Effects of an abasic site on triple helix formation characterized by affinity cleaving.

The stability of triple helical complexes of pyrimidine oligodeoxyribonucleotides containing one abasic 1,2-dideoxy-D-ribose (phi) residue was examined by affinity cleaving. Within a pyrimidine third strand, the triplets phi.AT, phi.GC, phi.TA and phi.CG are significantly less stable than the triplets, T.AT, C+GC and G.TA. The decrease in binding produced by an abasic residue is similar to that observed with imperfectly matched natural base triplets, with phi.AT and phi.GC being less stable than phi.TA and phi.CG triplets for the sequences studied.     

DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines.

We have used DNase I footprinting to assess the formation of triple helices at 15mer oligopurine target sites which are interrupted by several (up to four) adjacent central pyrimidine residues. Third strand oligonucleotides were designed to generate complexes containing central (X.TA)nor (X.CG)n triplets (X = each base in turn) surrounded by C+.GC and T.AT triplets. It has previously been shown that G.TA and T.CG are the most stable triplets for recognition of single TA and CG interruptions. We show that these triplets are the most useful for recognizing consecutive pyrimidine interruptions and find that addition of each pyrimidine residue leads to a 30-fold decrease in third strand affinity. The addition of 10 microM naphthylquinoline triplex-binding ligand stabilizes each complex so that all the oligonucleotides produce footprints at similar concentrations (0.3 microM). Targets containing two pyrimidines are only bound by oligonucleotides generating (G.TA)2 and (T.CG)2 with a further 30-fold decrease in affinity. (G.TA)2 is slightly more stable than (T.CG)2. In the presence of the triplex-binding ligand the order of stability is (G.TA)2 > (C.TA)2 > (T.TA)2 > (A.TA)2 and (T.CG)2 > (C.CG)2 > (G.CG)2 = (A.CG)2. No oligonucleotide footprints are generated at target sites containing three consecutive pyrimidines, though addition of 10 microM triplex-binding ligand produces stable complexes with oligonucleotides generating (G.TA)3, (T.CG)3 and (C.CG)3, with a further 30-fold reduction in affinity. No footprints are generated at targets containing four Ts, though the ligand induces a weak interaction with the oligonucleotide generating (T.CG)4.     

The influence of single base triplet changes on the stability of a pur.pur.pyr triple helix determined by affinity cleaving.

The influence of sixteen base triplet changes at a single position within a pur.pur.pyr triple helix was examined by affinity cleaving. For the 15 base pair target site studied here, G.GC, A.AT and T.AT triplets stabilize a triple helix to a greater extent than the other 13 natural triplets (pH = 7.4, 25 degrees C). Weaker interactions were detected for the C.AT, A.GC and T.CG triplets. The absence of specific, highly stabilizing interactions between third strand bases and the CG or TA base pairs demonstrates a current sequence limitation to formation of this structure. Models for the two dimensional base triplet interactions for all possible 16 natural triplets are presented.     

DNA triple helix formation at target sites containing several pyrimidine interruptions: stabilization by protonated cytosine or 5-(1-propargylamino)dU.

