A steroid cap adjusts the selectivity and accelerates the rates of nonenzymatic single nucleotide extensions of an oligonucleotide.
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Base Pair Mismatch↗
Biomedical subjects
C Richert
Publications and source records attributed to C Richert.
Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices.
A total of 16 oligodeoxyribonucleotides of general sequence 5'-TCTTCTZTCTTTCT-3', where Z denotes an N-acyl-N-(2-hydroxyethyl)glycine residue, were prepared via solid phase synthesis. The ability of these oligonucleotides to form triplexes with the duplex 5'-AGAAGATAGAAAGA-HEG-TCTTTCTATCTTCT-3', where HEG is a hexaethylene glycol linker, was tested. In these triplexes, an 'interrupting' T:A base pair faces the Z residue in the third strand. Among the acyl moieties of Z tested, an anthraquinone carboxylic acid residue linked via a glycinyl group gave the most stable triplex, whose UV melting point was 8.4 degrees C higher than that of the triplex with 5'-TCTTCTGTCTTTCT-3' as the third strand. The results from exploratory nuclease selection experiments suggest that a combinatorial search for strands capable of recognizing mixed sequences by triple helix formation is feasible.
Base Pairing↗
Synthesis and nuclease stability of trilysyl dendrimer-oligodeoxyribonucleotide hybrids.
Hybrids of oligonucleotides and trilysyl-dendrimers with terminal acyl groups were prepared via solid-phase synthesis, including a DNA hexamer bearing an additional 3'-appendage. These were shown to be degraded more slowly by nuclease S1 than control strands, particularly at low pH, and, in one case, to form a duplex with a complementary strand whose melting point at pH 7 was higher than that of the control duplex.
Hydrogen-Ion Concentration↗
A 2'-acylamido cap that increases the stability of oligonucleotide duplexes.
[structure: see text]. Reported here is the synthesis of oligonucleotides containing a 2'-acylamido-2'-deoxyuridine residue at their 3'-terminus. Compared to control sequences bearing a thymidine residue, the quinolone-capped duplexes give higher UV melting points. In the case of (5'-ACGCGU-NA-2')2, where NA denotes a nalidixic acid residue, the melting point increase is up to 22 degrees C over that of (ACGCGT)2.
Anti-Infective Agents↗
Synthesis and monitored selection of 5'-nucleobase-capped oligodeoxyribonucleotides.
Oligodeoxynucleotides bearing 5'-appendages consisting of a nucleobase and an amide linkage were prepared from 5'-amino-5'-deoxyoligonucleotides, amino acid building blocks and thymine or uracil derivatives. Small chemical libraries of 5'-modified oligonucleotides bearing the nucleobase moieties via five, three or two atom linkages were subjected to spectrometrically monitored nuclease selections to identify members with high affinity for target strands. The smallest of the appendages tested, a uracil acetic acid substituent, was found to convey the greatest duplex stabilizing effect on the octamer 5'-T*GGTTGAC-3', where T* denotes the 5'-amino-5'-deoxythymidine residue. Compared to 5'-TTGGTTGAC-3', the modified sequence 5'-u-T*GGTTGAC-3' gives a duplex with 5'-GTCAACCAA-3' that melts 4 degrees C higher. The duplex-stabilizing effect of this 5'-substituent does not require a specific residue at the 3'-terminus of the complement and the available data suggest that the uracil moiety is located in the major groove of the duplex.
Acetic Acid↗
Solid-phase synthesis of cyclic peptide-DNA hybrids.
[reaction: see text] A methodology for preparing cyclic peptide-DNA hybrids on controlled pore glass in high yield is reported. This methodology employs Fmoc/Alloc-protected amino acid and nucleoside phosphoramidites on an omega-hydroxylauric acid-derivatized support and is suitable for library synthesis. A cyclic hybrid of the sequence Glu-Leu-TT-DP-Lys, where Glu and Lys are linked and T denotes a 5'-amino-5'-deoxynucleotide, exhibited high resistance to exo- and endonucleases.
Animals↗
Rapid genotyping by MALDI-monitored nuclease selection from probe libraries.
Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with individual genes. Efficient technologies for rapidly detecting SNPs will therefore facilitate the mining of genomic information. Known methods for SNP analysis include restriction-fragment-length polymorphism polymerase chain reaction (PCR), allele-specific oligomer hybridization, oligomer-specific ligation assays, minisequencing, direct sequencing, fluorescence-detected 5'-exonuclease assays, and hybridization with PNA probes. Detection by mass spectrometry (MS) offers speed and high resolution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products, mass-tagged oligonucleotides, DNA created by restriction endonuclease cleavage, and genomic DNA. We have previously reported MALDI-TOF-monitored nuclease selections of modified oligonucleotides with increased affinity for targets. Here we use nuclease selections for genotyping by treating DNA to be analyzed with oligonucleotide probes representing known genotypes and digesting probes that are not complementary to the DNA. With phosphodiesterase I, the target-bound, complementary probe is largely refractory to nuclease attack and its peak persists in mass spectra (Fig. 1A). In optimized assays, both alleles of a heterozygote were genotyped with six nonamer DNA probes (> or = 125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).
