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1、A Two-stranded Template-based Approach to G?(C-A)Triad Formation: Designing Novel Structural Elementsinto an Existing DNA FrameworkAbdelali Kettani, Gautam Basu, Andrey Gorin, Ananya MajumdarEugene Skripkin and Dinshaw J. Patel*Cellular Biochemistry andBiophysics Program, MemorialSloan-Kettering Can
2、cer CenterNew York, NY 10021, USAWe have designed a DNA sequence, d(G-G-G-T-T-C-A-G-G), whichdimerizestoforma2-foldsymmetricG-quadruplexinwhichG(syn)?G(anti)?G(syn)?G(anti) tetrads are sandwiched between all transG?(C-A) triads. The NMR-based solution structural analysis was greatlyaided by monitori
3、ng hydrogen bond alignments across NH? ? ?N andNH? ? ?O1C hydrogen bonds within the triad and tetrad, in a uni-formly (13C,15N)-labeled sample of the d(G-G-G-T-T-C-A-G-G) sequence.The solution structure establishes that the guanine base-pairs with thecytosine through Watson-Crick G?C pair formation
4、and with adeninethrough sheared G?A mismatch formation within the G?(C-A) triad. Amodel of triad DNA was constructed that contains the experimentallydetermined G?(C-A) triad alignment as the repeating stacked unit.# 2000 Academic PressKeywords: G?(C-A) base triad; G-quadruplex; minor groove recognit
5、ion ofG?C pair; uniform13C,15N-labeled DNA*Corresponding authorIntroductionThe anti-parallel Watson-Crick-paired structureof DNA has played a key role in modern biology asa repository of genetic information and as asequence-specific target for proteins involved ingene packaging and regulation. Varia
6、nts of thisarchitecture include the recently elucidated struc-ture of stretched and over-wound DNA fibers(Allemand et al., 1998) where the phosphate groupsare in the interior and the bases are splayed out,and an earlier structure of the dinucleotide repeatZ-DNA (Wang et al., 1979) with its unanticip
7、atedleft-handed zig-zag repeat. It should be noted thatinside-out DNA, characteristic of the stretched andover-wound state had been previously proposed forthe architecture of filamentous bacteriophage pf1DNA bound to coat protein (Liu & Day. 1994) andthe recently solved structure of Z-DNA bound toad
8、enine deaminase (Schwartz et al., 1999) has impli-cations for transient left-handed DNA architecturesin actively transcribing genes (Herbert & Rich,1996). These two examples reinforce the premisethat unusual DNA architectures may contribute inimportant ways to gene function and regulation.An equally
9、 interesting situation arises when oneconsiders potential DNA architectures containingnon-Watson-Crick pairing alignments. Such struc-tural motifs could arise if individual strands ofduplex DNA undergo higher order folding invol-ving unanticipated multi-stranded pairing align-ments stabilized by mis
10、matches, triples, triads andtetrads (reviewed by Neidle, 1999). These architec-tures span the range from two-stranded zipper(Sheppard et al., 1998) and arrowhead (Kettani,et al., 1999) motifs, to three-stranded triplexes(reviewed by Radhakrishnan & Patel, 1944; Sunet al., 1996; Wang & Feigon, 1999)
11、and four-strandedquadruplex(reviewedbyRhodes&Giraldo, 1995; Patel et al., 1999) and i-motifs(Gehring et al., 1993; Chen et al., 1994). Even higherorderpairingalignmentsinvolvingpentads(Chaput & Switzer, 1999) and hexads (Kettam et al.,2000) have been reported recently. Many of thesemulti-stranded ar
12、chitectures are potential candi-dates for adaptation by DNA oligomers that con-tain elements of telomeric, centromeric and tripletrepeat disease sequences (reviewed by Patel et al.,1999).Present address: G. Basu, Department of Biophysics,Bose Institute, Calcutta, 700 054, India.E-mail address of the
13、 corresponding author:pateldmskcc.orgdoi:10.1006/jmbi.2000.3932 available online at http:/ onJ. Mol. Biol. (2000) 301, 1291460022-2836/00/01012918 $35.00/0# 2000 Academic PressOur laboratory has for some time focused con-siderable effort towards the identification of basetriads, a motif first propos
14、ed by Kuryavyi & Jovin(1995, 1996), and postulated by them as buildingblocks for architectural models of triplet-repeat dis-ease sequences. Both base triads and base triplesare formed through co-planar alignment of threebases, except that the former is a two-strandedstructure, while the latter is a
15、three-stranded struc-ture. In essence, two adjacent bases from onestrand adopt a co-planar platform arrangementand pair with a third base from the partner strandto form a base triad (Kuryavyi & Jovin, 1995,1996).The identification of new pairing alignments, bethey triads or other motifs, generally i
16、nvolves thescreening of scores of sequences for those rare can-didates that give NMR spectra containing unusualfeatures and which preferably adopt a single stableconformation with narrow resonances. A rationaldesign-based screening strategy for new alignmentmotifs could have advantages in both reducing thenumber of sequences evaluated and the potentialgenerality of inbuilt designed structural principlesassociatedwiththesuccessfulcandidates.We report below on the identification of theG?(C-A) tria
