Selection for thermodynamically stable DNA tetraloops using temperature gradient gel electrophoresis reveals four motifs: d(cGNNAg), d(cGNABg),d(cCNNGg), and d(gCNNGc).
Hairpins play important roles in the function of DNA, forming cruciforms and affecting processes such as replication and recombination. Temperature gradient gel electrophoresis (TGGE) and in vitro selection have been used to isolate thermodynamically stable DNA hairpins from a six-nucleotide random library. The TGGE-selection process was optimized such that known stable DNA tetraloops were recovered, and the selection appears to be exhaustive. In the selection, four families of exceptionally stable DNA loops were identified: d(cGNNAg), d(cGNABg), d(cCNNGg), and d(gCNNGc). (Lowercase denotes the closing base pair; N = A, C, G, or T; and B = C, G, or T.) It appears that the known stable d(cGNAg) triloop motif can be embedded into a tetraloop, with the extra nucleotide inserted into either the middle of the loop, d(cGNNAg), or at the 3'-end of the loop, d(cGNABg). For d(cGNNAg) and d(cGNABg), a CG closing base pair was strongly preferred over a GC, with DeltaDeltaG degrees (37) approximately 2 kcal/mol. Members of the two families, d(cCNNGg) and d(gCNNGc), are similar in stability. The loop sequences and closing base pairs identified for exceptionally stable DNA tetraloops show many similarities to those known for exceptionally stable RNA tetraloops. These data provide an expanded set of thermodynamic rules for the formation of tetraloops in DNA.
Nakano
Mariko
M
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Moody
Ellen M
EM
Liang
Jing
J
Bevilacqua
Philip C
PC
D020029:Base Pairing; D001483:Base Sequence; D002942:Circular Dichroism; D020650:Combinatorial Chemistry Techniques; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015723:Gene Library; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods; D013816:Thermodynamics; D014466:Ultraviolet Rays
D009841:Oligonucleotides; D004247:DNA