DNase I footprinting has been used to study the formation of parallel triplexes at oligopurine target sequences which are interrupted by pyrimidines at regular intervals. TA interruptions are targeted with third strand oligonucleotides containing guanine, generating G x TA triplets, while CG base pairs are targeted with thymine, forming T x CG triplets. We have attempted to optimize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third strand thymines with the positively charged analogue 5-(1-propargylamino)dU (U(P)). For the target sequence (AAAT)(5)AA, in which pyrimidines are positioned at every fourth residue, triplex formation with TG-containing oligonucleotides is only detected in the presence of a triplex-binding ligand, though stable triplexes were detected at the target site (AAAAAT)(3)AAAA. Triplex stability at targets containing pyrimidines at every fourth residue is increased by introducing guanines into the duplex repeat unit using the targets (AGAT)(5)AA and (ATGA)(5)AA. In contrast, placing C(+) x GC triplets on the 5'-side of G x TA, using the target (AGTA)(5)TT, produces complexes of lower stability. We have attempted further to increase the stability of these complexes by using the positively charged thymine base analogue U(P), and have shown that (TU(P)TG)(5)TT forms a more stable complex with target (AAAT)(5)AA than the unmodified third strand, generating a footprint in the absence of a triplex-binding ligand. Triplex formation at (AGTA)(5)AA is improved by using the modified oligonucleotide (TCGU(P))(5)TT, generating a complex in which the charged triplets C(+) x GC and U(P) x AT alternate with uncharged triplets. In contrast, placing U(P) x AT triplets adjacent to C(+) x GC, using the third strand oligonucleotide (U(P)CGT)(5)TT, reduces triplex formation, while the third strand with both substitutions, (U(P)CGU(P))(5)TT, produces a complex with intermediate stability. It appears that, although adjacent U(P) x AT triplets form stable triplexes, placing U(P) x AT adjacent to C(+) x GC is unfavorable. Similar results were obtained with fragments containing CG inversions within the oligopurine tract, though triplexes at (AAAAAC)(3)AA were only detected in the presence of a triplex-binding ligand. Placing C(+) x GC on the 5'-side of T x CG triplets also reduces triplex formation, while a 3'-C(+) x GC produces complexes with increased stability.     

Formation of DNA triple helices incorporating blocks of G.GC and T.AT triplets using short acridine-linked oligonucleotides.

We have used DNase I footprinting to assess triple helix formation at target sites containing the sequences A6G6.C6T6 and G6A6.T6C6. These sequences can be recognized by the acridine-linked oligopyrimidines Acr-T5C5 and Acr-C5T5 respectively at low pH, using well-characterised T.AT and C+.GC triplets. At pH 7.5 A6G6.C6T6 is specifically bound by Acr-G5T5, utilising G.GC and T.AT triplets in which the third strand runs antiparallel to the purine strand of the duplex. This interaction requires the presence of magnesium ions. No interaction was detected with Acr-T5G5, an oligonucleotide designed to form parallel G.GC and T.AT triplets. In contrast neither Acr-T5G5 nor Acr-G5T5 produced DNase I footprints with the target sequence G6A6.T6C6. These results suggest that, in an antiparallel R.RY triple helix, the T.AT triplet is weaker than the G.GC triplet. We find no evidence for the formation of structures containing parallel G.GC triplets.     

NMR structural studies of intramolecular (Y+)n.(R+)n(Y-)nDNA triplexes in solution: imino and amino proton and nitrogen markers of G.TA base triple formation.

We reported previously on NMR studies of (Y+)n.(R+)n(Y-)n DNA triple helices containing one oligopurine strand (R)n and two oligopyrimidine strands (Y)n stabilized by T.AT and C+.GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidine.adenosine base pair to form a G.TA triple in an otherwise (Y+)n.(R+)n(Y-)n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)n.(R+)n(Y-)n triplex with the strands linked through two T5 loops and that contains a central G.TA triple flanked by T.AT triples. The G.TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H2O solution, pH 4.85, at 5 degrees C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T.AT triples, as well as the guanosine imino and cytidine amino protons involved in Watson-Crick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C+.GC triples in the NOESY spectrum of the G.TA triplex. The NMR data are consistent with the proposed pairing alignment for the G.TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triple helix formation at (AT)n adjacent to an oligopurine tract.

