Stabilization factors affecting duplex formation of peptide nucleic acid with DNA.

Peptide nucleic acid (PNA) is an oligonucleotide analogue in which the sugar-phosphate backbone is replaced by an N-(2-aminoethyl)glycine unit to which the nucleobases are attached. We investigated the thermodynamic behavior of PNA/DNA hybrid duplexes with identical nearest neighbors but with different sequences and chain lengths (5, 6, 7, 8, 10, 12, and 16 mers) to reveal whether the nearest-neighbor model is valid for the PNA/DNA duplex stability. CD spectra of 6, 7, and 8 mer PNA/DNA duplexes showed similar signal, while 10, 12, and 16 mer duplexes did not. The average difference in Delta G degrees (37) for short PNA/DNA duplexes with identical nearest-neighbor pairs was only 3.5%, whereas that of longer duplexes (10, 12, and 16 mers) was 16.4%. Therefore, the nearest-neighbor model seems to be useful at least for the short PNA/DNA duplexes. Thermodynamics of PNA/DNA duplexes containing 1--3 bulge residues were also studied. While the stability of the 12 mer DNA/DNA duplex decreased as the number of bulge bases increases, the number of bulge bases in PNA/DNA unchanged the duplex stability. Thus, the influence of bulge insertion in the PNA/DNA duplexes is different from that of a DNA/DNA duplex. This might be due to the different base geometry in a helix which may potentially make hydrogen bonds in a base pair and stacking interaction unfavorable compared with DNA/DNA duplexes.     

Molecular dynamics structures of peptide nucleic acid x DNA hybrid in the wild-type and mutated alleles of Ki-ras proto-oncogene--stereochemical rationale for the low affinity of PNA in the presence of an AC mismatch

The low affinity of peptide nucleic acid (PNA) to hybridize with DNA in the presence of a mismatch endows PNA with a high degree of discriminatory capacity that has been exploited in therapeutics for the selective inhibition of the expression of point-mutated genes. To obtain a structural basis for this intriguing property, molecular dynamics simulations are carried out on PNA x DNA duplexes formed at the Ki-ras proto-oncogene, comprising the point-mutated (GAT), and the corresponding wild-type (GGT) codon 12. The designed PNA forms an A...C mismatch with the wild-type sequence and a perfect A...T pair with the point mutated sequence. Results show that large movements in the pyrimidine base of the A...C mismatch cause loss of stacking, especially with its penultimate base, concomitant with a variable mismatch hydrogen bond, including its occasional absence. These, in turn, bring about dynamic water interactions in the vicinity of the mismatch. Enthalpy loss and the disproportionate entropy gain associated with these are implicated as the factors contributing to the increase in free energy and diminished stability of PNA x DNA duplex with the A...C mismatch. Absence of these in the isosequential DNA duplex, notwithstanding the A...C mismatch, is attributed to the differences in topology of PNA x DNA vis-à-vis DNA duplexes. It is speculated that similar effects might be responsible for the reduced stability observed in PNA x DNA duplexes containing other base pair mismatches, and also in mismatch containing PNA x DNA duplexes.     

C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60–80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.     

Peptide nucleic acids: deoxyribonucleic acid mimics with a peptide backbone

This tutorial briefly describes a new class of synthetic biopolymer, which is referred to as peptide nucleic acid (PNA). In PNA, individual nucleobases are linked to an achiral neutral peptide backbone. PNA exhibits the hybridization characteristic (e.g., Watson—Crick duplex formation) of DNA. The achiral peptide backbone provides similar interbase distances as natural DNA, and adequate flexibility to permit base pair interactions with complementary RNA or DNA strands. Several potential applications of PNA oligomers in biotechnology are suggested. These include the use of PNAs as a probe for specific recognition of a DNA or RNA sequence selective, purification of nucleic acids via designed high affinity binding to PNA, screening for DNA mutations, and as possible therapeutic agents.     

