Biomedical subjects

Donald E Bergstrom

Publications and source records attributed to Donald E Bergstrom.

13 recordsLinked to original sources

PubMed2005

Efficient primer strand extension beyond oxadiazole carboxamide nucleobases.

Two oxadiazole carboxamide deoxyribonucleoside analogues are described that can be incorporated and efficiently extended by Taq DNA polymerase. The primer strand extension beyond oxadiazole nucleoside analogues occurs at rates similar to the values observed for the canonical Watson-Crick base pairs irrespective of the template nucleobase. These distinctive chemical effects in DNA polymerase extensions are attributed to the smaller size and unique electronic properties of the oxadiazole nucleobase.

Adenine↗

PubMed2005

(E)-5-[2-(methoxycarbonyl)ethenyl]cytidine as a chemical actinometer for germicidal UV radiation.

(E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine (S) was examined for use as a chemical actinometer for germicidal UV radiation. Its photoproduct, 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine (P), is strongly fluorescent with excitation and emission maxima at 330 and 385 nm, respectively. Experiments were conducted to characterize the dynamic behavior of aqueous solutions of S and P when subjected to UV radiation. UV sources used for these experiments included a low-pressure mercury lamp, a XeBr excimer lamp, and a KrCI excimer lamp; all three sources were mounted in collimating devices to provide incident beams that could be easily and accurately characterized by radiometry. These three sources each yielded essentially monochromatic outputwith characteristic wavelengths of 254, 282, and 222 nm, respectively. At practical concentrations, it was found that the absorbance of the actinometer solution was neither high enough to make the actinometer solutions optically opaque nor low enough to be optically transparent to UV. In addition, the photoproduct displayed a molar absorption coefficient that was similar in magnitude to that of the parent compound, thereby resulting in competitive absorption of UV energy between Sand Pduring irradiation. For purposes of evaluation of the results of irradiation, a mathematical model was developed to accountforthe nonideal optical characteristics of the system. The model is based on a description of local photochemical kinetics; predictions of overall reactor performance were developed by spatial and temporal integration of model results. The model was used to analyze the dynamic behavior of actinometer solutions during UV irradiation and to estimate the quantum yield for photoproduction of Pfrom S. This modeling approach is potentially applicable to other photochemical processes in which multiple compounds are present that absorb photoactive radiation; however, general application of this modeling approach to photochemical reactor systems will require inclusion of othertermsto describe relevanttransport behavior within the system.

Cytidine↗

PubMed2005

Alternative nucleic acid analogues for programmable assembly: hybridization of LNA to PNA.

Complementary locked nucleic acid (LNA) and peptide nucleic acid (PNA) hexamers bind to each other with significantly higher affinity than each binds to DNA, and with far greater affinity than DNA binds to complementary DNA. The hybridization is highly specific with a single mismatch causing decreases in T(m) values ranging from 12 (G/T) to 30 degrees C (A/A). Importantly, the hybridization of an LNA oligomer to a PNA oligomer is unaffected by the ionic strength of the buffer. These properties make the LNA/PNA pair an attractive candidate as a replacement for DNA in programmable assembly.

Buffers↗

PubMed2005

Synthesis and structural analysis of oxadiazole carboxamide deoxyribonucleoside analogs.

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2'-endo ("south") conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

Crystallography, X-Ray↗

PubMed2004

Protein-DNA footprinting by endcapped duplex oligodeoxyribonucleotides.

Oligodeoxyribonucleotides (5'-phosphorylated) of varying lengths were capped using a polyamide linker to form thermodynamically stable, endcapped DNA duplexes containing 8-14 bp. We have employed these endcapped DNA duplexes as tools to determine the DNA footprint of T4 DNA ligase. By high-performance liquid chromatography and PAGE analysis of the ligation mixtures of the endcapped DNA duplexes, we have found that by varying the lengths and the position of the nick, we can determine the minimal DNA-binding site as well as the mode of binding (symmetrical or asymmetrical binding) by the enzyme. The results of the study revealed that a 11 bp endcapped duplex was the shortest duplex effectively ligated. Dependence of ligation efficiency on nick position demonstrates that T4 DNA ligase bound asymmetrically to its DNA substrate. The use of a set of thermodynamically stable endcapped deoxyribonucleoside duplexes as a tool to elucidate the DNA footprint provides an efficient strategy for footprinting, which avoids ambiguities associated with chemical and biochemical footprinting methods.