We have used DNase I footprinting to investigate the recognition of (AT) n tracts in duplex DNA using GT-containing oligonucleotides designed to form alternating G.TA and T.AT triplets. Previous studies have shown that the formation of these complexes is facilitated by anchoring the triplex with a block of adjacent T.AT triplets, i.e. using T11(TG)6to recognize the target A11(AT)6. (AT)6T11. In the present study we have examined how the stability of these complexes is affected by the length of either the T.AT tract or the region of alternating G.TA and T.AT triplets, using oligonucleotides of type T x (TG) y to recognize the sequence A11(AT)11. We find that successful triplex formation at (AT)n (n = 3, 6 or 11) can be achieved with a stabilizing tail of 11xT.AT triplets. The affinity of the third strand increases with the length of the (GT) n tract, suggesting that the alternating G.TA and T.AT triplets are making a positive contribution to stability. These complexes are stabilized by the presence of manganese or a triplex-specific binding ligand. Shorter oligo-nucleotides, such as T7(TG)5, bind less tightly and require the addition of a triplex-binding ligand. T4(GT)5showed no binding under any conditions. Oligo-nucleotides forming a 3'-terminal T.AT are marginally more stable that those with a terminal G.TA. The stability of these complexes was further increased by replacing two of the T.AT triplets in the T n tail region with two C+.GC triplets.     

DNA triple-helix formation at target sites containing duplex mismatches

We have studied the formation of DNA triple helices at target sites that contain mismatches in the duplex target. Fluorescence melting studies were used to examine a series of parallel triple helices that contain all 64 N.XZ triplet combinations at the centre (where N, X and Z are each of the four natural DNA bases in turn). Similar experiments were also performed with N=bis-amino-U (BAU) (for stable recognition of AT base pairs) and N=S (for recognition of TA inversions). We find that the introduction of a duplex mismatch destabilises the C+.GZ, T.AZ and G.TZ triplets. A similar effect is seen with BAU.AZ triplets. In contrast, other base combinations, based on non-standard triplets such as C.AZ, T.TZ, G.CZ and A.CZ are stabilised by the presence of a duplex mismatch. In each case S binds to sites containing duplex mismatches better than the corresponding Watson-Crick base pairs.     

Stable recognition of TA interruptions by triplex forming oligonucleotides containing a novel nucleoside

We have prepared the 2'-aminoethoxy derivative of the S nucleoside ((2AE)S) and incorporated it into triplex-forming oligonucleotides for recognition of TA interruptions within a target oligopurine tract. Fluorescence melting, UV melting, and DNase I footprinting experiments show that (2AE)S has greater affinity than G or S for a single TA interruption. Stable triplexes are formed at pH 6.0 at an 18-mer target site containing two TA interruptions, even though this contains eight C(+).GC triplets. Although (2AE)S and S produce stable triplexes at TA interruptions, they also interact with other base pairs, in particular, CG, although the selectivity for TA improves with increased pH.( 2AE)S is the best nucleoside described so far for recognition of TA within a triple-helix target.     

Site-resolved energetics in DNA triple helices containing G*TA and T*CG triads

Recognition of specific sites in double-helical DNA by triplex-forming oligonucleotides has been limited until recently to sites containing homopurine-homopyrimidine sequences. G*TA and T*CG triads, in which TA and CG base pairs are specifically recognized by guanine or by thymine, have now extended this recognition code to DNA target sites of mixed base sequences. In the present work, we have obtained a characterization of the stabilities of G*TA and T*CG triads, and of the effects of these triads upon canonical triads, in triple-helical DNA. The three DNA triplexes investigated are formed by the folding of the 31-mers d(GAAXAGGT(5)CCTYTTCT(5)CTTZTCC) with X = G, T, or C, Y = C, A, or G, and Z = C, G, or T. We have measured the exchange rates of imino protons in each triad of the three triplexes using nuclear magnetic resonance spectroscopy. The exchange rates are used to map the local free energy of structural stabilization in each triplex. The results indicate that the stability of Watson-Crick base pairs in the G*TA and T*CG triads is comparable to that of Watson-Crick base pairs in canonical triads. The presence of G*TA and T*CG triads, however, destabilizes neighboring canonical triads, two or three positions removed from the G*TA/T*CG site. Moreover, the long-range destabilizing effects induced by the T*CG triad are larger than those induced by the G*TA triad. These findings reveal the molecular basis for the lower overall stability of G*TA- and T*CG-containing triplexes.     

