Synthesis of spin labelled deoxynucleotide analogues and their incorporation with terminal deoxynucleotidyl transferase into DNA

Toppin, Charles R.; Thomas, Ingrid E.; Bobst, Elisabeth V.; Bobst, Albert M.

doi:10.1016/0141-8130(83)90075-2

Cited by 12 publications

(9 citation statements)

References 9 publications

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“…To achieve site-specific labeling with respect to the nucleic acid base, spin-labeled ribonucleic and deoxyribonucleic acids were prepared with the aid of template-independent enzymes using as substrates unmodified nucleotides together with nucleotide analogue sites specifically spin-labeled at the 4or 5-position of a pyrimidine base. The copolymerization reactions were performed with polynucleotide phosphorylase and ribonucleotide diphosphates (Bobst & Torrence, 1978) to make RNA, and with terminal deoxynucleotidyltransferase and deoxyribonucleic triphosphates (Toppin et al, 1983) to make DNA. These enzymatic procedures yielded spin-labeled single-stranded nucleic acids which then were annealed with complementary unlabeled single strands to form RNA and DNA duplexes.…”

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confidence: 99%

Base dynamics of nitroxide-labeled thymidine analogs incorporated into (dA-dT)n by DNA polymerase I from E. coli

Pauly

Thomas²,

Bobst³

1987

Biochemistry

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Nitroxide-labeled thymidine substrates (dL) for Escherichia coli DNA polymerase I (pol I) were used to synthesize spin-labeled alternating double-stranded copolymers with (dA-dT)n as a template. All dL substrates use an alkane or alkene tether substituted into the 5-position of the pyrimidine ring to link a five- or six-membered ring nitroxide to the pyrimidine base. The kinetics of dL incorporation show some tether dependence with respect to tether length and tether geometry. The electron spin resonance (ESR) spectra of (dA-dT,dL)n duplexes directly formed by polymerization with pol I are compared with the ESR spectra of (dA)n(dT,dL)n duplexes, which are obtained after annealing of nitroxide-labeled single strands with complementary unlabeled single strands. The ESR spectra indicate that nitroxide-labeled analogues with tethers short enough to let the nitroxide ring reside in the major groove are excellent reporter groups for monitoring hybridization. A small difference between the ESR line shapes of the alternating duplexes (dA-dT,dL)n and the homopolymer duplexes (dA)n(dT,dL)n containing the same dL is detectable, suggesting the presence of subtle differences in the base dynamics between both systems. Computer simulation of the ESR spectra of the (dA-dT,dL)n duplexes was successful with the same motional model reported earlier [Kao, S.-C., & Bobst, A.M. (1985) Biochemistry 24, 5465-5469]. The thymidine motion arising from tilting and torsion of base pairs and base twisting in (dA-dT)n is similar to that in (dA)n(dT)n and is of the order of 4 ns.

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Base dynamics of nitroxide-labeled thymidine analogs incorporated into (dA-dT)n by DNA polymerase I from E. coli

Pauly

Thomas²,

Bobst³

1987

Biochemistry

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“…The methods of isolation and purification were analogous to those used by Tuppin et ul. [6]. UV (pH 7.0): 331 (22,000).…”

Section: -J/(4-mentioning

confidence: 99%

“…2'-Deoxyuridine (3) was first converted to 4 according to published procedures [%lo]. Next, 4 was phosphorylated to 5 by similar methods to those already described [6] [l 11 in 25% overall yield based on the amount of 4 used. Alkylation of 5 with the activated nitroxide-containing compound, a-iodoacetamido-Tempo, yielded the desired derivative 6.…”

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“…Of great interest is the synthesis of spin-labeled ribo-or deoxyribonucleotides which can serve as substrates for enzymes such as PNPase'), TdT, and Pol I in order to prepare enzymatically spin-labeled nucleic acids. The former two enzymes have no template requirement and were used in this laboratory to prepare a variety of RNA and DNA homopolymers containing different amounts of enzymatically incorporated spinlabeled ribo-or deoxyribonucleotides [5] [6]. Pol I, on the other hand, has a template requirement and is well suited to make template specific spin-labeled polynucleotides.…”

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Three Novel Spin‐Labeled Substrates for Enzymatic Incorporation into Nucleic Acid Lattices

Toppin

Pauly

Devanesan

et al. 1986

Helvetica Chimica Acta

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The synthesis of one C(4)-spin-laheled uridine-S'-diphosphate (C(4)-UDP and two nitroxide-containing 2'-deoxyuridine-S'-triphosphate (dUTP) analogs are reported. The C(4)-UDP derivative was incorporated into copolymers by polynucleotide phosphoxylase (PNPase); one of' the two dUTP analogs, substituted at position C(4), was a good substrate for TdT, whereas the other one, substituted at position C(5), served as substrate for E. coii DNA polymerase (Pol 1).Introduction. ~ The application of the spin-labeled nucleic acids to investigate problems of biological interest has been well established in this as well as other laboratories [ 1-41. Of great interest is the synthesis of spin-labeled ribo-or deoxyribonucleotides which can serve as substrates for enzymes such as PNPase'), TdT, and Pol I in order to prepare enzymatically spin-labeled nucleic acids. The former two enzymes have no template requirement and were used in this laboratory to prepare a variety of RNA and DNA homopolymers containing different amounts of enzymatically incorporated spinlabeled ribo-or deoxyribonucleotides [5] [6]. Pol I, on the other hand, has a template requirement and is well suited to make template specific spin-labeled polynucleotides.We wish to report here the synthesis of the three novel spin-labeled nucleotides pplss4U (2), pppls4dU (6), and pppDUAT (12). pplss4U and pppls4dU served as substrates for PNPase and TdT, respectively, and pppDUAT was readily incorporated by Pol I. The biophysical properties of (Iss4U,U),, (ls4dU,dT), and (DUAT,dT), were described in [7].

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“…4-(cr)-iodoacetamido-2,2.6,6-tetramethylpiperidino-l-oxy; (RUTT,U),, copolymer of RUTT and uridine; ls4U, 4-j [N-(2,2,6,6-tetranlethyl-4-piperidyl-l-oxy)-carbamoylmethyl]thiojuridine; (ls4U,U),, copolymer of ls4U and uridine; (ls4U,C),, copolymer of 2 as described in [6] and the thiolated nucleotide was isolated as disulfide 3. Compound 3 was quantitatively converted to 4 by alkylation with u-iodoacetamido tempo in the presence of dithiothreitol [7]. For the purpose of characterization the 5'-monophosphorylated analog of 4 was also synthesized.…”

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Synthesis of Unique Spin‐Labeled Nucleic Acids by Combined Enzymatic and Chemical Approaches

Toppin

Thomas

Bobst

1983

Helvetica Chimica Acta

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Synthesis of spin labelled deoxynucleotide analogues and their incorporation with terminal deoxynucleotidyl transferase into DNA

Cited by 12 publications

References 9 publications

Base dynamics of nitroxide-labeled thymidine analogs incorporated into (dA-dT)n by DNA polymerase I from E. coli

Base dynamics of nitroxide-labeled thymidine analogs incorporated into (dA-dT)n by DNA polymerase I from E. coli

Three Novel Spin‐Labeled Substrates for Enzymatic Incorporation into Nucleic Acid Lattices

Synthesis of Unique Spin‐Labeled Nucleic Acids by Combined Enzymatic and Chemical Approaches

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