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 singlestranded 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 lengthThis work was supported by grants from the National
Institutes of Health (NIGMS Grant 74564 to H.G.) and
Agilent Technologies. Dr Carl Fuller (GE Healthcare)
kindly provided us with dNTPs of 7-alkyl-7-deazaguanines
and 7-iodo-7-deazaguanine. We thank Dr Jeffrey Sampson
(Agilent Technologies) for his constant encouragement and
Caryn Evila (Idaho State University) for her early participation in the project. At TriLink Biotechnologies we are
grateful to Dr Richard Hogrefe, Dr Gerald Zon, Dr
Natasha Paul and Dr David Combs for support and helpful
discussions and Dr Inna Koukhareva and Stephanie Perry
for synthesis of dNTPs. Funding to pay the Open Access
publication charges for this article was provided by NIH
GM74564.Peer reviewe
