Heteronomous DNA.

A fibrous form of poly d(A):poly d(T) has a heteronomous secondary structure which is the first to be confirmed for a polynucleotide duplex: although both chains are 10(1) helices, mutually hydrogen-bonded in the standard (Watson-Crick) fashion, each has a quite different conformation. One chain -- probably poly d(A) -- has C3'-endo-puckered furanose rings characteristic of the A family of polynucleotide secondary structures while the other -- probably poly d(T) -- has the C2'-endo-puckered rings of the B family. Since analogous heteronomous structures could be assumed by DNA-DNA or DNA-RNA duplexes containing more general base sequences the polymorphic range of polynucleotide double-helices may be even greater than we have come to suppose.     

New wrinkles on polynucleotide duplexes

Most fibrous polynucleotides of general sequence exhibit secondary structures that are described adequately by regular helices with a repeated motif of only one nucleotide. Such helices exploit the fact that A:T, T:A, G:C, and C:G pairs are essentially isomorphous and have dyadically-related glycosylic bonds. Polynucleotides with regularly repeated base-sequences sometimes assume secondary structures with larger repeated motifs which reflect these base-sequences. The dinucleotide units of the Z-like forms of poly d(As4T):poly d(As4T), poly d(AC):poly d(GT) and poly d(GC):poly d(GC) are dramatic instances of this phenomenon. The wrinkled B and D forms of poly d(GC):poly d(GC) and poly d(AT):poly d(AT) are just as significant but more subtle examples. It is possible also to trap more exotic secondary structures in which the molecular asymmetric unit is even larger. There is, for example, a tetragonal form of poly d(AT):poly d(AT) which has unit cell dimensions a = b = 1.71nm, c = 7.40nm, gamma = 90 degrees. The c dimension corresponds to the pitch of a molecular helix which accommodates 24 successive nucleotide pairs arranged as a 4(3) helix of hexanucleotide duplexes. The great variety of nucleotide conformations which occur in these large asymmetric units has prompted us to describe them as pleiomeric, a term used in botany to describe whorls having more than the usual number of structures. Pleiomeric DNAs need not contain nucleotide conformations that are very different from one another. On the other hand, DNAs carrying nucleotides of very different conformation must be pleiomeric. This is because 4 nucleotides of different conformation are needed to join patches of secondary structure which are as different as A or B or Z. Differences in nucleotide structures may occur also between chains rather than within chains. In poly d(A):poly d(T), the purine nucleotides all contain C3'-endo furanose rings and the pyrimidine nucleotides C2'-endo rings. Analogous heteronomous structures may exist in DNA-RNA hybrids although these duplexes are also found to have symmetrical A-type conformations.     

New Wrinkles on Polynucleotide Duplexes

Abstract

Most fibrous polynucleotides of general sequence exhibit secondary structures that are described adequately by regular helices with a repeated motif of only one nucleotide. Such helices exploit the fact that A:T, T:A, G:C, and C:G pairs are essentially isomorphous and have dyadically-related glycosylic bonds. Polynucleotides with regularly repeated base-sequences sometimes assume secondary structures with larger repeated motifs which reflect these base-sequences. The dinucleotide units of the Z-like forms of poly d(As⁴T):poly d(As⁴T), poly d(AC):poly d(GT) and poly d(GC):poly d(GC) are dramatic instances of this phenomenon. The wrinkled B and D forms of poly d(GC):poly d(GC) and poly d(AT):poly d(AT) are just as significant but more subtle examples. It is possible also to trap more exotic secondary structures in which the molecular asymmetric unit is even larger. There is, for example, a tetragonal form of poly d(AT):poly d(AT) which has unit cell dimensions a = b = 1.71nm, c= 7.40nm, γ = 90°. The C dimension corresponds to the pitch of a molecular helix which accommodates 24 successive nucleotide pairs arranged as a 4₃ helix of hexanucleotide duplexes. The great variety of nucleotide conformations which occur in these large asymmetric units has prompted us to describe them as pleiomeric, a term used in botany to describe whorls having more than the usual number of structures. Pleiomeric DNAs need not contain nucleotide conformations that are very different from one another. On the other hand, DNAs carrying nucleotides of very different conformation must be pleiomeric. This is because 4 nucleotides of different conformation are needed to join patches of secondary structure which are as different as A or B or Z. Differences in nucleotide structures may occur also between chains rather than within chains. In poly d(A):poly d(T), the purine nucleotides all contain Ci'-endo furanose rings and the pyrimidine nucleotides C2 '-endo rings. Analogous heteronomous structures may exist in DNA-RNA hybrids although these duplexes are also found to have symmetrical A-type conformations.     

Structure of the alpha-form of poly[d(A)].poly[d(T)] and related polynucleotide duplexes

The alpha-form of poly[d(A)].poly[d(T)], observed in fibers at high (greater than 80%) relative humidity, is a 10-fold double-helical structure of pitch 3.2 nm. This new X-ray analysis shows that the two strands of the double helix are of the same kind conformationally and both B-like in containing C-2'-endo-puckered deoxyribose rings. Nevertheless, the two strands are different enough for the overall morphology of the duplex to resemble that of the heteromerous model for the drier (beta) form of poly[d(A)].poly[d(T)] in which one strand has C-2'-endo rings and the other C-3'-endo. Since the orientations of the bases in poly[d(A)].poly[d(T)] are persistently different from those of classical B-DNA it is likely that there will be local bending (about 10 degrees) at the junctions between general sequence tracts and the oligo[d(A)].oligo[d(T)] tracts that occur in some native DNAs. The conclusions about the structure of alpha-poly[d(A)].poly[d(T)] are reinforced by independent analyses of similar X-ray diffraction patterns from poly[d(A)].poly[d(U)] and poly[d(A-I)].poly[d(C-T)].     

Effect of environment, conformation, sequence and base substituents on the imino proton exchange rates in guanine and inosine-containing DNA, RNA, and DNA-RNA duplexes

High-resolution 1H nuclear magnetic resonance in H2O has been used to study the effect of sequence, conformation, environmental factors and base substituents on the exchange behavior of the hydrogen-bonded imino protons of guainine X cytosine and inosine X cytosine base-pairs in DNA, RNA, and DNA-RNA duplexes. The exchange rates were determined by measurement of the spin-lattice relaxation rates of the imino protons as a function of temperature. The exchange was not altered by the presence of high concentrations of salt, and the inability of phosphate to catalyze the exchange indicates that the exchange is limited by formation of a solvent-accessible "open" state. The exchange behavior depends on the duplex conformation and sequence. Exchange from the Z form polymers was orders of magnitude slower than the corresponding duplexes in the B conformation, and the A form RNA duplexes exchanged more slowly than the B form DNA polymers with the same sequence. The exchange behavior of the DNA-RNA hybrids was dependent on whether the purine or the pyrimidine strand contained the deoxyribose sugar. For both the guanine and inosine-containing duplexes, the homopolymer duplexes exchange more slowly than the more stable alternating copolymers. For the alternating duplexes, substitution of cytosine with 5-bromo- or 5-methylcytosine slowed the exchange and increased the activation energy for exchange. The inosine-containing duplexes exchanged more rapidly than the guanosine-containing duplexes, but both showed similar changes in exchange behavior in response to changes in sequence and base substituents. The activation energies for base-pair opening in B form DNA are correlated with the van der Waals contribution to the base-base interaction energy, suggesting that the purine base is partially unstacked in the open state. Using the relaxation measurements to set an upper limit on the exchange rate in poly(dG-dC) and the tritium exchange behavior at low temperature, we find that even though Z-DNA exchanges very slowly, the activation energy is similar to that observed in the A and B form duplexes, suggesting that exchange occurs from a similar open state.     

Production and characterization of anti-DNA-RNA monoclonal antibodies and their application in Listeria detection.

Murine monoclonal antibodies (MAbs) specific for DNA-RNA hybrids were successfully produced with two different heteropolymers as antigens, cDNA-mRNA and phi X174 DNA-RNA heteroduplexes. The former was simpler to prepare. Both had shown similar immunogenicities. Two different immunoglobulin M MAbs were isolated. The 20D3 MAb, generated with the phi X174 DNA-RNA hybrid, showed association constants of 1.05 x 10(12), 2.12 x 10(10), and 1.68 x 10(7) for the antigens phi X174 DNA-RNA, cDNA-mRNA, and poly(rA)-poly(dT), respectively. The 6B5 MAb, obtained with the cDNA-mRNA hybrid, showed association constants of 1.59 x 10(5), 5 x 10(12), and 7.1 x 10(8) for the above-described antigens, respectively. With the 20D3 MAb, an immunoassay was developed for the detection of Listeria DNA-RNA hybrids. In brief, a biotinylated rRNA gene probe specific for the genus Listeria was hybridized with rRNA in the solution phase. The hybrids thus formed were then captured in microtiter plate wells precoated with the purified 20D3 MAb, and the probe-target hybrids were detected with a streptavidin-alkaline phosphatase conjugate. This assay was shown to be specific for the genus Listeria and highly sensitive, allowing the detection of as little as 2.5 pg of target rRNA.     

CD of homopolymer DNA-RNA hybrid duplexes and triplexes containing A-T or A-U base pairs.

CD spectra and difference-CD spectra of (a) two DNA X RNA hybrid duplexes (poly[r(A) X d(U)] and poly[r(A) X d(T)]) and (b) three hybrid triplexes (poly-[d(T) X r(A) X d(T)], poly[r(U) X d(A) X r(U)], and poly[r(T) X d(A) X r(T)]) were obtained and compared with CD spectra of six A X U- and A X T-containing duplex and triplex RNAs and DNAs. We found that the CD spectra of the homopolymer duplexes above 260 nm were correlated with the type of base pair present (A-U or A-T) and could be interpreted as the sum of the CD contributions of the single strands plus a contribution due to base pairing. The spectra of the duplexes below 235 nm were related to the polypurine strands present (poly-[r(A)] or poly[d(A)]). We interpret the CD intensity in the intermediate 255-235 nm region of these spectra to be mainly due to stacking of the constituent polypurine strands. Three of the five hybrids (poly[r(A) X d(U)], poly[r(A) X d(T)], and poly[d(T) X r(A) X d(T)]) were found to have heteronomous conformations, while poly[r(U) X d(A) X r(U)] was found to be the most A-like and poly[r(T) X d(A) X r(T)], the least A-like.     

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.     

Assay of DNA-RNA hybrids by S1 nuclease digestion and adsorption to DEAE-cellulose filters.

A fast and accurate assay procedure for DNA-RNA hybrids is described in which exhaustive digestion of unhybridized DNA with S1 nuclease is followed by binding of hybrids to filter discs of DEAE-cellulose. The digested DNA can be efficiently washed from the filters so that background levels of 0.1-0.2% of input tracer DNA can be achieved, in contrast to the much higher (approximately 1-5%) backgrounds obtained using TCA precipitation procedures. Short duplexes, as small as 36 nucleotides in length, which are inefficiently bound to hydroxyapatite, are quantitatively bound to the DEAE-cellulose filters.     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

Structure of the beta-form of poly d(A).poly d(U)

The crystalline beta-form of the sodium salt of poly d(A).poly d(U) trapped in oriented fibers forms a Watson-Crick base-paired, 10(1) double-helix of pitch 3.2 nm. Two molecules are present in a monoclinic unit cell apparently isomorphous with beta-poly d(A).poly d(T). The two chains in each molecule both carry C2'-endo puckered furanose rings but are conformationally not identical. The orientations of the A:U base-pairs relative to the helix-axis are distinctly different from those in classical B-DNA and the overall morphology of the duplex in which they reside resembles that of the alpha-forms of poly (purine).poly (pyrimidine) DNA duplexes previously reported.     

Endoribonuclease from bovine adrenal cortex cytosol.

