmorphamine, which is a 2,6,9-trisubstituted purine compound, was discovered through cell-based high-throughput screening from a heterocycle combinatorial library. It differentiates multipotent mesenchymal progenitor cells into an osteoblast lineage. It will serve as a unique chemical tool to study the molecular mechanisms of osteogenesis of stem cells and bone development.

Alkaline Phosphatase↗
PubMed2004

Translation of DNA into synthetic N-acyloxazolidines.

The translation of DNA into synthetic molecules enables their manipulation by powerful evolution-based methods previously available only to proteins and nucleic acids. The development of increasingly sophisticated DNA-templated small-molecule syntheses is crucial to broadening the scope of this approach. Here, we report the translation of DNA templates into monocyclic and bicyclic N-acyloxazolidines using multistep DNA-templated organic synthesis. Second-generation template architectures, used for the first time in a multistep DNA-templated synthesis, together with reactions and linker cleavage strategies not previously described in a DNA-templated format, were crucial to the successful translation. The products generated in this work represent the most complex small molecules to date synthesized in a DNA sequence-programmed manner and provide the basis for DNA-templated synthetic heterocycle libraries.

Acylation↗
PubMed2004

Structural interaction fingerprint (SIFt): a novel method for analyzing three-dimensional protein-ligand binding interactions.

Representing and understanding the three-dimensional (3D) structural information of protein-ligand complexes is a critical step in the rational drug discovery process. Traditional analysis methods are proving inadequate and inefficient in dealing with the massive amount of structural information being generated from X-ray crystallography, NMR, and in silico approaches such as structure-based docking experiments. Here, we present SIFt (structural interaction fingerprint), a novel method for representing and analyzing 3D protein-ligand binding interactions. Key to this approach is the generation of an interaction fingerprint that translates 3D structural binding information from a protein-ligand complex into a one-dimensional binary string. Each fingerprint represents the "structural interaction profile" of the complex that can be used to organize, analyze, and visualize the rich amount of information encoded in ligand-receptor complexes and also to assist database mining. We have applied SIFt to tackle three common tasks in structure-based drug design. The first involved the analysis and organization of a typical set of results generated from a docking study. Using SIFt, docking poses with similar binding modes were identified, clustered, and subsequently compared with conventional scoring function information. A second application of SIFt was to analyze approximately 90 known X-ray crystal structures of protein kinase-inhibitor complexes obtained from the Protein Databank. Using SIFt, we were able to organize the structures and reveal striking similarities and diversity between their small molecule binding interactions. Finally, we have shown how SIFt can be used as an effective molecular filter during the virtual chemical library screening process to select molecules with desirable binding mode(s) and/or desirable interaction patterns with the protein target. In summary, SIFt shows promise to fully leverage the wealth of information being generated in rational drug design.

Binding Sites↗
PubMed2006

A pooling-deconvolution strategy for biological network elucidation.

The generation of large-scale data sets is a fundamental requirement of systems biology. But despite recent advances, generation of such high-coverage data remains a major challenge. We developed a pooling-deconvolution strategy that can dramatically decrease the effort required. This strategy, pooling with imaginary tags followed by deconvolution (PI-deconvolution), allows the screening of 2(n) probe proteins (baits) in 2 x n pools, with n replicates for each bait. Deconvolution of baits with their binding partners (preys) can be achieved by reading the prey's profile from the 2 x n experiments. We validated this strategy for protein-protein interaction mapping using both proteome microarrays and a yeast two-hybrid array, demonstrating that PI-deconvolution can be used to identify interactions accurately with fewer experiments and better coverage. We also show that PI-deconvolution can be used to identify protein-small molecule interactions inferred from profiling the yeast deletion collection. PI-deconvolution should be applicable to a wide range of library-against-library approaches and can also be used to optimize array designs.

Computational Biology↗
PubMed1999

Cytotoxic hammerhead ribozymes.

