Recognition of RNA duplex by a neomycin–Hoechst 33258 conjugate

DNA minor groove binding drugs such as Hoechst 33258 have been shown to bind to a number of RNA structures. Similarly, RNA binding ligands such as neomycin have been shown by us to bind to a number of A-form DNA structures. A neomycin–Hoechst 33258 conjugate was recently shown to bind B-DNA, where Hoechst exhibits high affinity for the minor groove of A/T tract DNA and neomycin docks into the major groove. Further studies now indicate that the Hoechst moiety of the conjugate can be driven to bind RNA duplex as a consequence of neomycin binding in the RNA major groove. This is the first example of Hoechst 33258 binding to RNA duplex not containing bulges or loop motifs.     

The hydration of nucleic acid duplexes as assessed by a combination of volumetric and structural techniques

Using high precision densimetric and ultrasonic measurements, we have determined, at 25°C, the apparent molar volumes ΦV and the apparent molar compressibilities ΦK_S of four nucleic acid duplexes—namely, the DNA duplex, poly(dIdC)poly(dIdC); the RNA duplex, poly(rA)poly(rU); and the two DNA/RNA hybrid duplexes, poly(rA)poly(dT) and poly(dA)poly(rU). Using available fiber diffraction data on these duplexes, we have calculated the molecular volumes as well as the solvent‐accessible surface areas of the constituent charged, polar, and nonpolar atomic groups. We found that the hydration properties of these nucleic acid duplexes do not correlate with the extent and the chemical nature of the solvent‐exposed surfaces, thereby suggesting a more specific set of duplex–water interactions beyond general solvation effects. A comparative analysis of our volumetric data on the four duplexes, in conjunction with available structural information, suggests the following features of duplex hydration: (a) The four duplexes exhibit different degrees of hydration, in the order poly(dIdC)poly(dIdC) > poly(dGdC)poly(dGdC) > poly(dAdT)poly(dAdT) ≈ poly(dA)poly(dT). (b) Repetitive AT and IC sequences within a duplex are solvated beyond general effects by a spine of hydration in the minor groove, with this sequence‐specific water network involving about 8 additional water molecules from the second and, perhaps, even the third hydration layers. (c) Repetitive GC and IC sequences within a duplex are solvated beyond general effects by a “patch of hydration” in the major groove, with this water network involving about 13 additional water molecules from the second and, perhaps, even the third hydration layers. (d) Random sequence, polymeric DNA duplexes, which statistically lack extended regions of repetitive AT, GC, or IC sequences, do not experience such specific enhancements of hydration. Consequently, consistent with our previous observations (T. V. Chalikian, A. P. Sarvazyan, G. E. Plum, and K. J. Breslauer, Biochemistry, 1994, Vol. 33, pp. 2394–2401), duplexes with approximately 50% AT content exhibit the weakest hydration, while an increase or decrease from this AT content causes enhancement of hydration, either due to stronger hydration of the minor groove (an increase in AT content) or due to stronger hydration of the major groove (an increase in GC content). (e) In dilute aqueous solutions, a B‐DNA duplex is more hydrated than an A‐DNA duplex, a volumetric‐based conclusion that is in agreement with previous results obtained on crystals, fibers, and DNA solutions in organic solvent–water mixtures. (f) the A‐like, RNA duplex poly(rA)poly(rU) and the structurally similar A‐like, hybrid duplex poly(rA)poly(dT), exhibit similar hydration properties, while the structurally distinct A‐like, hybrid duplex poly(rA)poly(dT) and non‐A‐like, hybrid duplex poly(dA)poly(rU) exhibit differential hydration properties, consistent with structural features dictating hydration characteristics. We discuss how volumetric characterizations, in conjunction with structural studies, can be used to describe, define, and resolve the general and sequence/conformation‐specific hydration properties of nucleic acid duplexes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 459–471, 1999     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.     

Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes

Saccharomyces cerevisiae Pif1, an SF1B helicase, has been implicated in both mitochondrial and nuclear functions. Here we have characterized the preference of Pif1 for RNA:DNA heteroduplexes in vitro by investigating several kinetic parameters associated with unwinding. We show that the preferential unwinding of RNA:DNA hybrids is due to neither specific binding nor differences in the rate of strand separation. Instead, Pif1 is capable of unwinding RNA:DNA heteroduplexes with moderately greater processivity compared with its duplex DNA:DNA counterparts. This higher processivity of Pif1 is attributed to slower dissociation from RNA:DNA hybrids. Biologically, this preferential role of the helicase may contribute to its functions at both telomeric and nontelomeric sites.     

NMR studies of fully modified locked nucleic acid (LNA) hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:DNA hybrid

LNA is a bicyclic nucleic acid analogue that contains one or more 2'-O,4'-C methylene linkage(s), which effectively locks the furanose ring in a C3'-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.     