Nuclear magnetic resonance structural studies of intramolecular purine.purine.pyrimidine DNA triplexes in solution. Base triple pairing alignments and strand direction

Recently, P.A. Beal and P.B. Dervan, expanding on earlier observations by others, have established the formation of purine.purine.pyrimidine triple helices stabilized by G.GC, A.AT and T.AT base triples where the purine-rich third strand was positioned in the major groove of the Watson-Crick duplex and anti-parallel to its purine strand. The present nuclear magnetic resonance (n.m.r.) study characterizes the base triple pairing alignments and strand direction in a 31-mer deoxyoligonucleotide that intramolecularly folds to generate a 7-mer (R/Y-)n.(R+)n(Y-)n triplex with the strands linked by two T5 loops and stabilized by potential T.AT and G.GC base triples. (R and Y stand for purine and pyrimidine, respectively, while the signs establish the strand direction.) This intramolecular triplex gives well-resolved exchangeable and non-exchangeable proton spectra with Li+ as counterion in aqueous solution. These studies establish that the T1 to C7 pyrimidine and the G8 to A14 purine strands are anti-parallel to each other and align through Watson-Crick A.T and G.C pair formation. The T15 to G21 purine-rich third strand is positioned in the major groove of this duplex and pairs through Hoogsteen alignment with the purine strand to generate T.AT and G.GC triples. Several lines of evidence establish that the thymidine and guanosine bases in the T15 to G21 purine-rich third strand adopt anti glycosidic torsion angles under conditions where this strand is aligned anti-parallel to the G8 to A14 purine strand. We have also recorded imino proton n.m.r. spectra for an (R-)n.(R+)n(Y-)n triplex stabilized by G.GC and A.AT triples through intramolecular folding of a related 31-mer deoxyoligonucleotide with Li+ as counterion. The intramolecular purine.purine.pyrimidine triplexes containing unprotonated G.GC, A.AT and T.AT triples are stable at basic pH in contrast to pyrimidine.purine.pyrimidine triplexes containing protonated C+.GC and T.AT triples, which are only stable at acidic pH.     

Thermodynamic contributions for the incorporation of GTA triplets within canonical TAT/TAT and C+GC/C+GC base-triplet stacks of DNA triplexes

Nucleic acid triple helices may be used in the control of gene expression. One limitation of using triplex-forming oligonucleotides as therapeutic agents is that their target sequences are limited to homopurine tracts. To increase the repertoire of sequences that can be targeted, it has been postulated that a guanine can target a thymidine forming a stable GTA mismatch triplet. In this work, we have used a combination of optical and calorimetric techniques to determine thermodynamic unfolding profiles of two triplexes containing a single GTA triplet, d(A(3)TA(3)C(5)T(3)AT(3)C(5)T(3)GT(3)) (ATA) and d(AGTGAC(5)TCACTC(5)TCGCT) (GTG), and their control triplexes, d(A(7)C(5)T(7)C(5)T(7)) (TAT7) and d(AGAGAC(5)TCTCTC(5)TCTCT) (AG5T). In general, the presence of a GTA mismatch in DNA triplexes is destabilizing; however, this destabilization is greater when placed in a C(+)GC/C(+)GC base-triplet stack than between a TAT/TAT stack. These destabilizations are accompanied by a reduced unfolding enthalpy of approximately 10 kcal/mol, suggesting a decrease in the base stacking contributions surrounding the mismatch. Relative to their corresponding control triplexes, the folding of ATA is accompanied by a lower counterion uptake and a similar proton uptake, while GTG folding is accompanied by an increase in the counterion and proton uptakes. These effects are consistent with the observed decrease in stacking interactions. The overall results indicate that the main difficulty of targeting pyrimidine interruptions is that the decrease in stacking contributions, due to the incorporation of a GTA mismatch, affects the stability of the neighboring base triplets. This suggests that nucleotide analogues that increase the strength of these base-triplet stacks will result in a more effective targeting of pyrimidine interruptions.     

