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Unnatural Nucleosides with Unusual Base UNIT 1.4 Pairing Properties Donald E. Bergstrom1 1Purdue University, West Lafayette, Indiana ABSTRACT Synthetic modified nucleosides designed to pair in unusual ways with natural nucleobases have many potential applications in biology and biotechnology. This overview lays the foundation for future protocol units on synthesis and application of unnatural bases, with particular emphasis on unnatural base analogs that mimic natural bases in size, shape, and biochemical processing. Topics covered include base pairs with alternative H-bonding schemes, dimensionally expanded base pairs, hydrophobic base pairs, metal-ligated bases, degenerate bases, universal nucleosides, and triplex constituents. Curr. Protoc. Nucleic Acid Chem. 37:1.4.1-1.4.32. C 2009 by John Wiley & Sons, Inc. Keywords: nucleoside r mimetic r nonpolar r degenerate r universal INTRODUCTION McCutcheon (2007). Extensive efforts over Synthetic modified nucleosides designed to the past decade on DNA-metal base pairs pair in unusual ways with the natural nucleic has spawned three recent reviews in this area acid bases have many potential applications (Shionoya and Tanaka, 2004; Clever et al., in biology and biotechnology. These range 2007; Muller, 2008). from biochemical tools for probing nucleic To design bases that mimic natural bases acid structure or protein-nucleic acid inter- in function, it is useful to consider the fac- actions to tools for re-engineering DNA and tors that are essential for effective base pair- ultimately proteins. Applications as compo- ing and stable duplex formation. In addition nents of nucleic acid—based diagnostic tools to structures that are configured to hydrogen for clinical analysis have been envisioned. Fur- bond and base stack within the spatial confines thermore, unnatural bases may be useful as of duplex DNA and RNA, any surrogate base components of synthetic antisense and anti- pair must also conform to specific dimensions gene nucleic acids, aptamers, and siRNA in and geometry if it is to function in roles that therapeutic applications. This unit serves to require recognition by nucleic acid-processing lay the foundation for future protocol units on enzymes. To be isosteric with A-T or G-C base unnatural base synthesis and application, with pairs, the C1 to C1 distance must be in the ◦ particular emphasis on unnatural base analogs range of 10.8 to 11.0 A, and λ1 and λ2 should that mimic natural bases in size, shape, and be ∼50◦ (see Fig. 1.4.1 and Saenger, 1984). biochemical processing. Just as it is important to know where to A much more extensive compilation of place hydrogen bond donor and acceptor sites, unnatural nucleobases has been published it is important to consider the availability of by Luyten and Herdewijn (1998). More re- surrounding free space when designing new cently, published overviews include those by nucleobases. This can generally be determined Kool (2002), Hunziker and Mathis (2005), by inspecting the groove regions of nucleic and Rozners (2005). Sekine and co-workers acid duplex and triplex models. Where the reviewed alternative Watson-Crick-type base foundation for the design is a natural nucle- pairs in 2008 (Sekine et al., 2008). In ad- obase, substitution is allowed at C5 of C, T, dition, Too and Loakes (2008) have written or U. N4 of C and N6 of A are also pos- about applications of universal nucleosides, sible attachment sites in the major groove, and Kool (2008) has reviewed nucleic acid but generally compromise base association. analogs as probes of DNA polymerases. The N2 of G is acceptable and, unlike all other latter theme has also been discussed in reviews sites, allows minor groove placement of an ap- by Krueger and Kool (2007) and Berdis and pendage. C8 of A and G have been used as sites Synthesis of Modified Nucleosides Current Protocols in Nucleic Acid Chemistry 1.4.1-1.4.32, June 2009 1.4.1 Published online June 2009 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142700.nc0104s37 Supplement 37 Copyright C 2009 John Wiley & Sons, Inc. λ C1' C1' - C1' ~10.8 to 11.0 Å λ λ ~50 - 54 C1' Figure 1.4.1 Watson-Crick base pair parameters. See APPENDIX 1B and Figure A.1B.4 for other base-pairing schemes. for attachment of appendages, but substitution DNA polymerases require the pyrimidine O2 here may influence the conformation prefer- and purine N3 as recognition features for effi- ence about the glycosidic bond. Replacement cient template-mediated