Spin label enhanced NMR screening.
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Nuclear Magnetic Resonance, Biomolecular↗
Biomedical subjects
S Rüdisser
Publications and source records attributed to S Rüdisser.
A simple and efficient method to transcribe RNAs with reduced 3' heterogeneity.
Ribose C2' methoxy groups (-OCH3) on the penultimate nucleotide or the last two nucleotides of DNA templates can dramatically improve the quality of transcripts produced by T7 RNA polymerase. This strategy can be adapted to generate transcripts of varying lengths and will allow greater ease in purification of the products.
DNA↗
Solution structure of Cobalt(III)hexammine complexed to the GAAA tetraloop, and metal-ion binding to G.A mismatches.
The solution structure of a 22 nt RNA hairpin and its complex with Co(NH(3))(6)(3+) bound to the GAAA tetraloop has been determined by NMR spectroscopy. Co(NH(3))(6)(3+) has a similar geometry to Mg(H(2)O)(6)(2+) and can be used as a probe for binding sites of completely solvated magnesium ions. The hairpin contains tandem G.A mismatches, similar to the P5abc region of a group I intron, and is closed by a GAAA tetraloop. The tandem G.A mismatches are imino hydrogen bonded in contrast with the sheared G.A mismatches found in a different context in the crystal structure of the P4-P6 domain. Chemical shift changes of the imino protons upon titration of the RNA hairpin with Mg(2+) and with Co(NH(3))(6)(3+) were used to identify ion-binding sites. Paramagnetic resonance broadening upon titration with Mn(2+) was also used. The titration curves gave dissociation binding constants for the magnesium ions in the millimolar range, similar to the binding in the major groove of RNA at tandem G.U base-pairs. Although the largest chemical shift change occurred at an imino proton of one of the G.A base-pairs, no nuclear Overhauser enhancement cross-peaks between the cobalt ligand and neighboring RNA protons were seen, presumably due to the high mobility of the Co(NH(3))(6)(3+) at this site. Nuclear Overhauser enhancement cross-peaks between Co(NH(3))(6)(3+) and the GAAA tetraloop were observed, which allowed the determination of the structure of the tetraloop binding site. The Co(NH(3))(6)(3+) is bound in the major groove of the GAAA tetraloop with hydrogen bonds to guanine base N7 and to phosphate oxygen atoms of the tetraloop.
Animals↗
Unexpected BII conformer substate population in unoriented hydrated films of the d(CGCGAATTCGCG)2 dodecamer and of native B-DNA from salmon testes.
Conformational substates of B-DNA had been observed so far in synthetic oligonucleotides but not in naturally occurring highly polymeric B-DNA. Our low-temperature experiments show that native B-DNA from salmon testes and the d(CGCGAATTCGCG)2 dodecamer have the same BI and BII substates. Nonequilibrium distribution of conformer population was generated by quenching hydrated unoriented films to 200 K, and isothermal structural relaxation toward equilibrium by interconversion of substates was followed by Fourier transform infrared spectroscopy. BI interconverts into BII on isothermal relaxation at 200 K, whereas on slow cooling from ambient temperature, BII interconverts into BI. Our estimation of the dodecamer's BI-to-BII conformer substate population by curve resolution of the symmetrical stretching vibration of the ionic phosphate is 2.4 +/- 0.5 to 1 at 200 K, and it is 1.3 +/- 0.5 to 1 between 270 and 290 K. Pronounced spectral changes upon BI-to-BII interconversion are consistent with base destacking coupled with migration of water from ionic phosphate toward the phosphodiester and sugar moieties. Nonspecific interaction of proteins with the DNA backbone could become specific by induced-fit-type interactions with either BI or BII backbone conformations. This suggests that the BI-to-BII substate interconversion could be a major contributor to the protein recognition process.
Animals↗
A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase.
DNA templates modified with C2'-methoxyls at the last two nucleotides of the 5' termini dramatically reduced nontemplated nucleotide addition by the T7 RNA polymerase from both single- and double-stranded DNA templates. This strategy was used to generate several different transcripts. Two of the transcripts were demonstrated by nuclear magnetic resonance spectroscopy to be unaffected in their sequence. Transcripts produced from the modified templates can be purified with greater ease and should be useful in a number of applications.
Base Sequence↗
Assignment of cytosine N3 resonances in nucleic acids via intrabase three-bond coupling to amino protons.
Coherences were observed between 15N3 of cytosine and its trans amino proton (H42) using a modified gradient-based heteronuclear single quantum coherence (HSQC) pulse sequence optimized for three-bond proton-nitrogen couplings. The method is demonstrated with a 22-nucleotide RNA fragment of the P5abc region of a group I intron uniformly labeled with 15N. Use of intraresidue 15N3-amino proton couplings to assign cytosine 15N3 signals complements the recently proposed JNN HNN COSY [Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293-8297] method of identifying hydrogen-bonded base pairs in RNA.
Animals↗