Unnatural Nucleosides with Unusual Base UNIT 1.4 Pairing Properties Synthetic modified nucleosides designed to rounding free space when designing new nu- pair in unusual ways with the natural nucleic cleobases. This can generally be determined by acid bases have many potential applications in inspecting the groove regions of nucleic acid nucleic acid biochemistry. These range from duplex and triplex models. Where the founda- biochemical tools for probing nucleic acid tion for the design is a natural nucleobase, structure or protein–nucleic acid interactions to substitution is allowed at C5 of C, T, or U. N4 tools for re-engineering DNA and ultimately of C and N6 of A are also possible attachment proteins. Applications as components of nu- sites, but generally compromise base associa- cleic acid–based diagnostic tools for clinical tion. N2 of G is acceptable and, unlike all other analysis have been envisioned. Furthermore, sites, allows minor groove placement of an unnatural bases may be useful as components appendage. C8 of A and G have been used as of antisense or antigene nucleic acid analogs in sites for attachment of appendages, but substi- therapeutic applications. This unit serves to lay tution here may influence the conformation the foundation for future protocol units on un- preference about the glycosidic bond. Replace- natural base synthesis and application, with ment of the purine N7 with a carbon provides particular emphasis on unnatural base analogs a site for attachment of appendages in a steri- that mimic natural bases in size, shape, and cally tolerated position. There are many exam- biochemical processing. A much more exten- ples of appendages that, when added to the sive compilation of unnatural nucleobases has natural nucleic acid bases, enhance base-pair recently been published by Luyten and Her- stability. These include substituents that in- dewijn (1998). crease the acidity of proton donor sites (Yu et To design bases that mimic natural bases in al., 1993) or increase hydrophobicity and aro- function, it is useful to consider the factors that matic surface area for enhanced base stacking are essential for effective base pairing and sta- (Inoue et al., 1985), as well as appended cations ble duplex formation. In addition to structures for electrostatic interactions with the phos- that are configured to hydrogen bond and base phodiester backbone (Ueno et al., 1998). stack within the spatial confines of duplex DNA and RNA, any surrogate base pair must also BASE PAIRS WITH ALTERNATIVE conform to specific dimensions and geometry HYDROGEN BONDING SCHEMES if it is to function in roles that require recogni- tion by nucleic acid–processing enzymes. To Purine-Pyrimidine-Like Base Pairs be isosteric with AT or GC base pairs, the C1′ Benner and co-workers have described a set to C1′ distance must be in the range of 10.8 to of nucleobase analogs that resemble natural 11.0 Å, and λ1 and λ2 should be ~50° (Fig. 1.4.1; bases, but have reconfigured hydrogen bonding Saenger, 1984). patterns (Piccirilli et al., 1990). Six orthogonal Just as it is important to know where to place base pairs (S.1 to S.6) are shown in Figure 1.4.2. hydrogen bond donor and acceptor sites, it is Extensive studies on these nucleosides have important to consider the availability of sur- revealed that even these subtle changes in struc- ture can cause profound effects on thermody- namic (Voegel and Benner, 1994) and bio- chemical properties (Switzer et al., 1993; Hor- lacher et al., 1995). For example, the Watson-Crick base pair geometry C-nucleoside pyrimidine mimics appear to C1' C1' - C1' ~ 10.8 to 11.0 Å λ base pair more weakly than equivalent base λ ~ 50 - 54° pairs composed of N-nucleosides. As far as biochemical properties, it appears that certain λ DNA polymerases require the pyrimidine O2 C1' and purine N3 as recognition features for effi- cient template-mediated oligonucleotide syn- Figure 1.4.1 Base pair parameters. See AP- thesis (Horlacher et al., 1995). PENDIX 1B and Figure A.1B.4 for other base Synthesis of pairing schemes. Modified Nucleosides Contributed by Donald E. Bergstrom 1.4.1 Current Protocols in Nucleic Acid Chemistry (2001) 1.4.1-1.4.13 Copyright © 2001 by John Wiley & Sons, Inc. Supplement 5 site itself, the duplex was destabilized more C-G than when the triazole was placed opposite each N H N O N of the natural bases. This result is in line with dR N N dR HH H N NN NMR studies on duplex DNA containing the H O NN H N N triazole carboxamide opposite either G or T HH N O N O H (Klewer et al., 2001). In both cases, the triazole N N dR 1 dR H 2 prefers