Conformation of nucleic acids and the analysis of the hypochromic effect

The spectra of two double-helical RNA species isolated from virus-like particles found in the mycelium of Penicillium chrysogenum were measured at about 25 degrees C, and at 95 degrees C after denaturation to a single-stranded form, and compared with the spectrum of the equivalent mixture of nucleotides. For both species the difference spectrum (epsilon(95 degrees C)-epsilon(25 degrees C)) when increased by 33% was very similar to the difference spectrum (epsilon(nucleotides)-epsilon(25 degrees C)). In contrast it was found for rat liver DNA that there was no simple relation between (epsilon(95 degrees C)-epsilon(25 degrees C)) and (epsilon(nucleotides)-epsilon(25 degrees C)). These observations were accounted for on the basis of the difference spectra for rA.rU, rG.rC, dA.dT and dG.dC base-pairs derived from model polyribonucleotides and polydeoxyribonucleotides. The difference spectra (epsilon(95 degrees C)-epsilon(25 degrees C)) found for rRNA and those calculated from a combination of the difference spectra for rA.rU and rG.rC base-pairs were in good agreement.     

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices.

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.     

C5-(1-propynyl)-2'-deoxy-pyrimidines enhance mismatch penalties of DNA:RNA duplex formation

UV melting experiments show that C5-(1-propynyl)ation of seven pyrimidines to give a fully propynylated oligodeoxynucleotide (PrODN) heptamer increases the thermodynamic stability of six Watson-Crick paired DNA:RNA duplexes by 8.2 kcal/mol, on average, at 37 degrees C. About 2.5 kcal/mol of this enhancement is due to long-range cooperativity between the propynylated pyrimidines, Y(p)'s. On average, penalties for dU(p):rG, dC(p):rA, dU(p):rC, and dC(p):rC mismatches are enhanced by 2.9 kcal/mol in PrODN:RNA duplexes over those in unmodified duplexes. This results in penalties as large as 10 kcal/mol for a single mismatch. Removing a single propyne two base pairs away from a mismatch in a PrODN:RNA duplex eliminates the enhancement in specificity. Evidently, enhanced specificity is directly linked to long-range cooperativity between Y(p)'s. In most cases, the enhanced specificity is larger for internal than for terminal mismatches. PrODN:RNA duplexes are destabilized by full phosphorothioate backbone substitution to give S-PrODN:RNA duplexes. The S-PrODN:RNA duplexes retain enhanced mismatch penalties, however. These results provide insight for utilizing long-range cooperativity and enhanced specificity to improve nucleic acid based probe and drug design.     

A second-generation copper(II)-mediated metallo-DNA-base pair

Metal-dependent pairing of nucleobases represents an alternative DNA base pairing scheme. Our first-generation copper(II)-mediated pyridine-2,6-dicarboxylate (Dipic) and pyridine (Py) metallo-base pair has a stability comparable to the natural base pairs dA:dT and dC:dG but does not have the selectivity of the Watson Crick base pairs. In order to increase the selectivity of base pair formation, a second-generation metallo-base pair was generated consisting of a pyridine-2,6-dicarboxamide (Dipam) and a pyridine (Py) nucleobase. This new metallo-base pair is more stable than the natural base pairs dA:dT and dC:dG and highly selective against mispairing. In addition, incorporation of multiple metallo-base pairs into DNA results in the formation of stable duplexes demonstrating that hydrogen bonding base pairs can efficiently be replaced by metal-dependent base pairs at multiple sites in DNA.     

Hydrogen exchange study of some polynucleotides and transfer RNA

The apparent disagreement between published transfer RNA hydrogen exchange results and the tRNA cloverleaf model, prompted a re-investigation of the relationship between hydrogen exchange data and nucleic acid structure. Hydrogen-tritium exchange experiments were carried out with samples of pure and mixed tRNA and with the synthetic polynucleotide bihelices: poly(rA) · poly(rU), poly(rI) · poly(rC), poly(rG) · poly(rC) and poly(dG) · poly (dC).     

Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases.

The thermal stability of DNA duplexes containing deoxyinosine in a pairing position in turn with each of the four major deoxynucleotides has been investigated by measuring ultraviolet-absorbance at different temperatures. d(G2A4 X A4G2) and d(C2T4YT4C2) were prepared by the solid-phase phosphotriester method. When X is deoxyinosine, the Tm values of the duplexes are in the order Y = dC greater than dA greater than dG greater than dT greater than dU. The Tm of other duplexes containing dG, dA and dT at X were also measured. Self-complementary duplexes d(GGGAAINTTCCC) showed the same order of stability with N being dC, dA, dG and dT. Thermal stabilities of duplexes containing dG instead of dI were compared with other matched and mismatched duplexes. The Tm values of sequence isomers containing purine-pyrimidine combinations were compared. Self-complementary duplexes containing G-C and A-T in the central positions showed Tm values ca. 10 degrees higher than those containing C-G and T-A in the same positions. Thermodynamic parameters and circular dichroism spectra of these oligonucleotides were compared.     

Spectroscopic studies on the interaction of aristololactam-beta-D-glucoside with DNA and RNA double and triple helices: A comparative study.

The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.     

Lipocortin (Annexin) I heterotetramer binds to purine RNA and pyrimidine DNA.

Lipocortin I-like protein with a molecular weight of 94,000 Da as judged by Western analysis was found to bind to ssDNA rather than to dsDNA in a Ca(2+)-dependent manner. This protein was also bound to [(32)P]poly(rA) and [(32)P]poly(rG) as measured by EMSA. Poly(rG), poly(rA), poly(dC), and poly(dT) were competitive against binding of either [(32)P]poly(rA) or [(32)P]poly(rG), while poly(rC), poly(rU), and poly(dA) were less effective binding competitors. The binding of this protein to poly(rA) or poly(rG) was inhibited by immunoprecipitable anti-lipocortin I (calpactin II) and anti-S100 protein antibodies, but not by an anti-Ig antibody. Phospholipids such as phosphatidylserine and phosphatidylinositol enhanced the binding of lipocortin I to poly(rA). Taken together, our present observations suggest that the lipocortin I-S100 protein heterotetramer binds to either purine RNAs or pyrimidine ssDNAs in a Ca(2+)- and phospholipid-dependent manner.     

Low-frequency Raman scattering study of six nucleosides

Raman spectroscopy was used to study the low-frequency (?200?cm^?1) vibrations in crystalline samples of six naturally occurring nucleosides: deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), uridine (rU), cytidine (rC), and adenosine (rA). Such low-frequency vibrations are important for biological processes in which the conformation of a nucleic acid molecule changes. These experiments also provide a test for the low-frequency vibrational modes of dT, dC, and dA predicted by Shishkin et al.     

Intracisternal A particles from FLOPC-1 BALB/c myeloma: presence of high-molecular-weight RNA and RNA-dependent DNA polymerase.

Intracisternal A particles from the FLOPC-1 line of BALB/c myeloma have been shown to contain high-molecular-weight RNA (60 to 70S) that is sensitive to RNase, alkali degradation, and heat but resistant to Pronase treatment. The intracisternal A-particle RNA contains tract of poly (A) approximately 180 nucleotides long. As shown in a reconstitution experiment, by antigenic analysis of A-particle preparation and the SC cytopathogenicity assay, the 70S RNA was not due to contamination by type C virus particles. The FLOPC-1 intracisternal A particles also possess an endogenous RNA-dependent DNA polymerase. The enzyme required Mn2+ or Mg2+, dithiothreitol, detergent, and four deoxyribonucleoside triphosphates for maximum activity. Enzymatic activity was maximally stimuated by poly (rC)-oligo (dG)12-18 and less with poly (rG)-oligo (dC)10 or poly (rA)-oligo (dT)12-18 as compare with synthetic DNA/DNA duplex templates such as poly (dA)-oligo (dT)12-18. The enzyme can utilize the A-particle endogenous RNA as template as shown by analysis of the early and late DNA products of the endogenous reaction by CsSO4 isopycnic gradient centrifuation and hybridization of purified 70S or 35S A-particle RNA with the purified complementary DNA product. Approximately 50% of the A-particle complementary DNA also hybridized with oncornavirus RNA.     

Structural requirements of the RNA precursor for the biosynthesis of the branched RNA-linked multicopy single-stranded DNA of Myxococcus xanthus

A precursor RNA molecule (pre-msdRNA) of approximately 375 bases is considered to form a stable secondary structure which serves as a primer as well as a template to synthesize the branched RNA-linked multicopy single-stranded DNA (msDNA) of Myxococcus xanthus. When 3-base mismatches were introduced into the stem structure immediately upstream of the branched rG residue to which msDNA is linked by a 2',5'-phosphodiester linkage, the production of msDNA was almost completely blocked. However, if additional 3-base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored, being consistent with the proposed model of msDNA synthesis. We also found that the branched rG residue of pre-msdRNA could not be replaced with either rC or rA, while the 5' end (dC) of msDNA which is linked to the branched rG could be substituted with a dG residue. Together with several other mutations, the structural requirements of pre-msdRNA are discussed with respect to the mechanism of msDNA biosynthesis.     

Influence of nucleotide sequence on dA.dT-specific binding of Netropsin to double stranded DNA.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.     

Hydration and conformational transitions in DNA, RNA, and mixed DNA-RNA triplexes studied by gravimetry and FTIR spectroscopy

We have studied by gravimetric measurements and FTIR spectroscopy the hydration of duplexes and triplexes formed by combinations of dA(n), dT(n), rA(n), and rU(n) strands. Results obtained on hydrated films show important differences in their hydration and in the structural transitions which can be induced by varying the water content of the samples. The number of water molecules per nucleotide (w/n) measured at high relative humidity (98% R.H.) is found to be 21 for dA(n).dT(n) and 15 for rA(n).rU(n). Addition of a third rU(n) strand does not change the number of water molecules per nucleotide: w/n=21 for rU(n)*dA(n).dT(n) and w/n=15 for rU(n)*rA(n).rU(n). On the contrary, the addition of a third dT(n) strand changes the water content but in a different way, depending whether the duplex is DNA or RNA. Thus, a loss of four water molecules per nucleotide is measured for dT(n)*dA(n).dT(n) while an increase of two water molecules per nucleotide is observed for dT(n)*rA(n).rU(n). The final hydration is the same for both triplexes (w/n=17). The desorption profiles obtained by gravimetry and FTIR spectroscopy are similar for the rA(n).rU(n) duplex and the rU(n)*rA(n).rU(n) triplex. On the contrary, the desorption profiles of the dA(n).dT(n) duplex and the triplexes formed with it (rU(n)*dA(n).dT(n) and dT(n)*dA(n).dT(n)) are different from each other. This is correlated with conformational transitions induced by varying the hydration content of the different structures, as shown by FTIR spectroscopy. Modifications of the phosphate group hydration and of the sugar conformation (S to N type repuckering) induced by decrease of the water content are observed in the case of triplexes formed on the dA(n).dT(n) duplex.     

Synthesis and properties of oligonucleotides containing a 7-membered (oxepane) sugar ring

Herein we describe the synthesis of novel 7-membered ring (oxepane) thymine and adenine nucleosides (oT and oA) and their corresponding 5'-O-phosphoramidite derivatives. Two homopolymeric sequences (oT(15) and oA(15)) were prepared via conventional solid-phase synthesis. The mutually complementary strands had the ability to form a duplex (oT(15):oA(15)) exhibiting a transition temperature of 12 degrees C. The oxepane oligonucleotides were also found to associate with their respective complementary RNA strands thus forming oT(15):rA(15) (13 degrees C) and oA(15):rU(15) (12 degrees C) hybrids. The corresponding native duplexes, namely dT(15):dA(15), dT(15):rA(15) and dA(15):rU(15) had melting temperatures of 37 degrees C, 32 degrees C and 16 degrees C, respectively. The CD spectrum of oT(15):rA(15) closely resembled that of the native dT(15):rA(15) hybrid and, in fact, both were found to be substrates for E. Coli RNase H. Thus the oxepane nucleic acids reported here are one of only a handful of DNA mimics capable of activating RNase H when bound to RNA.     

Binding of CC-1065 to poly- and oligonucleotides

The binding of the antitumor agent CC-1065 to a variety of poly- and oligonucleotides was studied by electronic absorption, CD, and resistance to removal by Sephadex column chromatography. Competitive binding experiments between CC-1065 and netropsin were carried out with calf-thymus DNA, poly(dI-dC) · poly(dI-dC), poly(dI) · poly(dC), poly(rA) · poly(dT), poly(dA- dC) · poly(dG-dT), and poly(dA) · 2poly(dT). CC-1065 binds to polynucleotides by three mechanisms. In the first, CC-1065 binds only weakly, as judged by the induction of zero or very weak CD spectra and low resistance to extraction of drug from the polynucleotide by Sephadex chromatography. In the second and third mechanisms, CC-1065 binds strongly, as judged by the induction of two distinct, intense CD spectra and high resistance to extraction of drug from the polynucleotide, by Sephadex chromatography in both cases. The species bound by the second mechanism converts to that bound by the third mechanism with varying kinetics, which depend both on the base-pair sequence and composition of the polynucleotide. Competitive binding experiments with netropsin show that CC-1065 binds strongly in the minor groove of DNA by the second and third mechanisms of binding. Netropsin can displace CC-1065 that is bound by the second mechanism but not that bound by the third mechanism. CC-1065 binds preferentially to B-form duplex DNA and weakly (by the first binding mechanism) or not at all to RNA, DNA, and RNA–DNA polynucleotides which adopt the A-form conformation or to single-strand DNA. This correlation of strong binding of CC-1065 to B-form duplex DNA is consistent with x-ray data, which suggest an anomalous structure for poly(dI) · poly(rC), as compared with poly(rI) · poly(dC) (A-form) and poly(dI) · poly(dC) (B-form). The binding data indicate that poly(rA) · poly(dU) takes the B-form secondary structure like poly(rA) · poly(dT). Triple-stranded poly(dA) · 2poly(dT) and poly(dA) · 2poly(dU), which are considered to adopt the A-form conformation, bind CC-1065 strongly. Netropsin, which also shows a binding preference for B-form polynucleotides, also binds to poly(dA) · 2poly(dT) and occupies the same binding site as CC-1065. These binding studies are consistent with results of x-ray studies, which suggest that A-form triplex DNA retains some structural features of B-form DNA that are not present in A-form duplex DNA; i.e., the axial rise per nucleotide and the base tilt. Triple-stranded poly(dA) · 2poly(rU) does not bind CC-1065 strongly but has nearly the same conformation as poly(dA) · 2poly(dT) based on x-ray analysis. This suggests that the 2′-OH group of the poly(rU) strands interferes with CC-1065 binding to this polynucleotide. The same type of interference may occur for other RNA and DNA–RNA polynucleotides that bind CC-1065 weakly.     

