Enzymatic synthesis of structure-free DNA with pseudo-complementary properties.
Long single-stranded DNAs and RNAs possess considerable secondary structure under conditions that support stable hybrid formation with oligonucleotides. Consequently, different oligomeric probes can hybridize to the same target with efficiencies that vary by several orders of magnitude. The ability to enzymatically generate structure-free single-stranded copies of any nucleic acid without impairing Watson-Crick base pairing to short probes would eliminate this problem and significantly improve the performance of many oligonucleotide-based applications. Synthetic nucleic acids that exhibit these properties are defined as pseudo-complementary. Previously, we described a pseudo-complementary A-T couple consisting of 2-aminoadenine (nA) and 2-thiothymine (sT) bases. The nA-sT couple is a mismatch even though nA-T and A-sT are stable base pairs. Here we show that 7-alkyl-7-deazaguanine and N(4)-alkylcytosine (where alkyl = methyl or ethyl) can be used in conjunction with nA and sT to render DNA largely structure-free and pseudo-complementary. The deoxynucleoside triphosphates (dNTPs) of these bases are incorporated into DNA by selected mesophilic and thermophilic DNA polymerases and the resulting primer extension products hybridize with good specificity and stability to oligonucleotide probes composed of the standard bases. Further optimization and characterization of the synthesis and properties of pseudo-complementary DNA should lead to an ideal target for use with oligonucleotide probes that are <25 nt in length.
Lahoud
Georges
G
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Timoshchuk
Victor
V
Lebedev
Alexandre
A
de Vega
Miguel
M
Salas
Margarita
M
Arar
Khalil
K
Hou
Ya-Ming
YM
Gamper
Howard
H
D020029:Base Pairing; D003596:Cytosine / Q000031:analogs & derivatives*; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006147:Guanine / Q000031:analogs & derivatives*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D013696:Temperature
D004277:DNA, Single-Stranded; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D015345:Oligonucleotide Probes; D006147:Guanine; D003596:Cytosine; D004259:DNA-Directed DNA Polymerase