Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion

Abstract

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental 13C/. 15N/. 2H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2′ of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively 13C. 9/. 15N. 3/. 2H. (1',2",3',4',5',5")-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively 13C. 9/. 15N. 3/. 2H. (1',2",3',4',5',5")-dC and 13C. 9/. 15N. 2/. 2H. (1',2",3',4',5',5")-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective 2H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1'-H2' and C2'-H2' resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments. © 2009 The Author(s).