Thermodynamic contributions of single internal rA·dA, rC·dC, rG·dG and rU·dT mismatches in RNA/DNA duplexes

The thermodynamic contributions of rA·dA, rC·dC, rG·dG and rU·dT single internal mismatches were measured for 54 RNA/DNA duplexes in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using the curve-fitting program Meltwin. The weighted average thermodynamic data were fit using singular value decomposition to determine the eight non-unique nearest-neighbor parameters for each internal mismatch. The new parameters predict the ΔG°₃₇, ΔH° and melting temperature (T_m) of duplexes containing these single mismatches within an average of 0.33 kcal/mol, 4.5 kcal/mol and 1.4°C, respectively. The general trend in decreasing stability for the single internal mismatches is rG·dG > rU·dT > rA·dA > rC·dC. The stability trend for the base pairs 5′ of the single internal mismatch is rG·dC > rC·dG > rA·dT > rU·dA. The stability trend for the base pairs 3′ of the single internal mismatch is rC·dG > rG·dC >> rA·dT > rU·dA. These nearest-neighbor values are now a part of a complete set of single internal mismatch thermodynamic parameters for RNA/DNA duplexes that are incorporated into the nucleic acid assay development software programs Visual oligonucleotide modeling platform (OMP) and ThermoBLAST.     

Copper insertion facilitates water-soluble porphyrin binding to rA.rU and rA.dT base pairs in duplex RNA and RNA.DNA hybrids

The binding of the copper(II) complex of water-soluble meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) to double-helical polynucleotides has been studied by optical absorption, circular dichroism (CD), and resonance Raman spectroscopic methods. The target polymers were RNA and RNA.DNA hybrids consisting of rA.rU, rI.rC, rA.dT, and rI.dC base pairs. Relative to the metal-free H(2)TMPyP [Uno, T., Hamasaki, K., Tanigawa, M., and Shimabayashi, S. (1997) Inorg. Chem. 36, 1676-1683], CuTMPyP binds to poly(rA).poly(dT) and poly(rA).poly(rU) with a greatly increased binding constant. The external self-stacking of the porphyrin on the surface of the polymers was evident from the strong conservative-type induced CD signals. The signal intensity correlated almost linearly with the number of stacking sites on the polymer except for poly(rA).poly(dT), which showed extraordinarily strong CD signals. Thus, the bound porphyrin may impose an ordered architecture on the polymer surface, the stacking being facilitated by the more planar nature of the CuTMPyP than the nonmetal counterpart. Resonance Raman spectra of the stacked CuTMPyP were indistinguishable from those of the intercalated one with positive delta(Cbeta-H) and negative delta(Cm-Py) bending shifts, and hence the stacked porphyrins are suggested to adopt a similar structure to that of intercalated ones. Porphyrin flattening by copper insertion opens a new avenue for medical applications of porphyrins, blocking biological events related to RNA and hybrids in malignant cells.     

The poly dA strand of poly dA.poly dT adopts an A-form in solution: a UV resonance Raman study.

The study by resonance Raman spectroscopy with a 257 nm excitation wave-length of adenine in two single-stranded polynucleotides, poly rA and poly dA, and in three double-stranded polynucleotides, poly dA.poly dT, poly(dA-dT).poly(dA-dT) and poly rA.poly rU, allows one to characterize the A-genus conformation of polynucleotides containing adenine and thymine bases. The characteristic spectrum of the A-form of the adenine strand is observed, except small differences, for poly rA, poly rA.poly rU and poly dA.poly dT. Our results prove that it is the adenine strand which adopts the A-family conformation in poly dA.poly dT.