Relative specificities in binding of Watson-Crick base pairs by third strand residues in a DNA pyrimidine triplex motif.

The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.     

Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites.

The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrimidine.purine.pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 degrees C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3' (1) and 5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3' (2, m5C is 2'-deoxy-5-methylcytidine) increased by 10- and 20-fold, respectively, from pH 7.6 to 5.8, indicating that the corresponding triple-helical complexes are stabilized by 1.4 and 1.7 kcal.mol-1, respectively, at the lower pH. Replacement of the five cytosine residues in 1 with 5-methylcytosine residues to yield 2 affords a stabilization of the triple helix by 0.1-0.4 kcal.mol-1 over the pH range 5.8-7.6. An analysis of these data in terms of a quantitative model for a general pH-dependent equilibrium transition revealed that pyrimidine oligonucleotides with cytidine and 5-methylcytidine form local triple-helical structures with apparent pKa's of 5.5 (C+GC triplets) and 5.7 (m5C+GC triplets), respectively, and that the oligonucleotides should bind to single sites on large DNA with apparent affinity constants of approximately 10(6) M-1 even above neutral pH.     

Comparison of antiparallel A.AT and T.AT triplets within an alternate strand DNA triple helix.

We have examined the formation of alternate strand triple-helices at the target sequence A11(TC)6.(GA)6T11 using the oligonucleotides T11(AG)6 and T11(TG)6, by DNase I footprinting. These third strands were designed so as to form parallel T.AT triplets together with antiparallel G.GC and A.AT or T.AT triplets. We find that, although both oligonucleotides yield clear footprints at similar concentrations (0.3 microM) in the presence of manganese, only T11(TG)6 forms a stable complex in magnesium-containing buffers, albeit at a higher concentration (10-30 microM). Examination of the interaction of (AG)6 and (TG)6 with half the target site confirmed that the complex containing A.AT triplets was only stable in the presence of manganese. In contrast no binding of (TG)6 was detected in the presence of either metal ion, suggesting that the reverse-Hoogsteen T.AT triplet is less stable that G.GC. We suggest that, within the context of G.GC triplets, the rank order of antiparallel triplet stability is A.AT (Mn2+) > T.AT (Mn2+) > T.AT (Mg2+) > A.AT (Mg2+). Third strands containing a single base substitution in the centre of either the parallel or antiparallel portion showed a (10-fold) weaker interaction in manganese-containing buffers, and no interaction in the presence of magnesium.     

Sequence specificity of the non-natural pyrido[2,3-d]pyrimidine nucleoside in triple helix formation.

The non-natural pyrido[2,3-d]pyrimidine nucleoside F, which pairs preferentially with guanine (G) and adenine (A) within double-helical DNA, recognizes with high selectivity AT base pairs within triple-helical complexes. These observations suggest that F may exist in different tautomeric forms within double-helical and triple-helical complexes. Analysis of the base stacking properties of this extended ring system using two oligodeoxyribonucleotides containing terminal thymines and/or pyrido[2,3-d]pyrimidines bound to adjacent sites showed a decrease in free energy of binding in a triple-helical complex in the order (5'-3') TT > FT > TF > FF.     

Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I): effects of single base pair transitions on the cleavage of preferred pyrimidine-purine-pyrimidine triplets