Enhanced base-pair opening in the adenine tract of a RNA double helix

Proton exchange and nuclear magnetic resonance spectroscopy are being used to characterize the kinetics and energetics of base-pair opening in two nucleic acid double helices. One is the RNA duplex 5'-r(GCGAUAAAAAGGCC)-3'/5'-r(GGCCUUUUUAUCGC)-3', which contains a central tract of five AU base pairs. The other is the homologous DNA duplex with a central tract of five AT base pairs. The rates and the equilibrium constants of the opening reaction of each base pair are measured from the dependence of the exchange rates of imino protons on ammonia concentration, at 10 °C. The results reveal that the tract of AU base pairs in the RNA duplex differs from the homologous tract of AT base pairs in DNA in several ways. The rates of opening of AU base pairs in RNA are high and increase progressively along the tract, reaching their largest values at the 3'-end of the tract. In contrast, the opening rates of AT base pairs in DNA are much lower than those of AU base pairs. Within the tract, the largest opening rate is observed for the AT base pair at the 5'-end of the tract. These differences in opening kinetics are paralleled by differences in the stabilities of individual base pairs. All AU base pairs in the RNA are less stable than the AT base pairs in the DNA. The presence of the tract enhances these differences by increasing the stability of AT base pairs in DNA while decreasing the stability of AU base pairs in RNA. Due to these divergent trends, along the tracts, the AU base pairs become progressively less stable than AT base pairs. These findings demonstrate that tracts of AU base pairs in RNA have specific dynamic and energetic signatures that distinguish them from similar tracts of AT base pairs in DNA.     

Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes

Peptide nucleic acids (PNAs) have been developed for applications in biotechnology and therapeutics. There is great potential in the development of chemically modified PNAs or other triplex-forming ligands that selectively bind to RNA duplexes, but not single-stranded regions, at near-physiological conditions. Here, we report on a convenient synthesis route to a modified PNA monomer, thio-pseudoisocytosine (L), and binding studies of PNAs incorporating the monomer L. Thermal melting and gel electrophoresis studies reveal that L-incorporated 8-mer PNAs have superior affinity and specificity in recognizing the duplex region of a model RNA hairpin to form a pyrimidine motif major-groove RNA₂–PNA triplex, without appreciable binding to single-stranded regions to form an RNA–PNA duplex or, via strand invasion, forming an RNA–PNA₂ triplex at near-physiological buffer condition. In addition, an L-incorporated 8-mer PNA shows essentially no binding to single-stranded or double-stranded DNA. Furthermore, an L-modified 6-mer PNA, but not pseudoisocytosine (J) modified or unmodified PNA, binds to the HIV-1 programmed −1 ribosomal frameshift stimulatory RNA hairpin at near-physiological buffer conditions. The stabilization of an RNA₂–PNA triplex by L modification is facilitated by enhanced van der Waals contacts, base stacking, hydrogen bonding and reduced dehydration energy. The destabilization of RNA–PNA and DNA–PNA duplexes by L modification is due to the steric clash and loss of two hydrogen bonds in a Watson–Crick-like G–L pair. An RNA₂–PNA triplex is significantly more stable than a DNA₂–PNA triplex, probably because the RNA duplex major groove provides geometry compatibility and favorable backbone–backbone interactions with PNA. Thus, L-modified triplex-forming PNAs may be utilized for sequence-specifically targeting duplex regions in RNAs for biological and therapeutic applications.     

Selective catalysis of A.T base pair proton exchange in DNA complexes: imino proton NMR analysis.

Polyaromatic molecules with amino chain substituents, upon binding with DNA, selectively catalyze exchange of the A.T base pair protons with bulk water protons. The amine-catalyzed exchange is mediated by compounds which are A.T and G.C base sequence specific, intercalators, and outside binders. A mechanism for the selective exchange, involving transient opening and closing of individual A.T base pairs in the duplex, is discussed.     

Peptide nucleic acid-DNA duplexes containing the universal base 3-nitropyrrole

A peptide nucleic acid (PNA) monomer containing the universal base 3-nitropyrrole was synthesized by coupling 1-carboxymethyl-3-nitropyrrole to ethyl N-[2-(tert-butoxycarbonylamino)ethyl]glycinate. The PNA sequence H-TGTACGTXACAACTA-NH2 (X = 3-nitropyrrole and C) and DNA sequence 5'-TGTACGTXACAACTA-3' were synthesized and thermal melting studies with the complementary DNA sequence 5'-TAGTTGTYACGTACA-3' (Y = A,C, G, T) compared. The T(m) data show that 3-nitropyrrole pairs indiscriminately with all four natural nucleobases as a constituent of either DNA or PNA. However, 3-nitropyrrole-containing PNA-DNA (average T(m) value = 51.1 degrees C) is significantly more thermally stable than 3-nitropyrrole-containing DNA-DNA (average T(m) value = 39.6 degrees C). From circular dichroism measurements, it is apparent that 3-nitropyrrole in the PNA strand causes a significant change in duplex structure.     

