Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs.
DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR reaction, DNA is obtained in which the natural hydrogen bonding rule is inversed. When PCR is performed at limiting denaturation temperatures, it is possible to recover GC-rich viral genomes and inverted Alu elements embedded in cellular mRNAs resulting from editing by dsRNA dependent host cell adenosine deaminases. The editing of Alu elements in cellular mRNAs was strongly enhanced by type I interferon induction indicating a novel link mRNA metabolism and innate immunity.
Suspène
Rodolphe
R
Unité de Rétrovirologie Moléculaire, CNRS URA 3015, Unité de Génétique Moléculaire des Bunyaviridés and Laboratoire de Génomique Virale et Vaccination, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15.
Renard
Myrtille
M
Henry
Michel
M
Guétard
Denise
D
Puyraimond-Zemmour
David
D
Billecocq
Agnès
A
Bouloy
Michèle
M
Tangy
Frederic
F
Vartanian
Jean-Pierre
JP
Wain-Hobson
Simon
S
D015075:2-Aminopurine / Q000031:analogs & derivatives / Q000737:chemistry; D020856:AT Rich Sequence; D000243:Adenosine Deaminase / Q000378:metabolism*; D020087:Alu Elements; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry*; D020862:GC Rich Sequence; D015322:Gene Rearrangement, B-Lymphocyte; D006860:Hydrogen Bonding; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007293:Inosine Triphosphate / Q000737:chemistry; D008459:Measles virus / Q000235:genetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D017393:RNA Editing; D012333:RNA, Messenger / Q000737:chemistry; D016601:RNA-Binding Proteins; D014709:Vero Cells
D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D007293:Inosine Triphosphate; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D004247:DNA; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase