Nucleic Acid Triplexes Research Articles

Parallel and antiparallel (G.GC)2 triple helix fragments in a crystal structure.

Nucleic acid triplexes are formed by sequence-specific interactions between single-stranded polynucleotides and the double helix. These triplexes are implicated in genetic recombination in vivo and have application to areas that include genome analysis and antigene therapy. Despite the importance of the triple helix, only limited high-resolution structural information is available. The x-ray crystal structure of the oligonucleotide d(GGCCAATTGG) is described; it was designed to contain the d(G middle dotGC)2 fragment and thus provide the basic repeat unit of a DNA triple helix. Parameters derived from this crystal structure have made it possible to construct models of both parallel and antiparallel triple helices.

A nucleic acid triple helix formed by a peptide nucleic acid-DNA complex.

The crystal structure of a nucleic acid triplex reveals a helix, designated P-form, that differs from previously reported nucleic acid structures. The triplex consists of one polypurine DNA strand complexed to a polypyrimidine hairpin peptide nucleic acid (PNA) and was successfully designed to promote Watson-Crick and Hoogsteen base pairing. The P-form helix is underwound, with a base tilt similar to B-form DNA. The bases are displaced from the helix axis even more than in A-form DNA. Hydrogen bonds between the DNA backbone and the Hoogsteen PNA backbone explain the observation that polypyrimidine PNA sequences form highly stable 2:1 PNA-DNA complexes. This structure expands the number of known stable helical forms that nucleic acids can adopt.

Determination of the hydrogen‐bonded structure of CGG trimers in chloroform solution by vibrational spectroscopy

AbstractInfrared and Raman spectra of 2′‐deoxy‐3′,5′‐bis (triisopropylsilyl) guanosine (G) and its 2 : 1 mixture with 2′‐deoxy‐3′,5′‐bis (triisopropylsilyl) cytidine (C) have been studied in chloroform solution. Strong interactions occur between CG base pair and a second G whose association constant was first determined. CGG trimer is stabilized by two hydrogen bonds between a G which is in a Watson‐Crick base pair and a second G. These interguanine hydrogen bonds involve the N(7) and O(6) guanine acceptors and N(1)H and N(2)H guanine donors. The 1570 cm−1 band of guanine is sensitive to hydrogen bonding at the N(3) position. Since this band is unchanged upon CGG formation, it is concluded that no binding at the guanine N(3) position occurs in the trimer. The pattern of hydrogen bonding in CGG trimer in chloroform solution here appears to correspond with patterns of hydrogen bonding in nucleic acid triplexes. These spectroscopic measurements constitute the first application of vibrational spectroscopy to the structural study of CGG trimer and self‐associated G in a nonaqueous solvent, and these nucleosides can be used as molecular models to know the hydrogen bonding in the hydrophobic interior of nucleic acid double and triple helices. © 1995 John Wiley & Sons, Inc.

Triplex Formation of Oligonucleotides Containing 2′‐O‐Methylurldine, 5‐Bromo‐2′‐OMethyluridine and 2′‐0‐Methycytidine

AbstractThe influence of 5‐bromo and 2′‐O‐methyl substitution of pyrimidine bases on nucleic acid triplex formation was examined by thermal denaturation and circular dichroism spectra with the following oligomers: 5′‐(Um) (I, Um = 2′‐O‐methyluridine), 5′‐(BUm)15T (II, BUm = 5‐bromo2′‐O‐metnyluridine), 5′‐(CmUm)6Cm‐T (III, Cm = 2′‐O‐methylcytidine), and 5′‐(CmUm)6Cm T (IV). Both I and II form triplexes with dA16 (A) or 5′‐d(C2A16C2)‐5′‐d(G2T16G2) (D), but III and IV form triplexes with 5′‐d(AG)8 (G). Triplex formation involving two unmodified oligomers, dT16 (T) and 5′‐d(CT)7 (C), was used for comparison. The orders of melting temperatures (Tm) of triplexes were II A II (Tm = 48 °C) > I A I (34 °C) > T A T (22 °C); II D (Tm= 30 °C) > I D (22 °) > T D (12 °C); and IV G IV (Tm= 82 °C) > III G III (72 °C) > C G C (55 °C). The pKa of IVG IV, III G III, and C G C were determined by CD to be 6.5,6.3, and 6.2, respectively. Triplexes formed by oligonucleotides containing both 2′‐O‐methyl and 5‐bromo substitutions are more stable and have a greater pKa than those formed by oligonucleotides with 2′‐O‐methyl substitution only; which in turn are more stable and have a greater pKa than those formed by unmodified oligonucleotides.