oligonucleotide syn- of the purine N7 with a carbon provides a site thesis (Horlacher et al., 1995). for attachment of appendages in a sterically tolerated position. There are many examples Base Pairs with 4-Hydrogen Bonds of appendages that, when added to the nat- Matsuda and co-workers have con- ural nucleobases, enhance base-pair stability. structed four bases (S.7 to S.10) that These include substituents that increase the pair as shown in Figure 1.4.3 (Minakawa acidity of proton donor sites (Yu et al., 1993) et al., 2003). When incorporated into or increase hydrophobicity and aromatic sur- the middle of the complementary DNA face area for enhanced base stacking (Inoue sequences 5-GCACCGAAXAAACCACG-3 et al., 1985), as well as appended cations for and 5-CGTGGTTTYCGGTGC-3 the du- electrostatic interactions with the phosphodi- plexes were destabilized based on a decrease ester backbone (Ueno et al., 1998). in Tm values. This is to be expected given the greater separation between the two C-1 of the opposing nucleosides. However, when the se- BASE PAIRS WITH ALTERNATIVE quences were extended to include three tan- HYDROGEN-BONDING PATTERNS dem base pairs [3 NN (S.7) opposite 3 OO Purine-Pyrimidine-Like Base Pairs (S.8) and 3 NO (S.9) opposite 3 ON (S.10)] Benner and co-workers originally de- the duplexes were substantially more stable scribed a set of nucleobase analogs that re- than the corresponding sequences containing semble natural bases, but have reconfigured G-C and A-T base pairs. However, NN and OO hydrogen-bonding patterns (Piccirilli et al., also pair effectively with G, ON pairs with A 1990). Six orthogonal base pairs (S.1 to S.6) and T, and NO pairs with T. The OO-G pair are shown in Figure 1.4.2. In later studies, may involve an alternative tautomeric form Benner and co-workers found that the origi- of OO. nal concept for the pyDDA base, S.3a,was The C-1 to C-1 distance in opposing nu- compromised because of the ease of epimer- cleosides is reduced when one of the imida- ization at C-1 of the pyrazine nucleoside at zopyridopyrimdine is replaced by naphthyri- pH 7. They found a suitable replacement with dine base analogs. Pairs between S.11 and the nitropyridone, S.3b, which is stable and S.12 and between S.13 and S.10 are illustrated still pairs effectively with the puAAD compo- in Figure 1.4.4. Duplex DNA from hybridiza- nent (Hutter and Benner, 2003; von Krosigk tion of 5-GCACCGAAXAAACCACG-3 and and Benner, 2004; Yang et al., 2006, 2007). 5-CGTGGTTTYCGGTGC-3 containing the Extensive studies of these nucleosides have base pairs shown in the figure have higher Tm revealed that even these subtle changes in values compared to the natural sequences with structure can have profound effects on ther- G-C and A-T base pairs (Hikishima et al., modynamic (Voegel and Benner, 1994) and 2005). When base pair S.10-S.13 was incor- biochemical properties (Switzer et al., 1993; porated at both ends of a DNA duplex, duplex Horlacher et al., 1995). For example, the C- stability and resistance to degradation by snake nucleoside pyrimidine mimics appear to base venom phosphodiesterase were increased sig- Unnatural pair more weakly than equivalent base pairs nificantly. The end-modified duplexes proved Nucleosides with composed of N-nucleosides. As far as bio- to be effective as decoy oligonucleotides tar- Unusual Base chemical properties, it appears that certain geted to NF-κB (Hikishima et al., 2006). Pairing Properties 1.4.2 Supplement 37 Current Protocols in Nucleic Acid Chemistry C-G Iso-C - Iso-G N H N O N dR N N dR HH H N NN O NN H H N N HH N O N O H N N dR 1 dR H 2 pyDAA - puADD pyAAD - puDDA T - Amino-A H N N N O N dR N N dR O N dR H HH HH N NN O NN N NN O2N H H H N N N N N N HH HH HH N O N O O dR 3b dR 4 dR 3a pyDDA - puAAD pyADA - puDAD N H N O N dR N N dR HH H N NN HH O NN CH3 O H NN N O H N H N N dR H 5 6 dR H pyDAD - puADA pyADD - puDAA Figure 1.4.2 Structures of six Watson-Crick-type base pairs utilizing mutually exclusive hydrogen-bonding schemes. The alternative hydrogen-bonding schemes are symbolized by the combinations, pu, py, A, and D, where pu represents purine analog, py represents pyrimidine analog, A represents H-bond acceptor, and D represents H-bond donor. Abbreviation: dR, deoxyribose. H H N N H O N N N H O N N N N N H N N N H N dR dR dR dR N H N N H N O N N OO N O N N N N N N H O O H N 7 H 8 9 H 10 Figure 1.4.3 Base pairs derived from imidazopyridopyrimidine nucleosides containing four hydro- gen bonds.
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