Imidazole-imidazole pair as a minor groove recognition motif for T:G mismatched base pairs.

The T:G mismatched base pair is associated with many genetic mutations. Understanding its biological consequences may be aided by studying the structural perturbation of DNA caused by a T:G base pair and by specific probing of the mismatch using small molecular ligands. We have shown previously that AR-1-144, a tri-imidazole (Im-Im-Im) minor groove binder, recognizes the sequence CCGG. NMR structural analysis of the symmetric 2:1 complex of AR-1-144 and GAACCGGTTC revealed that each AR-1-144 binds to four base pairs with the guanine N2 amino group forming a bifurcated hydrogen bond to a side-by-side Im/Im pair. We predicted that the free G-N2 amino group in a T:G wobble base pair can form two individual hydrogen bonds to a side-by-side Im/Im pair. Thus an Im/Im pair may be a good recognition motif for a T:G base pair in DNA. Cooperative and tight binding of an AR-1-144 homodimer to GAACTGGTTC permits a detailed structural analysis by 2D NOE NMR refinement and the refined structure confirms our prediction. Surprisingly, AR-1-144 does not bind to GAATCGGTTC. We further show that both the Im-Im-Im/Im-Py-Im heterodimer and the Im-Im-Im/Im-Im-Im homodimer bind strongly to the CACGGGTC + GACTCGTG duplex. These results together suggest that an Im/Im pair can specifically recognize a single T:G mismatch. Our results may be useful in future design of molecules (e.g. linked dimers) that can recognize a single T:G mismatch with specificity.     

Constrained flexibility in PNA: DNA binding studies with bridged aminopropylglycyl PNA

Introduction of methylene bridges in aegPNA and apgPNA molecules give rise to cyclic five and six membered ring structures. Synthesis of a new six membered cyclic PNA monomer, aminopipecolyl PNA (pipPNA) is reported. Incorporation of pipPNA into PNA oligomers and comparative binding with target DNA sequences is studied.     

Using NMR and molecular dynamics to link structure and dynamics effects of the universal base 8-aza, 7-deaza,N8 linked adenosine analog

A truly universal nucleobase enables a host of novel applications such as simplified templates for PCR primers, randomized sequencing and DNA based devices. A universal base must pair indiscriminately to each of the canonical bases with little or preferably no destabilization of the overall duplex. In reality, many candidates either destabilize the duplex or do not base pair indiscriminatingly. The novel base 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin- 4-amine) N⁸-(2′deoxyribonucleoside), a deoxyadenosine analog (UB), pairs with each of the natural DNA bases with little sequence preference. We have utilized NMR complemented with molecular dynamic calculations to characterize the structure and dynamics of a UB incorporated into a DNA duplex. The UB participates in base stacking with little to no perturbation of the local structure yet forms an unusual base pair that samples multiple conformations. These local dynamics result in the complete disappearance of a single UB proton resonance under native conditions. Accommodation of the UB is additionally stabilized via heightened backbone conformational sampling. NMR combined with various computational techniques has allowed for a comprehensive characterization of both structural and dynamic effects of the UB in a DNA duplex and underlines that the UB as a strong candidate for universal base applications.     

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.     

Structure of the DNA interstrand cross-link of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) cross-links a 5' TpA or a 5' ApT site by photoreacting with one thymine moiety in each DNA strand. We are interested in whether psoralen interstrand cross-links all share one structure or whether there are significant differences. In this paper, we employed a rapid method for probing the structure of the cross-link by making a series of TMP cross-linked duplexes containing specific base-pair mismatches. The relative stability provided by a base pair can be correlated with neighboring base pairs by comparing the extents of gel retardation when base-pair mismatches happen in each position. From our studies, we infer that with respect to the furan-side strand, the 5'T.A base pair of the two T.A base pairs in the TpA site is not hydrogen bonded. Immediately on each side of the cross-linked TpA site is a highly stabilized base pair. Next, a region of decreased stability occurs in each arm of a cross-linked duplex and these base pairs of least stability are located farther away from the cross-linked thymines as the lengths of the arms of the cross-linked helix increase. Finally, even in 7 M urea at 49 degrees C the cross-linked helix is hydrogen bonded at both ends of a duplex of 22 base pairs. We propose that the structures of interstrand cross-links in DNA vary appreciably with the DNA sequence, the length of the DNA duplex, and the structures of the DNA cross-linking agents.     