Small catalytic RNA molecules of the hammerhead ribozyme type were found to have cytotoxic effects unrelated to their intended activity. An expression library of ribozyme sequence variants was constructed in a recA-deficient strain of Escherichia coli such that individual library members differed in regions designed to form base pairs with human immunodeficiency virus-1 (HIV-1) tat mRNA. The parental ribozyme and many variants exhibited a bacteriostatic effect. One variant studied in detail was also bactericidal. When its expression was induced, ribozyme-dependent inhibition of bacterial growth was not observed in recA+ or recA+ lexA3 (Ind-) cells, suggesting that the recombination function of the RecA protein, not the absence of the SOS response, is sufficient to alleviate the cytotoxic effect. These data document the need for careful testing for toxic effects during intracellular studies of ribozyme action.

Base Sequence↗
PubMedDate not supplied

Nucleic acid aptamers in human viral disease.

Nucleic acid aptamers are short, single-stranded oligonucleotides or their modified analogues which avidly and specifically interact with targeted ligands through their 3-dimensional structure. Aptamers can be selected out of a large combinatorial oligonucleotide library through an in vitro evolution process termed SELEX. Since 1990, a wide variety of aptamers targeted to ligands ranging from small molecules to complex mixtures have been isolated. Most selected aptamers have shown high specificity to and affinity for their ligands and are potential detection and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, resulting in potent therapeutic candidates in disease models. Some recent advances to increase the stability of aptamers, extend their in vivo circulation time and their in vivo expression have pushed aptamers closer to therapeutic applications. This review presents recent developments in the field of aptamer research and focuses on their applications to human viral diseases, particularly HIV induced diseases.

HIV Infections↗
PubMed2004

Dual surface selection methodology for the identification of thrombin binding epitopes from hotspot biased phage-display libraries.

Protein libraries biased towards amino-acid residues found at so-called 'hotspots' were incorporated into the beta-sheet region of the thermostable variant (HTB1) of the B1 domain of the immunoglobulin (IgG) binding protein G and expressed as gene 3 fusions on M13 bacteriophage. The HTB1 library (2.2 x 10(9)) variants with a minimal 12 amino acid basis set were selected for binding IgG, to ensure structural conservation, and subsequently to thrombin to evolve a thrombin-binding function. We believe that this dual surface selection strategy will have great utility in evolving new bi-functional proteins without compromising structure. Furthermore the discrete beta-sheet epitopes identified by our methodology will lend itself to small-molecule mimicry of beta-sheets.

Amino Acid Sequence↗
PubMed1992

CD24, a signal-transducing molecule expressed on human B cells, is a major surface antigen on small cell lung carcinomas.

Cell lines derived from human small cell carcinoma of the lung express high levels of a surface polypeptide termed the cluster-w4 antigen, which was previously identified as a potential target for toxin-based immunotherapy of lung cancer. We have cloned a complementary DNA encoding the cluster-w4 antigen from COS-1 fibroblasts transfected with a SW2 small cell carcinoma library, by panning with a mixture of the cluster-w4-specific monoclonal antibodies SWA11, SWA21, and SWA22. The sequence of the cluster-w4 complementary DNA encodes an unusually short (80-amino acid) protein identical to that recently reported for the leukocyte activation molecule CD24 except for a single valine-alanine substitution due to a single-base polymorphism within the region of the gene coding for the extracellular domain. Biochemical analyses of the cloned cluster-w4 antigen confirmed both the presence of the phosphatidylinositol tail and the extensive glycosylation reported for the CD24 molecule. Furthermore, the cloned cluster-w4 antigen expressed on COS cells was shown to react with a comprehensive panel of CD24-specific monoclonal antibodies, as assessed by indirect immunofluorescence staining. Northern blot hybridization indicated the presence of several transcript sizes for the cluster-w4 antigen that were greatly overexpressed in small cell carcinoma cell lines, compared with normal hemopoietic cells and CD24-positive cell lines. Southern blot hybridization of restriction digests of genomic DNA identified a complex pattern of bands consistent with either a complex gene structure containing many exons or the presence of a family of closely related genes.

