Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion.
We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental (13)C/(15)N/(2)H 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 (13)C(9)/(15)N(3)/(2)H((1',2'',3',4',5',5''))-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively (13)C(9)/(15)N(3)/(2)H((1',2'',3',4',5',5''))-dC and (13)C(9)/(15)N(2)/(2)H((1',2'',3',4',5',5''))-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective (2)H-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.
Nelissen
Frank H T
FH
Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, the Netherlands.
Girard
Frederic C
FC
Tessari
Marco
M
Heus
Hans A
HA
Wijmenga
Sybren S
SS
D011088:DNA Ligases; D017931:DNA Primers / Q000737:chemistry; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000737:chemistry*; D003854:Deoxyribonucleotides / Q000096:biosynthesis; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D003854:Deoxyribonucleotides; D004262:DNA Restriction Enzymes; D011088:DNA Ligases