Hidden in plain sight: subtle effects of the 8-oxoguanine lesion on the structure, dynamics, and thermodynamics of a 15-base pair oligodeoxynucleotide duplex

The base lesion 8-oxoguanine is formed readily by oxidation of DNA, potentially leading to G → T transversion mutations. Despite the apparent similarity of 8-oxoguanine-cytosine base pairs to normal guanine-cytosine base pairs, cellular base excision repair systems effectively recognize the lesion base. Here we apply several techniques to examine a single 8-oxoguanine lesion at the center of a nonpalindromic 15-mer duplex oligonucleotide in an effort to determine what, if anything, distinguishes an 8-oxoguanine-cytosine (8oxoG-C) base pair from a normal base pair. The lesion duplex is globally almost indistinguishable from the unmodified parent duplex using circular dichroism spectroscopy and ultraviolet melting thermodynamics. The DNA mismatch-detecting photocleavage agent Rh(bpy)(2)chrysi(3+) cleaves only weakly and nonspecifically, revealing that the 8oxoG-C pair is locally stable at the level of the individual base pairs. Nuclear magnetic resonance spectra are also consistent with a well-conserved B-form duplex structure. In the two-dimensional nuclear Overhauser effect spectra, base-sugar and imino-imino cross-peaks are strikingly similar between parent and lesion duplexes. Changes in chemical shift due to the 8oxoG lesion are localized to its complementary cytosine and to the 2-3 bp immediately flanking the lesion on the lesion strand. Residues further removed from the lesion are shown to be unperturbed by its presence. Notably, imino exchange experiments indicate that the 8-oxoguanine-cytosine pair is strong and stable, with an apparent equilibrium constant for opening equal to that of other internal guanine-cytosine base pairs, on the order of 10(-6). This collection of experiments shows that the 8-oxoguanine-cytosine base pair is incredibly stable and similar to the native pair.     

Acid-induced exchange of the imino proton in G.C pairs.

Acid-induced catalysis of imino proton exchange in G.C pairs of DNA duplexes is surprisingly fast, being nearly as fast as for the isolated nucleoside, despite base-pair dissociation constants in the range of 10(-5) at neutral or basic pH. It is also observed in terminal G.C pairs of duplexes and in base pairs of drug-DNA complexes. We have measured imino proton exchange in deoxyguanosine and in the duplex (ATATAGATCTATAT) as a function of pH. We show that acid-induced exchange can be assigned to proton transfer from N7-protonated guanosine to cytidine in the open state of the pair. This is faster than transfer from neutral guanosine (the process of intrinsic catalysis previously characterized at neutral ph) due to the lower imino proton pK of the protonated form, 7.2 instead of 9.4. Other interpretations are excluded by a study of exchange catalysis by formiate and cytidine as exchange catalysts. The cross-over pH between the regimes of pH-independent and acid-induced exchange rates is more basic in the case of base pairs than in the mononucleoside, suggestive of an increase by one to two decades in the dissociation constant of the base pair upon N7 protonation of G. Acid-induced catalysis is much weaker in A.T base pairs, as expected in view of the low pK for protonation of thymidine.     

Structure of DNA sequence d-TGATCA by two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics

The 5' d-TpG 3' element is a part of DNA sequences involved in regulation of gene expression and is also a site for intercalation of several anticancer drugs. Solution conformation of DNA duplex d-TGATCA containing this element has been investigated by two-dimensional NMR spectroscopy. Using a total of 12 torsional angles and 121 distance constraints, structural refinement has been carried out by restrained molecular dynamics (rMDs) in vacuum up to 100 ps. The structure is characterized by a large positive roll at TpG/CpA base pair step and large negative propeller twist for AT and TA base pairs. The backbone torsional angle, gamma(O5'-C5'-C4'-C3'), of T1 residue adopts a trans-conformation which is corroborated by short intra nucleotide T1H6-T1H5' (3.7A) distance in nuclear overhauser effect spectroscopy (NOESY) spectra while the backbone torsional angle, beta(P-O5'-C5'-C4'), exists in trans as well as gauche state for T1 and C5 residues. There is evidence of significant flexibility of the sugar-phosphate backbone with rapid inter-conversion between two different conformers at TpG/CpA base pair step. The base sequence dependent variations and local structural heterogeneity have important implications in specific recognition of DNA by ligands.     

