A Parallel Screen for the Discovery of Novel DNA Base Pairs

Abstract

The genetic information in DNA is encoded in the sequence of the four nucleobases, and its readout is based on the highly specific Watson-Crick pairing A-T and G-C. Over the years there has been growing interest in expanding this genetic alphabet by adding novel, orthogonal base pairs. Such additional base pairs could be used in biotechnology to introduce nonnatural amino acids into engineered proteins, [1][2][3] and to create novel tools for identifying aberrant or exogenous, disease-related nucleic acids. [4][5][6][7][8] The degree of freedom in designing novel base pairs, however, is limited by the structural scaffold of the double helix. The most logical approach is based on structurally isomorphic bases with an alternative Watson-Crick-like hydrogen-bonding network. [6,9,10] Alternatively, nonisomorphic aromatic heterocycles can serve as base replacements; they recognize each other by means other than classical hydrogen bonding and can be replicated and transcribed with high fidelity and similar kinetic properties. [11][12][13][14][15][16] This latter approach provides access to a larger structural space for potential base replacements. Unfortunately this approach is hampered by a lack of design rules which makes it necessary to screen a large number of potential candidates. From each potential base analogue the corresponding nucleoside must first be synthesized and incorporated into oligonucleotides before its coding properties can be evaluated. This is associated with multistep syntheses for each nucleoside candidate which is a timeconsuming procedure.To address these drawbacks we set out to design an assay for the rapid parallel screening of novel potential base candidates out of a library of heterocyclic amines. In a first step we identified selective, high-affinity binders to natural nucleobases. The assay is based on a dodecamer DNA duplex which carries an abasic site X in the center and a fluorescencequencher pair on one end of the duplex (Scheme 1). X structurally deviates from a natural DNA abasic site by the replacement of O(3') by a CH 2 group which renders it chemically stable towards base-or heat-induced strand cleavage at the 3'-end.[17] These duplexes were then incubated with a library of heterocyclic amines in a parallel fashion. During this treatment the amines become covalently attached to X through the formation of a hemiaminal, resulting in exoamino nucleosides that are structurally similar to the natural nucleosides. The thermal stabilities of these duplexes, which reflect the relative affinity of each amine to its target, were then determined by parallel fluorescence measurements of the melting temperature T m (see the Supporting Information).To validate the assay we searched a library of 34 randomly chosen, commercially available heterocyclic amines (Scheme 2) for individuals that bind selectively with high affinity to each of the four natural nucleobases. Duplexes (0.6 mm) were incubated with a 1000-fold excess of the amines in buffer at pH 8 for two days at 55 8C to form the hemiamin...

Synthesis and Incorporation into Oligodeoxynucleotides of Carbocyclic exo-Amino Nucleosides

“…In terms of molecular recognition in duplex structures we find in general that the T m -values observed here with the carbocyclic exo-amino nucleosides match well with those observed previously in the parallel screen 25 with the (noncarbocyclic) exo-amino nucleosides. This validates the methodology of the screen and suggests that there are no significant differences in complementary base recognition as a function of whether there is a CH 2 group or an oxygen atom in the five-membered ring.…”

“…28 The aromatic amines chosen, namely aniline (AN), naphthalene-1,5-diamine (DAN), benzene-1,3-diamine (DAB), pyridin-3-amine (AP), and 6-aminoflavone (CHR) have been selected from the previously described combinatorial assay. 25 Ozlem (1987)(1988) as a postdoctoral fellow to investigate catalytic antibodies (Prof. Peter G. Schultz). In 1988 he returned to ETH Zürich to start his independent career.…”

“…The fluorescence measurements (Supporting Information) were performed to comply with the measurements of the previous assay. 25 The UV-measurements were carried out to validate the fluorescence measurements. As expected, we found no significant differences between the T m -values determined by the two methods.…”

“…In this context, we recently developed a combinatorial assay for the discovery of novel base surrogates out of a library of aromatic amines. 25 The assay is based on the reversible formation of hemiaminals of primary aromatic amines with a chemically functional abasic site in the center of a DNA duplex in a parallel fashion. Matches were identified by fluorescence-based thermal melting experiments.…”

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

“…Although many candidate unnatural base pairs have been reported (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21), only a few are actually replicable by DNA polymerases (10,11,13,16). Moreover, it is clear that most applications will require that the unnatural base pair not only be replicated with high efficiency and fidelity but also, that replication be at least approximately independent of sequence context.…”