Conformational transitions of duplex and triplex nucleic acid helices: thermodynamic analysis of effects of salt concentration on stability using preferential interaction coefficients

For order-disorder transitions of double- and triple-stranded nucleic acid helices, the midpoint temperatures Tm depend strongly on a +/-, the mean ionic activity of uniunivalent salt. Experimental determinations of dTm/d ln a +/- and of the enthalpy change (delta H(o)) accompanying the transition in excess salt permit evaluation of delta gamma, the stoichiometrically weighted combination of preferential interaction coefficients, each of which reflects thermodynamic effects of interactions of salt ions with a reactant or product of the conformational transition (formula; see text) Here delta H(o) is defined per mole of nucleotide by analogy to delta gamma. Application of Eq. 1 to experimental values of delta H(o) and Tm yields values of delta gamma for the denaturation of B-DNA over the range of NaCl concentrations 0.01-0.20 M (Privalov et al. (1969), Biopolymers 8,559) and for each of four order-disorder transitions of poly rA.(poly rU)n, n = 1, 2 over the range of NaCl concentrations 0.01-1.0 M (Krakauer and Sturtevant (1968), Biopolymers 6, 491). For denaturation of duplexes and triplexes, delta gamma is negative and not significantly dependent on a +/-, but delta gamma is positive and dependent on a +/- for the disproportionation transition of poly rA.poly rU duplexes. Quantitative interpretations of these trends and magnitudes of delta gamma in terms of coulombic and excluded volume effects are obtained by fitting separately each of the two sets of thermodynamic data using Eq. 1 with delta gamma PB evaluated from the cylindrically symmetric Poisson-Boltzmann (PB) equation for a standard model of salt-polyelectrolyte solutions. The only structural parameters required by this model are: b, the mean axial distance between the projections of adjacent polyion charges onto the cylindrical axis; and a, the mean distance of closest approach between a salt ion center and the cylindrical axis. Fixing bMS and aMS for the multi-stranded (ordered) conformations, we determined the corresponding best fitted values of bSS and aSS for single-stranded RNA and DNA. The resulting best fitted values of aSS are systematically less than aDS by 2-4 A. Uncertainty in the best-fitted values of bSS is significantly lower than in the aSS, because bMS is known with relatively high precision and because the larger uncertainty in aMS has a relatively small effect on the best-fitted values of bSS:bSS = 3.2 +/- 0.6 A for single-stranded poly rA and poly rU; and bSS = 3.4 +/- 0.2 A for single-stranded DNA. These values are approximately one-halt of those expected for a fully extended single-stranded conformation. With the best fitted values of ass and bss, our calculations of delta gamma PB are in close quantitative agreement with experimental observations on each of five nucleic acid order-disorder transitions.

Inhibition of transcription of HIV-1 in infected human cells by oligodeoxynucleotides designed to form DNA triple helices.

The effect on human immunodeficiency virus 1 (HIV-1) viral transcription and subsequent gene expression mediated by mixed purine-pyrimidine oligodeoxyribonucleotides (oligodeoxynucleotides) designed to form collinear DNA triplexes with purine-rich elements in the viral promoter was evaluated in intact mammalian cell lines (MT4 and U937). Oligonucleotides HIV31 (5'-GTTTTTGGGTGTTGTGGGTGTGTGTGGTTTG-3') and HIV38 (5'-TGGGTGGGGTGGGGTGGGGGGGTGTGGGGTGTGGGGTG-3') were designed to interact with the transcription initiation site (-16 to + 13) and nuclear factor Sp1 binding site (-81 to -44) of HIV-1, respectively. Oligonucleotides, synthesized with a 3' amine blocking group (5'-R-O-PO2-OCH (CHOH)-CH2-NH+3-3') to prevent degradation by cellular nucleases, were readily taken up by MT4 cells from the culture medium, achieving measured intranuclear concentrations higher than the medium in less than 2 h of incubation. The 3' amine modified oligonucleotides were recoverable from the cells after 24 h as greater than 90% intact material. Treatment of acutely infected MT4 cells with either HIV31 or HIV38 significantly inhibited viral-associated cytopathology and P24 antigen production (p less than 0.001). Additionally, inhibition of P24 antigen release, culture supernatant viral titer, and expression of the intact 9.2-kb HIV-1 mRNA was observed when the chronically infected promonocyte cell line, U937, was treated with 10 microM HIV38. Control oligonucleotides with similar base composition did not inhibit virus expression in either cell line. Furthermore, inhibition of viral expression was not due to alpha-interferon induction resulting from oligonucleotide treatment. Both HIV31 and HIV38 associate with their respective DNA target duplexes at micromolar concentrations, and a strong negative ellipticity near 210 nm, characteristic of DNA triplexes, was observed in the circular dichroism spectrum of either target-oligonucleotide complex. These observations suggest that oligonucleotides, designed to form nucleic acid triplexes with specific proviral target sequences, can selectively inhibit transcription of viral mRNA in intact cells and suppress accumulation of viral products.