Linear free energy correlations for enzymatic base flipping: how do damaged base pairs facilitate specific recognition?

To efficiently maintain their genomic integrity, DNA repair glycosylases must exhibit high catalytic specificity for their cognate damaged bases using an extrahelical recognition mechanism. One possible contribution to specificity is the weak base pairing and inherent instability of damaged sites which may lead to increased extrahelicity of the damaged base and enhanced recognition of these sites. This model predicts that the binding affinity of the enzyme should increase as the thermodynamic stability of the lesion base pair decreases, because less work is required to extrude the base into its active site. We have tested this hypothesis with uracil DNA glycosylase (UDG) by constructing a series of DNA duplexes containing a single uracil (U) opposite a variety of bases (X) that formed from zero to three hydrogen bonds with U. Linear free energy (LFE) relationships were observed that correlated UDG binding affinity with the entropy and enthalpy of duplex melting, and the dynamic accessibility of the damaged site to chemical oxidation. These LFEs indicate that the increased conformational freedom of the damaged site brought about by enthalpic destabilization of the base pair promotes the formation of extrahelical states that enhance specific recognition by as much as 3000-fold. However, given the small stability differences between normal base pairs and U.A or U.G base pairs, relative base pair stability contributes little to the >10(6)-fold discrimination of UDG for uracil sites in cellular DNA. In contrast, the intrinsic instability of other more egregious DNA lesions may contribute significantly to the specificity of other DNA repair enzymes that bind to extrahelical bases.     

Peptide nucleic acid (PNA)/DNA hybrid duplexes: intercalation by an internally linked anthraquinone.

Peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing. A set of reporter compounds that bind to DNA by intercalation are known, but these compounds do not intercalate in PNA/DNA hybrid duplexes. Analysis of the hybrid PNA duplexes requires development of reporter compounds that probe their chemical and physical properties. We prepared a series of anthraquinone (AQ) derivatives that are linked to internal positions of a PNA oligomer. These are the first non-nucleobase functional groups that have been incorporated into a PNA. The resulting PNA(AQ) conjugates form stable hybrids with complementary DNA oligomers. We find that when the AQ groups are covalently bound to PNA that they stabilize the hybrid duplex and are, at least partially, intercalated.     

Structural pre-organization of peptide nucleic acids

Introduction of constraint via chemical bridging in the aegPNA leads to the five or six membered cyclic structures that may contribute towards maintaining the balance between rigidity and flexibility of the PNA backbone. The significant promise of our approach to use the naturally occurring trans-4-hydroxy-L-proline to arrive at different chirally pure cyclic PNA analogs and their DNA binding properties will be presented.     

Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I

We describe studies aimed at evaluating the physical factors governing the rate of 3'-end proofreading by the Klenow fragment of E. coli DNA polymerase I. Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene (F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mimics of thymine and adenine, respectively, are used to investigate the effects of base pair geometry and stability on the rate of this exonuclease activity. Steady-state kinetics measurements show that complementary T.A base pairs at the end of a primer-template duplex are edited 14-40-fold more slowly than mismatches. By contrast, a 3'-end T residue in a T. Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact that it resembles a T.A pair in structure. Similarly, the A in an A.F pair is edited as rapidly as a mismatched pair despite its close structural mimicry of an A.T pair. Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicating specific interactions between the exonuclease domain and the base of the nucleotide being edited. Finally, thermal denaturation studies are carried out to investigate the relationship between editing and the ease of unwinding of the duplex. The rapid editing of bases opposite F or Z residues at the duplex terminus seems to correlate well with the stability of these base pairs when placed in a context resembling a primer-template duplex. In general, the rate of 3'-end editing appears to be governed by the rate of fraying of the DNA terminal pair, and base pair geometry appears to have little effect.