High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG)

The nonsymmetrical double-helical hybrid dodecamer d(CGTTATAATGCG).r(CGCAUUAUAACG) was synthesized with solid-phase phosphoramidite methods and studied by high-resolution 2D NMR. The imino protons were assigned by one-dimensional nuclear Overhauser methods. All the base protons and H1', H2', H2", H3', and H4' sugar protons of the DNA strand and the base protons, H1', H2', and most of the H3'-H4' protons of the RNA strand were assigned by 2D NMR techniques. The well-resolved spectra allowed a qualitative analysis of relative proton-proton distances in both strands of the dodecamer. The chemical shifts of the hybrid duplex were compared to those of the pure DNA double helix with the same sequence (Wemmer et al., 1984). The intrastrand and cross-strand NOEs from adenine H2 to H1' resonances of neighboring base pairs exhibited characteristic patterns that were very useful for checking the spectral assignments, and their highly nonsymmetric nature reveals that the conformations of the two strands are quite different. Detailed analysis of the NOESY and COSY spectra, as well as the chemical shift data, indicate that the RNA strand assumes a normal A-type conformation (C3'-endo) whereas the DNA strand is in the general S domain but not exactly in the normal C2'-endo conformation. The overall structure of this RNA-DNA duplex is different from that reported for hybrid duplexes in solution by other groups (Reid et al., 1983a; Gupta et al., 1985) and is closer to the C3'-endo-C2'-endo hybrid found in poly(dA).poly(dT) and poly(rU).poly(dA) in the fiber state (Arnott et al., 1983, 1986).     

Recognition of the unique structure of DNA:RNA hybrids

Targeting nucleic acids using small molecules routinely uses the end products in the conversion pathway of "DNA to RNA, RNA to protein". However, the intermediate processes in this path have not always been targeted. The DNA-RNA interaction, specifically DNA:RNA hybrid formation, provides a unique target for controlling the transfer of genetic information through binding by small molecules. Not only do DNA:RNA hybrids differ in conformation from widely targeted DNA and RNA, the low occurrence within biological systems further validates their therapeutic potential. Surprisingly, a survey of the literature reveals only a handful of ligands that bind DNA:RNA hybrids; in comparison, the number of ligands designed to target DNA is in the thousands. DNA:RNA hybrids, from their scientific inception to current applications in ligand targeting, are discussed.     

Structural Studies of Heterochiral DNA/DNA,RNA/RNA,AND DNA/RNA Duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

Characterization of RNA primers synthesized by the human breast cancer cell DNA synthesome

We previously reported on the purification and characterization of a functional multi‐protein DNA replication complex (the DNA synthesome) from human cells and tissues. The synthesome is fully competent to carry‐out all phases of the DNA replication process in vitro. In this study, DNA primase, a component of the synthesome, is examined to determine its activity and processivity in the in vitro synthesis and extension of RNA primers. Our results show that primase activity in the P4 fraction of the synthesome is 30‐fold higher than that of crude cell extracts. The synthesome synthesizes RNA primers that are 7–10 ribonucleotides long and DNA primers that are 20–40 deoxyribonucleotides long using a poly(dT) template of exogenous single‐stranded DNA. The synthesome‐catalyzed RNA primers can be elongated by E. coli DNA polymerase I to form the complementary DNA strands on the poly(dT) template. In addition, the synthesome also supports the synthesis of native RNA primers in vitro using an endogenous supercoiled double‐stranded DNA template. Gel analysis demonstrates that native RNA primers are oligoribonucleotides of 10–20 nt in length and the primers are covalently link to DNA to form RNA‐primed nascent DNA of 100–200 nt. Our study reveals that the synthesome model is capable of priming and continuing DNA replication. The ability of the synthesome to synthesize and extend RNA primers in vitro elucidates the organizational and functional properties of the synthesome as a potentially useful replication apparatus to study the function of primase and the interaction of primase with other replication proteins. J. Cell. Biochem. 106: 798–811, 2009. © 2009 Wiley‐Liss, Inc.     

Triple helices containing arabinonucleotides in the third (Hoogsteen) strand: effects of inverted stereochemistry at the 2'-position of the sugar moiety.

Arabinonucleic acid, the 2'-stereoisomer of RNA, was tested for its ability to recognize double-helical DNA, double-helical RNA and RNA-DNA hybrids. A pyrimidine oligoarabinonucleotide (ANA) was shown to form triple-helical complexes only with duplex DNA and hybrid DNA (Pu):RNA (Py) with an affinity that was slightly lower relative to the corresponding pyrimidine oligodeoxynucleotide (DNA) third strand. Neither the ANA nor DNA third strands were able to bind to duplex RNA or hybrid RNA (Pu):DNA (Py). In contrast, an RNA third strand recognized all four possible duplexes (DD, DR, RD and RR), as previously demonstrated. Such an understanding can be applied to the design of sequence-selective oligonucleotides which interact with double-stranded nucleic acids and emphasizes the role of the 2'-OH group as a general recognition and binding determinant of RNA.     