Alleles↗
Solution structure of the aminoacyl-capped oligodeoxyribonucleotide duplex (W-TGCGCAC)(2).
Reported here is the solution structure of the aminoacyl-DNA duplex (W-TGCGCAC)(2). This duplex forms a continuously pi-stacked helix consisting of both nucleobases and amino acid side chains. According to NMR and UV analyses, the duplex melts in a cooperative transition and with 1.3-1.8% greater hyperchromicity than the control duplex (TGCGCAC)(2). A van't Hoff analysis of UV melting points at different concentrations shows that the two tryptophan residues contribute 4.8 kcal/mol to the DeltaH degrees of complex formation at 10 mM salt concentration and less than 1 kcal/mol at 150 mM salt. The entropic cost for duplex association in the presence of the amino acid residues is 13 cal/molK greater than that for the control at 10 mM salt concentration, and 3 cal/molK lower than that of the control at 0.15 ionic strength. The conformation of W-TGCGCAC in duplex form, determined via restrained torsion angle molecular dynamics, shows an undisturbed B-form DNA duplex with dangling 3'-termini. The tryptophanyl residue at the 5'-terminus packs tightly against T2 and the proximal part of adenine, without engaging in hydrogen bonding. While not providing strong enthalpic net stabilization of the duplex, the tryptophan "cap" on the duplex does seem to reduce the fraying at the termini, indicating a subtle balance of entropic and enthalpic factors contributing to the molecular dynamics. The structure also shows that, at least in the present sequence context, stacking on the terminal base pair is more favorable than intercalation, probably because the enthalpic cost associated with breaking up the stacking between DNA base pairs cannot be paid for by favorable pi-stacking interactions with the indole ring of tryptophan. These results are of importance for understanding stacking interactions in protein-DNA complexes, particularly those in enzyme-substrate complexes involving exposed nucleobases.
Base Sequence↗
5'-Peptidyl substituents allow a tuning of the affinity of oligodeoxyribonucleotides for RNA.
The affinity of amide-linked 5'-aminoacyl and 5'-dipeptidyl DNA octamers for two RNA undecamers with 3'-overhangs was measured via UV melting analysis. A sequence-dependent increase in melting points was observed. At low ionic strength, two appended lysine residues elevate melting points more than two additional A:U base pairs.
Circular Dichroism↗
Monitoring the hybridization of the components of oligonucleotide mixtures to immobilized DNA via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Reported here is how the hybridization of individual components of oligonucleotide mixtures to solid-phase bound complementary strands can be monitored simultaneously by quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Three oligonucleotides, a DNA heptamer, a DNA octamer and a DNA octamer with a terminal cholic acid appendage were used as the test mixture. Upon cooling in the presence of a complementary undecamer on controlled pore glass, depletion of the components from the solution was observed. The resulting hybridization curves show the same relative affinities as traditional UV melting curves with single components and their complement. Assays of the kind described here may be used to select high affinity binders from combinatorial libraries of modified antisense oligonucleotides.
Calibration↗
Are porphyrin mixtures favorable photodynamic anticancer drugs? A model study with combinatorial libraries of tetraphenylporphyrins.
Reported here is the preparation of tetraphenylporphyrin libraries via efficient combinatorial solution-phase syntheses, their purification, and preliminary results from a bioorganic study on their uptake in liposome membranes. Libraries with up to 666 components were prepared with substituents including Br, CF3, Cl, CN, CO2Me, Et, F, OAc, and Ph. Further, a first example for the synthesis of more diverse libraries via a "latent libraries" approach is presented. This involves masking polar groups with lipophilic protecting groups. After purification of the latent library, the masking protecting groups are removed in a quantitative reaction that produces the library compounds as the only non-volatile components. Libraries were characterized by laser desorption time-of-flight mass spectrometry, NMR, and UV-vis spectroscopy. In vitro uptake into membranes of small sonicated liposomes was measured, both in terms of total porphyrin incorporation and in terms of structure-incorporation relationships. The latter were determined from isotopically-resolved laser-desorption mass spectra under conditions that yield quantitative results. Smaller libraries showed increased uptake of porphyrins bearing OH and CF3 substituents and lower uptake of ester-, alkyl-, and halide-bearing porphyrins. This structure-dependent selectivity disappears for larger libraries, however, where uniformly high uptake is observed, i.e., at a constant lipid:porphyrin ratio the total porphyrin incorporation is higher for libraries than for single compounds of similar polarity. We propose that the decreased concentration of individual compounds in large libraries is responsible for this effect. Membrane incorporation has previously been shown to correlate with photodynamic activity in vitro and in vivo.16 Therefore, these results may help to explain why photodynamic therapy of tumors, a modern anti-cancer treatment modality, is successfully performed with a complex mixture of porphyrins.