The cleavage of DNA restriction fragments by bis(1,10-phenanthroline)copper(I) [[(OP)2CuI]+] is sequence dependent: the trimer TAT is most strongly preferred, while the trimer TGT and tetramers TAAT, TAGT, and CAGT are strongly to moderately preferred [Veal, J. M., & R. L. (1988) Biochemistry 27, 1822-1827]. [(OP)2CuI]+ cleavage of a series of oligonucleotide duplexes of the type 5'-CCCTPyPuPyCCCC-3'/3'-GGGAPuPyPuGGGG-5' (Py = pyrimidine; Pu = purine) was examined to determine the effects of purine substituents in the central triplet on specificity. The relative cleavage rates of different PyPuPy triplets in oligomers were similar to those observed for restriction fragments. The undecamer duplex containing the trimer TAT (TTATC) was most preferentially cleaved, predominantly at the central adenosine and the adjacent 3'-thymidine. Duplexes differing from TTATC by a single A.T----G.C transition in the central triplet were cleaved at significantly reduced rates relative to TTATC, the order of preference being TAT greater than TGT greater than TAC greater than CAT. By contrast, duplexes differing from TTATC by a single A.T----I.C transition were cleaved at rates similar to those for TTATC when the transition occurred at the 5'-pyrimidine or central purine [i.e., C(.I)AT and TIT]. A duplex containing the trimer TAC(.I) was cleaved at a reduced rate similar to the duplex containing TAC(.G). The guanine 2-amino group at positions 1 and 2, but not position 3, of a 5'-PyPuPy-3' trimer is therefore implicated as a strong inhibitor of DNA binding by the copper-phenanthroline complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Base triplet nonisomorphism strongly influences DNA triplex conformation: effect of nonisomorphic G* GC and A* AT triplets and bending of DNA triplexes

Structural understanding of DNA triplexes is grossly inadequate despite their efficacy as therapeutic agents. Lack of structural similarity (isomorphism) of base triplets that figure in different DNA triplexes brings in an added complexity. Recently, we have shown that the residual twist (Deltat degrees ) and the radial difference (Deltar A) adequately define base triplet nonisomorphism in structural terms and allow assessment of their role in conferring stability as well as sequence-dependent structural variations in DNA triplexes. To further corroborate these, molecular dynamics (MD) simulations are carried out on DNA triplexes comprising nonisomorphic G* GC and A* AT base triplets under different sequential contexts. Base triplet nonisomorphism between G* GC and A* AT triplets is dominated by Deltat degrees (9.8 degrees ), in view of small Deltar (0.2 A), and is in contrast to G* GC and T* AT triplets where both Deltat degrees (10.6 degrees ) and Deltar (1.1A) are prominent. Results show that Deltat degrees alone enforces mechanistic influence on the triplex-forming purine strand so as to favor a zigzag conformation with alternating conformational features that include high (40 degrees ) and low (20 degrees ) helical twists, and high anti(G) and anti(A) glycosyl conformation. Higher thermal stability of this triplex compared to that formed with G* GC and T* AT triplets can be traced to enhanced base-stacking and counterion interactions. Surprisingly, it is found for the first time that the presence of a nonisomorphic G* GC or A* AT base triplet interrupting an otherwise mini A* AT or G* GC isomorphic triplex can induce a bend/curvature in a DNA triplex. These observations should prove useful in the design of triplex-forming oligonucleotides and in the understanding the binding affinities of this triplex with proteins.     

Structural consequences of D-amino acids in collagen triple-helical peptides

The effects of racemization of aspartic acid on triple-helical formation have been studied using a "host-guest" peptide approach where selected guest Gly-Xaa-Yaa triplets were included within a common acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide frame-work. Four guest triplets, Gly-Asp-Hyp and Gly-Asp-Ala where Asp is either L-Asp or D-Asp were studied. Thermal stability data indicated that incorporation of D-Asp residues prevented triple-helix formation in phosphate buffered saline, although triple-helical structures were formed in a stabilizing solvent, 67% aqueous ethylene glycol. In this solvent the melting temperatures of D-Asp containing peptides were more than 30 degrees C lower than the corresponding peptides containing L-Asp. For Gly-Asp-Ala peptides, but not Gly-Asp-Hyp, peptides, melting profiles indicated that a mixture of the D- and L-Asp containing peptides were able to form heterotrimer triple-helical molecules. These studies illustrate the dramatic destabilizing effect of D-amino acids on the triple-helix stability, but indicate that they can be accommodated in this conformation.