Triple helix formation at A₈XA₈·T₈YT₈

Abstract

We have examined the formation of DNA triple he&es between the oligonucleotides T,XTs (X = A,C,G,T) and DNA fragments containing the target sequences A,XA,.T,YT, (X=T,C,G; Y=A,G,C), by DNase I footprinting. We find that A8GA8-T&T* yields a footprint with T&T8 and shows a weaker interaction with T,, and T,GTB. AsC&*T,GT, yields a footprint with T,,, and shows weaker interaction with T,CTs. &TAs.T,AT, yields a footprint with T,GT, and shows weaker interaction with T,,. Each of the successful complexes is character&d by enhanced DNase I cleavage at the 3' end of the purine strand of the target, as well as protection at the 5' end. We have been unable to form triplexes with third strands of the type A,%.Triple helix; Sequence recognition; Stringency; DNase I footprinting One means of achieving long-range DNA sequence recognition is by oligonucleotide-directed triple helix formation [1,2]. To date the formation of these structures has been restricted to homopurine * homopyrimidine sequences. Two major types of intermolecular triplexes have been characterized. In one the third strand contains only pyrimidines (YYR type); AT is recognised by T while GC is recognised by C+, requiring PI-I'S less than 5.5 [3-5J The other motif has a p~ne~on~~g third strand (RRY type); AT is recognised by A while GC is recognised by G [6,7]. In each case the identical strands run antiparallel to each other [3]. These two motifs cannot be easily combined within a single molecule [S]. In each case recognition is achieved by the formation of specific hydrogen bonds between the third strand bases and major groove substituents on the purines of the target site.Although triple helices show exquisite sequence specificity we are interested to examine how single base changes, in both the target sequence and the third strand o~gonucleotide, affect triplex formation. In this paper we investigate how single base changes are tolerated within triple helices containing T*AT triplets. We have prepared DNA fragments containing the target sites AsXAs -T,YT, and examined their interaction with each oligonucleotide of the type TsNT8, where N = G,C,T or A, in turn. MATERIALS AND ~E~ODS Chemicals and enzymesDeoxyoligonucleotides were synthesised on an Applied Biosystems 380 B DNA synthesizer using standard phosphotamidite chemistry and used without further purification.DNase I was purchased from Sigma and stored at -20°C at a concentration of 7,200 U/ml. Reverse transcriptase was purchased from Promega; restriction enzymes were purchased from Pharmacia or New England Biolabs. Plasmids @UC19 and SmaI-cut, alkaline phosphatase-treated pUC18) were purchased from Pharmacia. DM fkagmerttsThe ~lf~rnpl~~~~ o~gonucleoti~s A8NA8 and T&T, (N = any base) were treated with polynucleotide kinase and ATP, annealed and ligated into &a1 (CCCIGGG)-cut pUCl9 or pUCl8. The ligation mixtures were transformed into E. co& TG2. White colonies were picked from X-gal, IPTG plates containing lOO~g/ml ampicillin. The sequences of successful clones were detemined by dideoxy sequencing u...

Specificity of Antiparallel DNA Triple Helix Formation

“…Before discussion of the effect of various central triplet combinations on the antiparallel triplexes studied in this paper, it should be noted that all the oligonucleotides produce footprints at each target at concentrations of 10 µM, and many complexes are still stable at 1.0-3.0 µM, even for those containing non-standard central triplets. This is in marked contrast to the results for parallel triple helix formation at A 8 XA 8 sites using T 8 XT 8 oligonucleotides which showed that a mismatch in the center of the third strand could totally abolish triplex formation, even at oligonucleotide concentrations as high as 100 µM (Chandler & Fox, 1993). This lower stringency and tighter binding could be a general property of antiparallel triplexes, but may reflect the greater stability of the antiparallel G‚-GC over parallel T‚AT triplets.…”

Effect of a triplex-binding ligand on triple helix formation at a site within a natural DNA fragment

“…We have recognized the single thymine that interrupts the block of purines with guanine, forming a G:TA triplet. The formation of G:TA triplets has been described by several groups [23][24][25][26], though it is thought to be less stable than T:AT and C + :GC. In addition we have changed the central thymine to adenine, by sitedirected mutagenesis, generating a perfect homopurine sequence GAGAAAAAAGAA [tyrT(46A)], targeting this with the oligonucleotide CTCTTTTTTCTT (12T), forming a structure containing 9iT:AT and 3iC + :GC triplets.…”

Extension of DNA triple helix formation to a neighbouring (AT)n site

“…Secondly recognition is generally limited to the duplex purine strand, so that triplexes can only be formed at homopurine.homopyrimidine sequences. Recent work has demonstrated that, within the parallel motit, it is also possible to form G'TA [6][7][8][9][10][11][12][13][14] and T-CG [7,8,15] triplets, although these each contain only one hydrogen bond and are thought to be less stable than T. AT and C ÷. GC.…”