to adopt a conformation in which the amide group points out into the major groove rather than inward towards the opposing base O N N dR H N pair. To achieve specific association through HH N N dR N NN H hydrogen bond interactions, one of the two H O NN N N triazole carboxamides in a self-pair would have HH H N N N O HH to face with the amide projecting inward to- dR 3 N O wards the opposing base. This example reflects dR 4 parameters that one must consider when de- N signing base pairs; the interior of the helix is a H N O N dR N N dR less hydrophilic environment and, without suf- HH H N NN HH O NN ficient compensation, highly hydrophilic CH3 O H NN N O groups will prefer to assume positions that H N N H N place them in a more hydrophilic environment. 5 6 dR H dR H HYDROPHOBIC BASE PAIRS There are now a significant number of ex- Figure 1.4.2 Structures of six Watson-Crick- amples of hydrophobic unnatural bases that type base pairs utilizing mutually exclusive hy- pair with other hydrophobic bases or with drogen bonding schemes. themselves in duplex DNA with higher affinity than with any of the natural bases. 3-Nitropyr- Self-Complementary Nucleobases role deoxyribonucleoside (S.26; Fig. 1.4.8), Another concept for new base pair develop- which was originally designed as a universal ment was proposed by Pochet and Marliére nucleobase, pairs with almost equal affinity to (1996). Based on the known ability of the mu- each of the natural bases, but a 12-mer duplex tagenic base 8-oxoguanine to base pair with A with nitropyrrole opposite itself is significantly from a syn conformation, these researchers re- more stable than the same duplex with nitropyr- designed the base by removing the 6-oxo group. role opposite each of the natural bases This yields the unnatural base 2-amino-8- (Bergstrom et al., 1995; Zhang et al., 1998). oxopurine (S.7), which they postulate would The significance of the hydrophobic substi- pair with itself according to the arrangement tuent (nitro) in mediating this effect is clear shown in Figure 1.4.3. The results of biochemi- when one compares pyrrole-3-carboxamide, cal studies with this unusual base have not yet which in duplex DNA yields far more stable been reported. One can imagine similar self- duplexes when paired opposite each of the pairing potential for azole carboxamide nucleo- natural bases than when paired opposite itself. sides, as illustrated in Figure 1.4.3 for 1,2,4- Because of these results, hydrophobic base pairs are considered to be attractive candidates triazole-3-carboxamide (S.8). However, Tm studies on a duplex containing 1,2,4-triazole- for extension of the genetic alphabet. Two 3-carboxamide showed that when placed oppo- themes are possible: (1) the development of a H N H N N O N H dR N N N H H N H N H N O H O N N dR N N O N N N dR N H dR H Unnatural 7 8 Nucleosides with Unusual Base Pairing Properties Figure 1.4.3 Self-pairing bases. 1.4.2 Supplement 5 Current Protocols in Nucleic Acid Chemistry complementary hydrophobic pair, and (2) the The nonpolar nucleobase difluorotoluene creation of a single self-complementary hydro- (S.14), a thymine isostere lacking hydrogen- phobic base. The latter possibility is more at- bonding functionality, can effectively substi- tractive, because one need contend with the tute for thymine in both the template strand and optimization of DNA replication with only one as the incoming nucleoside triphosphate. These unnatural nucleoside. results suggest that shape recognition in the A series of recent papers from a research absence of hydrogen bonding is an important effort at Scripps Research Institute led by factor in base pair recognition (Guckian and Romesberg and Schultz has described consid- Kool, 1997; Moran et al., 1997a,b; Guckian et erable progress in the development of hydro- al., 1998; Kool, 1998). Similarly, 4-methyl- phobic self-pairing bases (McMinn et al., 1999; benzimidazole (S.12) is an effective surrogate Berger et al., 2000a; Ogawa et al., 2000; Wu et for adenine when matched opposite the di- al., 2000). Three of these nucleoside analogs, fluorotoluene nucleobase (Morales and Kool, 1-β-D-deoxyribosyl-7-azaindole (S.10; Ogawa 1999). et al., 2000) and two different 7-propynyl iso- carbostyril deoxyribonucleosides (S.9; R = H METAL-MEDIATED ASSOCIATION and CH3; McMinn et al., 1999) are shown in OF LIGAND NUCLEOBASE Figure 1.4.4. These nucleoside analogs, as as- MIMICS sessed by Tm measurements, self-pair signifi- Another way of mediating specific associa- cantly more effectively than they pair with any tion between nucleobase-like molecules con- of the natural bases.