title: α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.
abstract: (S(C5'), R(P)) α,β-D- Constrained Nucleic Acids (CNA) are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.
authors: Institut de Pharmacologie et de Biologie Structurale, Unité Mixte de Recherche 5089, Centre National de la Recherche Scientifique, Toulouse, France.
authors: Olivier
authors: O
authors: Martínez
authors: Vincent
authors: V
authors: Ecochard
authors: Sabrina
authors: S
authors: Mahéo
authors: Grégori
authors: G
authors: Gross
authors: Pierre
authors: P
authors: Bodin
authors: Justin
authors: J
authors: Teissié
authors: Jean-Marc
authors: JM
authors: Escudier
authors: Laurent
authors: L
authors: Paquereau
mesh_terms: D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D058105:Polymerization; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D009696:Nucleic Acids; D004259:DNA-Directed DNA Polymerase