N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38(2001)83-114 Culture of cells withC carbon source [6]. Labeled ribonucleotides are prepared from the and Nitrogen sourc isolation of bacterial rna from in labeled medium, the hydrolysis of RNA and the separation phenolextraction of the ribonucleotides [7]. They are then chemi tized into nucleoside 3-phosphoramidites which DNA and RNA are used for preparing oligonucleotides on a DNA onthe Nucleic acids hydrolysis Using this method, a 14-base pair DNA dupl ully C, n doubly-labeled as well as partially 5 monophosphate nucleotide labeled at those nucleotides that form the prote DNA interface has been prepared to study its inter action with the antennapedia homeodomain [8] The general procedure for the production of uniformly C, N-labeled DNA by enzymatic synth- dNMPs rAMPs esis is described in Fig. 1. Zimmer and Crothers have shown that milligram quantities of material can be synthesized using this procedure [9]. Their method dNTP comprises the production of uniformly,N- labeled deoxynucleotides from enzymatic hydrolysis of the dNA of bacteria grown on 99%CH3OH and >98%NHCI as sole carbon and nitrogen sources DNA oligonucleotid The labeled DNa are then converted enzymatically to the triphosphates and used in a DNA polymerization I General procedure for the enzymatic synthesis reaction that utilizes an oligonucleotide hairpin labeled DNA primer-template containing a ribonucleotide at the 3 terminus. Alkaline hydrolysis of the ribonucleotide of coupling constants and of relaxation parameters not linkage between the labeled DNA and the unlabeled accessible by proton resonance spectroscopy. It is primer-template followed by purification yields the only recently that efficient labeling of DNA [6-9] labeled DNA. More recently variations of this method has been published thus opening applications of have been proposed by two other groups [10,11] heteronuclear spectroscopy to DNA. We will present Masse and coworkers [10] proposed three modifica- briefly the new labeling methods proposed for DNA. tions. First, the mixed dNTPs are separated from one We will also give an overview of the NMr techniques another so that the ratio of the four dNTPs correspond used to extract structural information about protei ing to the sequence of the deoxyoligonucleotide are DNA complexes including chemical shift changes, used in the reaction. Secondly, Taq polymerase is hydrogen exchange rates, isotope editing and filtering used instead of Klenow fragment of DNA polymerase echniques and methods for measuring protein I in the polymerization step. Third, an additional step dynamics to study the changes in protein flexibility is used to remove non-templated addition at the 3 upon binding. end. Louis and coworkers [11] used the same mole cule for the primer and template in the bidirectional 2.1. Labeling of dNA polymerase chain reaction thus obtaining an exponen- tial growth in the length of the double strand that matic methods. The chemical synthesis of DNA oligo- and coworkers [ll]. It comprises the growth of a mers involves the solid-phase phosphoramidite suitable plasmid containing mutiple copies of theof coupling constants and of relaxation parameters not accessible by proton resonance spectroscopy. It is only recently that ef®cient labeling of DNA [6±9] has been published thus opening applications of heteronuclear spectroscopy to DNA. We will present brie¯y the new labeling methods proposed for DNA. We will also give an overview of the NMR techniques used to extract structural information about protein± DNA complexes including chemical shift changes, hydrogen exchange rates, isotope editing and ®ltering techniques and methods for measuring protein dynamics to study the changes in protein ¯exibility upon binding. 2.1. Labeling of DNA Large quantities of labeled DNA fragments for NMR studies can be synthesized by chemical or enzy￾matic methods. The chemical synthesis of DNA oligo￾mers involves the solid-phase phosphoramidite method using isotopically labeled monomer units [6]. Labeled ribonucleotides are prepared from the isolation of bacterial RNA from cells grown in labeled medium, the hydrolysis of RNA and the separation of the ribonucleotides [7]. They are then chemi￾cally converted to deoxynucleotides and deriva￾tized into nucleoside 30 -phosphoramidites which are used for preparing oligonucleotides on a DNA synthesizer. Using this method, a 14-base pair DNA duplex fully 13C,15N doubly-labeled as well as partially labeled at those nucleotides that form the protein± DNA interface has been prepared to study its inter￾action with the antennapedia homeodomain [8]. The general procedure for the production of uniformly 13C,15N-labeled DNA by enzymatic synth￾esis is described in Fig. 1. Zimmer and Crothers have shown that milligram quantities of material can be synthesized using this procedure [9]. Their method comprises the production of uniformly 13C,15N￾labeled deoxynucleotides from enzymatic hydrolysis of the DNA of bacteria grown on 99% 13CH3OH and .98% 15NH4Cl as sole carbon and nitrogen sources. The labeled DNA are then converted enzymatically to the triphosphates and used in a DNA polymerization reaction that utilizes an oligonucleotide hairpin primer-template containing a ribonucleotide at the 30 terminus. Alkaline hydrolysis of the ribonucleotide linkage between the labeled DNA and the unlabeled primer-template followed by puri®cation yields the labeled DNA. More recently variations of this method have been proposed by two other groups [10,11]. Masse and coworkers [10] proposed three modi®ca￾tions. First, the mixed dNTPs are separated from one another so that the ratio of the four dNTPs correspond￾ing to the sequence of the deoxyoligonucleotide are used in the reaction. Secondly, Taq polymerase is used instead of Klenow fragment of DNA polymerase I in the polymerization step. Third, an additional step is used to remove non-templated addition at the 30 end. Louis and coworkers [11] used the same mole￾cule for the primer and template in the bidirectional polymerase chain reaction thus obtaining an exponen￾tial growth in the length of the double strand that contains two repeats of the desired DNA sequence. An additional method has been presented by Louis and coworkers [11]. It comprises the growth of a suitable plasmid containing mutiple copies of the N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 85 Culture of cells with 13C carbon source and 15N nitrogen source cell lysis phenol extraction DNA and RNA proteins Nucleic acids hydrolysis 5’ monophosphate nucleotide nucleotide separation dNMPs rNMPs dNTPs DNA oligonucleotide Fig. 1. General procedure for the enzymatic synthesis of 13C,15N￾labeled DNA