Base Sequence↗

PubMed2004

Orthogonal base pairs continue to evolve.

Recent developments in the design and construction of unusual analogs of the natural nucleic acid bases have reached a milestone with the report (in this issue of Chemistry & Biology) of a new orthogonal base pair that allows site-specific introduction of a photo-crosslinkable modified base into an RNA molecule by T7 RNA polymerase-mediated transcription of DNA containing the base-pairing partner.

Base Pairing↗

PubMed2003

A combination chemical and enzymatic approach for the preparation of azole carboxamide nucleoside triphosphate.

Alternative substrates for DNA and RNA polymerases offer an important set of biochemical tools. Many of the standard methods for nucleoside triphosphate synthesis fail in the cases of nonpurine and nonpyrimidine nucleosides. An efficient preparation of the 5'-O-tosylates for both the deoxy- and ribonucleosides enabled preparation of the diphosphate esters by displacement with tris(tetra-n-butylammonium) pyrophosphate. Enzymatic synthesis of the azole carboxamide deoxyribonucleoside triphosphate was based on ATP as the phosphate donor, nucleoside diphosphate kinase as the catalyst, coupled with phosphoenol pyruvate (PEP) and pyruvate kinase as an ATP regeneration system. Ribonucleoside triphosphate synthesis required PEP as the phosphate donor and pyruvate kinase as the catalyst. An optimized purification procedure based upon boronate affinity gel was developed to yield highly purified nucleoside triphosphates. The strategy outlined here provides a new and efficient method for preparation of nucleoside 5'-triphosphate and is likely applicable to a broad variety of base and sugar modified nucleoside analogues.

Adenosine Triphosphate↗

PubMed2003

Fluoride-cleavable biotinylation phosphoramidite for 5'-end-labeling and affinity purification of synthetic oligonucleotides.

A fluoride-cleavable phosphoramidite for biotinylation was designed, synthesized and coupled efficiently to the 5'-end of DNA on an automatic synthesizer. The diisopropylsilyl acetal functionality was used to link the biotin moiety through a tertiary hydroxide group to the 5'-end of DNA. This linkage proved to be completely stable under certain post-synthetic DNA cleavage/deprotection conditions [0.05 M K(2)CO(3) in MeOH, room temperature, 24 h and MeNH(2) (approximately 40%)/NH(4)OH (approximately 29%), 1:1 v/v, 65 degrees C, 30 min] while it can be readily broken by fluoride ion, releasing unmodified DNA. To demonstrate the use of this DNA biotinylation method, we applied this method in affinity purification of synthetic DNA. As revealed by HPLC analysis, biotinylated full-length DNA can be efficiently attached to NeutrAvidin coated microspheres, and failure sequences can be readily removed. Subsequent treatment of the microspheres with pyridine/HF released high quality full-length unmodified DNA in good yield.

Avidin↗

PubMed2003

DNA polymerase template interactions probed by degenerate isosteric nucleobase analogs.

The development of novel artificial nucleobases and detailed X-ray crystal structures for primer/template/DNA polymerase complexes provide opportunities to assess DNA-protein interactions that dictate specificity. Recent results have shown that base pair shape recognition in the context of DNA polymerase must be considered a significant component. The isosteric azole carboxamide nucleobases (compounds 1-5; ) differ only in the number and placement of nitrogen atoms within a common shape and therefore present unique electronic distributions that are shown to dictate the selectivity of template-directed nucleotide incorporation by DNA polymerases. The results demonstrate how nucleoside triphosphate substrate selection by DNA polymerase is a complex phenomenon involving electrostatic interactions in addition to hydrogen bonding and shape recognition. These azole nucleobase analogs offer unique molecular tools for probing nonbonded interactions dictating substrate selection and fidelity of DNA polymerases.