Antineoplastic Agents↗
Redesigning nucleic acids.
A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.
Catalysis↗
Targeting of the tumor microcirculation by photodynamic therapy with a synthetic porphycene.
9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) is a chemically pure substance with fast pharmacokinetics and superior photodynamic properties in vitro as compared to Photofrin. In this study the pharmacokinetics, photodynamic efficacy and tissue localization of ATMPn were investigated in vivo. Amelanotic melanomas (A-Mel-3) were implanted in dorsal skin fold chambers fitted to Syrian Golden hamsters. Fluorescence kinetics of ATMPn (1.4 mumol kg-1 b.w.i.v.; n = 8) were monitored by intravital microscopy. Quantitative measurements of fluorescence intensity were carried out by digital image analysis. For tumor growth studies 1.4 mumol kg-1 was injected 24 h (n = 3), 3 h (n = 3), 1 min (n = 6) and 2.8 mumol kg-1 1 min (n = 6) before PDT (Laser (630 nm) or lamp (600-750 nm), 100 mW cm-2, 100 J cm-2). Tumor volume was measured for 28 d. Solid tumors (n = 3) were excised 1 min after injection of ATMPn (2.8 mumol kg-1) and cryostat sections (20 mm) were analyzed by confocal laser scanning microscopy (CLSM) for tissue localization of the dye. Maximal fluorescence (mean +/- S.E.) arose in the tumor (94 +/- 7%) and surrounding host tissue (67 +/- 5%) 30 s post injection followed by a rapid decrease. Hardly any fluorescence was detectable 12 h after administration. Only PDT 1 min after injection of ATMPn was effective yielding 3/6 complete remissions (2.8 mmol kg-1, laser) and 6/6 complete remissions (2.8 mumol kg-1, lamp), respectively. One minute after injection the dye is primarily localized in the vascular wall of normal and tumor vessels as shown by CLSM. PDT at a time, when the dye is localized primarily in the tumor microcirculation, exhibits the best tumor killing effects showing that vascular targeting is effective in treating solid malignant tumors. ATMPn in liposomes makes administration and light irradiation in one session possible due to its fast pharmacokinetics. Thus, using ATMPn as a photosensitizer may provide more flexibility to perform PDT after surgical exploration and debulking as adjuvant therapy.
Animals↗
Spectrometrically monitored selection experiments: quantitative laser desorption mass spectrometry of small chemical libraries.
BACKGROUND: Selection experiments involving chemical libraries are routinely used in the pharmaceutical industry for finding and optimizing lead compounds. In principle, almost any process involving a binding event or a reaction could be probed systematically with chemical libraries prepared by combinatorial synthesis. Traditionally, however, the vast majority of library members cannot be monitored during the selection, making a systematic correlation of structure and activity difficult. To interpret selection experiments on the level of all library components, monitoring technologies are required that give a unique and quantitative spectroscopic signal for every compound in a mixture. RESULTS: Quantitative matrix-assisted laser desorption mass spectrometry of libraries of porphyrins and peptide-DNA hybrids consisting of 2-35 compounds is described. Porphyrin libraries were subjected to in vitro selections for liposome incorporation and binding to a protein pocket. It was shown that mesohydroxyphenyl substituted porphyrins, known high activity photosensitizers of tumors, are preferentially incorporated in liposome membranes. A mixture of peptide-DNA hybrids was assayed for the nuclease stability of its components. CONCLUSIONS: Small libraries of non-isobaric compounds can be exhaustively or near-exhaustively monitored by mass spectrometry. Monitored selection experiments can yield detailed structure-activity maps in a single experiment, speeding up drug discovery and the probing of biochemically relevant recognition events. It is proposed that monitored assays for target binding, membrane partitioning, and biostability could be run in parallel, to select drug candidates combining several favorable properties in 'multidimensional' selection experiments.
DNA↗
Analysis of chronic rejection and obliterative arteriopathy. Possible contributions of donor antigen-presenting cells and lymphatic disruption.