An endoribonuclease which digests a variety of synthetic homoribopolymers and poly(A)-rich mRNA has been identified and purified greater than 500-fold with respect to specific activity from bovine adrenal cortex cytosol. Enzymatic digestion of synthetic poly(riboadenylic acid) was stimulated by Mn-2+ and Mg-2+ and the enzyme exhibited broad pH and salt optima. Poly(cytidylic acid) and poly(uridylic acid), but not poly(guanylic acid), served as substrates for the enzyme preparation; double-stranded RNA, DNA, and DNA-RNA hybrids were not digested by the enzyme. Digestion generated oligonucleotides with 3-hydroxyl and 5'-monophosphoester termini. On isoelectric focusing, the enzymatic activity banded at pH 8.3 plus or minus 0.2. An initial preferential cleavage of the poly(A) tract of poly(A)-rich RNA is suggested by the rapid appearance of a 4-6S digestion product highly enriched for adenylic acid; however, progressive digestion of the RNA occurs with additional incubation.     

The bacterial histone-like protein HU specifically recognizes similar structures in all nucleic acids. DNA,RNA, and their hybrids

HU, a major component of the bacterial nucleoid, shares properties with histones, high mobility group proteins (HMGs), and other eukaryotic proteins. HU, which participates in many major pathways of the bacterial cell, binds without sequence specificity to duplex DNA but recognizes with high affinity DNA repair intermediates. Here we demonstrate that HU binds to double-stranded DNA, double-stranded RNA, and linear DNA-RNA duplexes with a similar low affinity. In contrast to this nonspecific binding to total cellular RNA and to supercoiled DNA, HU specifically recognizes defined structures common to both DNA and RNA. In particular HU binds specifically to nicked or gapped DNA-RNA hybrids and to composite RNA molecules such as DsrA, a small non-coding RNA. HU, which modulates DNA architecture, may play additional key functions in the bacterial machinery via its RNA binding capacity. The simple, straightforward structure of its binding domain with two highly flexible beta-ribbon arms and an alpha-helical platform is an alternative model for the elaborate binding domains of the eukaryotic proteins that display dual DNA- and RNA-specific binding capacities.     

Reassociation of nucleic acids in solutions containing formamide

The effect of formamide on the thermal stability of native and reassociated DNA-DNA duplexes and DNA-RNA hybrids has been reexamined. In contrast to McConaughy et al. (1) it was found that the Tm for native DNA of E. coli, calf and P. pallidum was reduced by 0.60°C per each 1% increase in formamide concentration. As measured by thermal stability there is no loss of specificity of the reassociation and hybridization in the formamide system under our conditions.     

Structure of poly (I).poly (A).poly (I)

The molecular structure of poly (I).poly (A).poly (I) has been determined and refined using the continuous intensity data on layer lines in the x-ray diffraction pattern obtained from an oriented fiber of this polymorphic RNA complex. The polymer forms a 12-fold right-handed triple-helix of pitch 39.7A and each base-triplet is stabilized by quasi Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in all the three strands have C3'-endo conformations. The final R-value for this best structure is 0.24 and the x-ray fit is significantly superior to all the alternative structures where the different chains might have different furanose conformations. This all-purine triple-helix, counter-intuitively, has a diameter roughly 3A shorter than that of DNA and RNA triple-helices containing a homopurine and two complementary homopyrimidine strands. Its compact, grooveless cylindrical shape is consistent with the lack of lateral organization.     

A base-pairing model of duplex formation. I. Watson-Crick pairing geometries

We present a base-pairing model of oligonucleotide duplex formation and show in detail its equivalence to the nearest-neighbor dimer methods from fits to free energy of duplex formation data for short DNA-DNA and DNA-RNA hybrids containing only Watson-Crick pairs. For completeness, the corresponding RNA-RNA parameters are included. In this approach, the connection between rank-deficient polymer and rank-determinant oligonucleotide parameter sets for DNA duplexes is transparent. The method is generalized to include RNA-DNA hybrids where the rank-deficient model with 11 dimer parameters in fact provides slightly improved predictions relative to the standard method with 16 independent dimer parameters (DeltaG mean errors of 4.5 and 5.4%, respectively).     

Synthesis and spectroscopic studies of the aminoglycoside (neomycin)--perylene conjugate binding to human telomeric DNA

Synthesis of a novel perylene-neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5'-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA-RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV-vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand-quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.