Alanine↗
PubMed1997

Comparison of fusion phage libraries displaying VH or single-chain Fv antibody fragments derived from the antibody repertoire of a vaccinated melanoma patient as a source of melanoma-specific targeting molecules.

A single-chain Fv (scFv) fusion phage library derived from random combinations of VH and VL (variable heavy and light chains) domains in the antibody repertoire of a vaccinated melanoma patient was previously used to isolate clones that bind specifically to melanoma cells. An unexpected finding was that one of the clones encoded a truncated scFv molecule with most of the VL domain deleted, indicating that a VH domain alone can exhibit tumor-specific binding. In this report a VH fusion phage library containing VH domains unassociated with VL domains was compared with a scFv fusion phage library as a source of melanoma-specific clones; both libraries contained the same VH domains from the vaccinated melanoma patient. The results demonstrate that the clones can be isolated from both libraries, and that both libraries should be used to optimize the chance of isolating clones binding to different epitopes. Although this strategy has been tested only for melanoma, it is also applicable to other cancers. Because of their small size, human origin and specificity for cell surface tumor antigens, the VH and scFv molecules have significant advantages as tumor-targeting molecules for diagnostic and therapeutic procedures and can also serve as probes for identifying the cognate tumor antigens.

Amino Acid Sequence↗
PubMed2026

Ligand-Mediated Reprogramming Redirects Liver-Tropic Ionizable Lipid Nanoparticles for Lung-Selective mRNA Delivery.

Systemic delivery of messenger RNA (mRNA) to target tissues and cells using lipid nanoparticles (LNPs) holds transformative potential for gene therapy. However, most clinically validated LNP exhibit strong liver tropism, and redirecting their organ specificity without redesigning entirely new chemistries remains challenging. Here we present a ligand-mediated lipid reprogramming approach that repurposes chemically defined, liver-tropic, ionizable lipids (lipidoids) for mRNA delivery beyond the liver. From a library of 90 degradable lipidoids, we identified 2-t6b as a potent liver-targeting platform. By site-specific displaying of small molecule ligands onto 2-t6b headgroup, we engineered a series of reconfigured lipidoids that achieve lung-specific targeting while retaining the parent delivery scaffold. Ligand7-2-t6b-lipid-functionalized LNP achieved over 200-fold higher mRNA translation in the lungs compared to the parent liver-tropic LNP. Proteomics and molecular docking analysis revealed enhanced binding of the modified lipid to vitronectin, a serum glycoprotein that improves integrin binding and thus promotes cellular uptake and translation efficiency. Ligand-mediated 2-t6b/ligand7 LNPs achieved outperformed efficacy and therapeutic potential in lung-specific genome editing relative to SORT-constructed 2-t6b LNP system. Our modular reprogramming strategy provides a generalizable framework to upgrade existing liver-biased LNPs into lung-selective mRNA carriers, advancing next-generation tissue-specific mRNA therapies for gene editing, protein replacement therapy, and regenerative medicine.

RNA, Messenger↗
PubMed2006

Identification of new compounds that trigger apoptosome-independent caspase activation and apoptosis.

Identification of alternative pathways of caspase activation is an important step to develop new antitumor treatments. We report here the result of a screening with a small chemical library, the Developmental Therapeutics Program-National Cancer Institute "challenge set," on cells expressing mutated caspase-9. We have identified two molecules capable of activating an apoptosome-independent apoptotic pathway. These compounds, named F6 and G5, target the ubiquitin-proteasome system by inhibiting the ubiquitin isopeptidases. We have shown that F6 and G5 induce a rather unique apoptotic pathway, which includes a Bcl-2-dependent but apoptosome-independent mitochondrial pathway with up-regulation of the BH3-only protein Noxa, stabilization of the inhibitor of apoptosis antagonist Smac, but also the involvement of the death receptor pathway. Noxa plays an important role in the induction of mitochondrial fragmentation and caspase activation, whereas the death receptor pathway becomes critical in the absence of a functional apoptosome. This study suggests that screening of chemical libraries on cancer cells with defined mutations in apoptotic key elements can lead to the identification of compounds that are useful to characterize alternative pathways of caspase activation.