Sequential analysis of changes that lead to chronic rejection was undertaken in an animal model of chronic rejection and obliterative arteriopathy. Brown Norway rats are pretreated with a Lewis bone marrow infusion or a Lewis orthotopic liver allograft and a short course of immunosuppression. They are challenged 100 days later with a Lewis heterotopic heart graft without immunosuppression. The heart grafts in both groups undergo a transient acute rejection, but all rats are operationally tolerant; the heart grafts are accepted and remain beating for more than 100 days. Early arterial remodeling, marked by arterial bromodeoxyuridine incorporation, occurred in both groups between 5 and 30 days during the transient acute rejection. It coincided with the presence of interstitial (but not arterial intimal) inflammation and lymphatic disruption and resulted in mild intimal thickening. Significant arterial narrowing occurred only in the bone-marrow-pretreated rats between 60 and 100 days. It was associated with T lymphocyte and macrophage inflammation of the heart graft that accumulated in the endocardium and arterial intima and adventitia near draining lymphatics. There also was loss of passenger leukocytes from the heart graft, up-regulation of cytokine mRNA and major histocompatibility class II on the endothelium, and focal disruption of lymphatics. In contrast, long-surviving heart grafts from the Lewis orthotopic liver allograft pretreated group are near normal and freedom from chronic rejection in this group was associated with persistence of donor major histocompatibility class-II-positive hematolymphoid cells, including OX62+ donor dendritic cells. This study offers insights into two different aspects of chronic rejection: 1) possible mechanisms underlying the persistent immunological injury and 2) the association between immunological injury and the development of obliterative arteriopathy. Based on the findings, it is not unreasonable to raise the testable hypothesis that direct presentation of alloantigen by donor antigen-presenting cells is required for long-term, chronic-rejection-free allograft acceptance. In addition, chronic intermittent lymphatic disruption is implicated as a possible mechanism for the association between chronic interstitial allograft inflammation and the development of obliterative arteriopathy.
Animals↗
Continuing medical education on the World Wide Web (WWW). Interactive pathology case studies on the Internet.
OBJECTIVE: To present interactive online continuing medical education (CME) over the World Wide Web as a more efficient alternative to traditional modes of CME delivery. DESIGN: A departmental Web site available to those with access to the Internet. SETTING: A tertiary-care teaching hospital in the United States. RESULTS: Comprehensive case studies have been developed and are complete with images, text, and questions, including explanations for correct and incorrect responses. The images are linked to pertinent text to maximize their educational value. The cases are easily accessible, user friendly, and fully referenced. The system became operational in January 1996, and the first CME certificate was awarded to a participant shortly thereafter. CONCLUSIONS: Continuing medical education over the World Wide Web is an efficient means of delivering CME to the community at large; it allows participating physicians the latitude to obtain convenient CME credit at their leisure, in contrast to the regimented experience of formal CME conferences or symposiums. The interactive format of the CME cases allows the participant to submit immediate comments or criticism to case authors and receive instant feedback on their own performance, features unavailable in comparable educational software packages. The dynamic environment of the World Wide Web lends itself to the production and dissemination of such flexible forms of CME for the physician and will continue to expand in this capacity into the foreseeable future.
Certification↗
Photodynamic eradication of amelanotic melanoma of the hamster with fast acting photosensitizers.
Three porphycenes with fast pharmacokinetics were tested for their ability to photosensitize amelanotic hamster melanoma A-Mel-3 at short time intervals after injection. Laser light irradiation was performed at the time of maximal photosensitizer level in tumor tissue. Photodynamic therapy as short as 5 min after injection led to complete local tumor remission at a dosage of 1.4 mumol/kg for the porphycene CBPn. In comparison, Photofrin required 8.4 mumol/kg for local tumor remission in 5 of 6 animals with 24 hr accumulation time after injection. We propose a swift photodynamic protocol which can compete favorably with conventional techniques of tumor treatment.
Animals↗
X-ray crystal structure of a dimethylene sulfone-bridged ribonucleotide dimer in a single-stranded state.
A crystal structure has been solved for an analog of the r(ApU) ribodinucleotide, r(Aso2U), where a bridging non-ionic dimethylene sulfone linker replaces the phosphodiester linking group found in natural RNA. Crystals of the single-stranded state of r(Aso2U) were obtained from water at 50 degrees C. In these crystals, one hydrogen bond is formed between bases from different strands and base stacking occurs in intermolecular 'homo-A' and 'homo-U' stacks. Similar to typical oligoribonucleotides, the ribose rings adopt N-type conformations and dihedral angles chi are in the anti range. The all-trans rotamer of the CH2-SO2-CH2-CH2 bridge was found, which leads to a large adenine-uracil distance. Qualitative analysis of a NOESY spectrum of the Aso2U part in r(Uso2Cso2Aso2U) dissolved in a dimethylsulfoxide-D2O mixture indicates that the conformation observed in the crystal is also populated in solution. Comparison with the structure of r(Gso2C), which has been crystallized in the Watson-Crick paired state, shows that a rotation around zeta by +112 degrees leads from the observed, single-stranded state to a conformation that is compatible with formation of a duplex. A concerted trans/gauche flip of alpha and gamma then yields the standard conformer of A-type RNA helices. From the observed structure of r(Gso2C) and other oligonucleotides it is anticipated that this flip will also revert the ribose pucker from C2'-exo to C3'-endo.
Adenine↗