Azoles↗

PubMed2002

Polysulfonates derived from metal thiolate complexes as inhibitors of HIV-1 and various other enveloped viruses in vitro.

Sodium 2-mercaptoethanesulfonate reacts with the metal ions Pd(II), Pt(II), Ag(I), Cd(II) and Zn(II) to yield complexes containing multiple anionic sulfonate sites. On the basis of spectroscopic and other analytical data the complexes were assigned the tentative molecular formulas: Pd6(SCH2CH2SO3Na)12, Ptn(SCH2CH2SO3Na)2n+2, Agn(SCH2CH2SO3Na)n, Na2Zn4(SCH2CH2SO3Na)10, and Na2Cd4(SCH2CH2SO3Na)10. The complexes displayed a variety of differences in activity towards DNA and RNA viruses. The platinum complex showed no measurable cytotoxicity and exhibited a spectrum of antiviral activity resembling that of dextran sulfate. It was active against HIV-1 and HIV-2, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), thymidine kinase-deficient HSV-1, human cytomegalovirus, vesicular stomatitis virus (VSV), influenza A virus, respiratory syncytial virus (RSV), Sindbis virus, Junin virus and Tacaribe virus. The palladium complex also showed no measurable cytotoxicity, but was completely inactive against most viruses, with one notable exception: both HIV-1 and HIV-2 were substantially inhibited by the palladium complex. The silver complex showed significantly less antiviral activity and greater cytotoxicity than the platinum complex but did show some selectivity against RSV. The zinc complex showed only modest activity against VSV, RSV, Junin virus, and Tacaribe virus, and like the silver compound was more cytotoxic than either the platinum or palladium complex. The cadmium complex was toxic to all of the cell lines used for in vitro evaluation of antiviral activity. Based on these results, the platinum and palladium compounds appear to be promising candidates for further studies, that is, as vaginal microbicides in the prevention of genital HIV and/or HSV transmission.

Anti-HIV Agents↗

PubMedDate not supplied

Reversible biotinylation phosphoramidite for 5'-end-labeling, phosphorylation, and affinity purification of synthetic oligonucleotides.

A fluoride/amine-cleavable phosphoramidite designed for biotinylation, phosphorylation, and affinity purification of synthetic oligonucleotides was synthesized and coupled efficiently to the 5'-end of DNA on a solid-phase automatic synthesizer. The two hydroxyl groups of diethyl bis(hydroxymethyl)malonate were used to link biotin and the 5'-end of DNA together through a diisopropylsilyl acetal functionality and a phosphate ester group, respectively. The DNA was cleaved from solid support and fully deprotected by treating with a mixture of MeNH(2) ( approximately 40%) and NH(4)OH ( approximately 29%) (1:1, v/v, 65 degrees C, 30 min), and the linkage between biotin and DNA was found completely stable under these conditions. The biotinylated full-length DNA was efficiently attached to NeutrAvidin coated microspheres and failure sequences and other impurities were simply removed by washing with buffer and water. The microspheres were then treated with HF/pyridine/THF (rt, 1 h) and MeNH(2) ( approximately 40%, rt, 15 min) sequentially to yield high quality full-length 5'-end phosphorylated unmodified DNA as revealed by HPLC analysis. It is anticipated that this method will find applications in areas that require efficient isolation of 5'-end phosphorylated DNA from a complex mixture.

Affinity Labels↗

PubMedDate not supplied

Development of a nucleoside analog UV light sensor.

Conjugation of the photosensitive nucleoside (E)-5-(2-methoxycarbonylethenyl)cytidine to biotin provided a means to attach this analogue to microparticles for dosimetry applications that require UV sensor mobility.

Biosensing Techniques↗

PubMedDate not supplied

Endcaps for stabilizing short DNA duplexes.

The syntheses of endcaps for covalently linking the 3' and 5' hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by Tm experiments on the self-complementary octamer.

Base Sequence↗