Apoptosis↗
PubMed1985

cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen.

Sera from patients with systemic lupus erythematosus and other autoimmune disorders contain antibodies against nuclear proteins. One such autoantibody system, known as Sm, reacts with antigens associated with small nuclear RNA molecules. In this paper we report the use of Sm autoantibodies to isolate a cDNA clone for the mRNA of one of these nuclear antigens. A HeLa cell cDNA library was screened by message selection followed by autoantibody reaction of cell-free translation products. This led to the identification of a cDNA clone, p281, containing sequences complementary to mRNA for an Sm autoantibody-reactive, 11,000 Mr protein. This cloned Sm antigen comigrated with the small nuclear RNA-associated protein known as "E" and reacted with four out of four Sm autoantibodies that precipitate E protein from total mRNA translation products. RNA gel blot hybridization with clone p281 DNA revealed a poly(A)+ mRNA of approximately equal to 600 nucleotides in human and marmoset (New World primate) cells. Southern blot hybridization of HeLa cell and human lymphocyte DNA indicated the presence of 6-10 copies of p281-homologous sequences. Similar copy numbers were observed with genomic DNA from baboon, cat, and mouse, indicating that the Sm antigen mRNA sequence represented in p281 is conserved across three classes of the Mammalia (primates, carnivores, and rodents). However, no cross-hybridization of p281 was observed with frog or Drosophila DNA. In light of existing evidence that the mammalian Sm antigen E is a weaker autoantigen than other small nuclear RNA-associated proteins, these results suggest a possible correlation between a protein's capacity to serve as an autoantigen during breakdown of the host's immunological tolerance and its extent of evolutionary conservation, whereas the inverse relationship applies to conventional immunity. We suspect, as have others, that this is a clue to the mechanism of autoimmunity.

Antigens↗
PubMed2002

Experimental RNomics: identification of 140 candidates for small non-messenger RNAs in the plant Arabidopsis thaliana.

BACKGROUND: Genomes from all organisms known to date express two types of RNA molecules: messenger RNAs (mRNAs), which are translated into proteins, and non-messenger RNAs, which function at the RNA level and do not serve as templates for translation. RESULTS: We have generated a specialized cDNA library from Arabidopsis thaliana to investigate the population of small non-messenger RNAs (snmRNAs) sized 50-500 nt in a plant. From this library, we identified 140 candidates for novel snmRNAs and investigated their expression, abundance, and developmental regulation. Based on conserved sequence and structure motifs, 104 snmRNA species can be assigned to novel members of known classes of RNAs (designated Class I snmRNAs), namely, small nucleolar RNAs (snoRNAs), 7SL RNA, U snRNAs, as well as a tRNA-like RNA. For the first time, 39 novel members of H/ACA box snoRNAs could be identified in a plant species. Of the remaining 36 snmRNA candidates (designated Class II snmRNAs), no sequence or structure motifs were present that would enable an assignment to a known class of RNAs. These RNAs were classified based on their location on the A. thaliana genome. From these, 29 snmRNA species located to intergenic regions, 3 located to intronic sequences of protein coding genes, and 4 snmRNA candidates were derived from annotated open reading frames. Surprisingly, 15 of the Class II snmRNA candidates were shown to be tissue-specifically expressed, while 12 are encoded by the mitochondrial or chloroplast genome. CONCLUSIONS: Our study has identified 140 novel candidates for small non-messenger RNA species in the plant A. thaliana and thereby sets the stage for their functional analysis.

Arabidopsis↗