Selective labeling and detection of specific RNAs in an RNA mixture

We report here a unique approach to selectively label and detect specific RNA in an RNA mixture (without separation or purification) using DNA polymerase, dNTP labels, and a short synthetic DNA template complementary to the 3(')-terminus of the RNA. The detection sensitivity is high, at attomole level (10-18 mole). The selective principle was demonstrated by individually labeling and detecting RNAs in a RNA mixture when different templates were provided. By taking advantage of the template-directed selectivity, poly(A) tail-containing mRNA in total RNA was detected and labeled at the 3(')-terminal on a poly(T) template. Nonradioactive labels, such as fluorophore and antigen labels, may also be used; this method can be applied in methodology for direct detection and quantification of viral RNAs.     

Binding of nucleic acids to E. coli RNase HI observed by NMR and CD spectroscopy.

To clarify the mechanism by which the RNA portion of a DNA/RNA hybrid is specifically hydrolyzed by ribonuclease H (RNase H), the binding of a DNA/RNA hybrid, a DNA/DNA duplex, or an RNA/RNA duplex to RNase HI from Escherichia coli was investigated by 1H-15N heteronuclear NMR. Chemical shift changes of backbone amide resonances were monitored while the substrate, a hybrid 9-mer duplex, a DNA/DNA 12-mer duplex, or an RNA/RNA 12-mer duplex was titrated. The amino acid residues affected by the addition of each 12-mer duplex were almost identical to those affected by the substrate hybrid binding, and resided close to the active site of the enzyme. The results reveal that all the duplexes, hybrid-, DNA-, and RNA-duplex, bind to the enzyme. From the linewidth analysis of the resonance peaks, it was found that the exchange rates for the binding were different between the hybrid and the other duplexes. The NMR and CD data suggest that conformational changes occur in the enzyme and the hybrid duplex upon binding.     

The isolation of plasmids containing dna complementary to messenger rna for variant surface glycoproteins of Trypanosoma brucei

We have isolated poly(A)⁺ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)⁺ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs.The total translation products from the four poly(A)⁺ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins.We have made duplex DNA copies of these poly(A)⁺ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)⁺ RNA showed that 7–10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)⁺ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation.Poly(A)⁺ RNA from each of the variants only hybridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.     

Thermodynamic study of hybridization properties of heterochiral nucleic acids

Heterochiral DNA and RNA heptamers, which contained an unnatural L-nucleotide, were synthesized, and thermodynamic analyses of their hybridization properties with complementary DNA and RNA strands were systematically conducted by UV melting experiments. The results clearly demonstrated that the incorporation of an L-ribonucleotide into the RNA strand leads to more significant destabilization of the duplexes than that of an L-deoxyribonucleotide into the DNA strand, regardless of whether the complementary strand is DNA or RNA. The destabilization of the duplexes by the substitution of D-thymidine with L-thymidine in the DNA strand is entropically driven, whereas that by the substitution of D-uridine with L-uridine in the RNA strand is enthalpically driven. The thermodynamic characteristic that the stability of homochiral duplex is far superior to that of heterochiral duplex is much more remarkable in RNA than in DNA. Thus, RNA might have been a self-replicating system superior to DNA to exclude the chiral antipode.     

Kinetic hybrid i-motifs: intercepting DNA with RNA to form a DNA(2)-RNA(2) i-motif

We have constructed and characterized a long-lived hybrid DNA(2)-RNA(2) i-motif that is kinetically formed by mixing equivalent amount of C-rich RNA (R) and C-rich DNA (D). Circular dichroism shows that these hybrids are distinct from their parent DNA(4) or RNA(4) i-motif. pH dependent CD and UV thermal melting experiments showed that the complexes were maximally stable at pH 4.5, the pK(a) of cytosine, consistent with the complex being held by CH(+)-C base pairs. Fluorescence studies confirmed their tetrameric nature and established the relative strand polarities of the RNA and DNA strands in the complex. These showed that in a hybrid D(2)R(2) i-motif two DNA strands occupy one narrow groove and the two RNA strands occupy the other. This suggests that even the sugar-sugar interactions are highly specific. Interestingly, this hybrid slowly disproportionates into DNA(4) i-motifs and ssRNA which would be valuable to study intermediates in DNA(4) i-motif formation.     

Immunochemical detection of multiple conformations within a 36 base pair oligonucleotide.

A 36 base pair chimeric oligonucleotide containing a central core of DNA duplex flanked by RNA/DNA hybrid at each end was synthesized. These distinct regions of the oligonucleotide adopt different conformations which were detected with antibody probes. Enzyme linked immunosorbent assays (ELISA) and a gel electrophoresis retardation assay were used to demonstrate the binding of antibodies which recognize B-DNA, Z-DNA and RNA/DNA hybrid. The DNA duplex core of this oligonucleotide adopts the B-conformation in 0.14 M NaCl. In high salt solution (4 M NaCl) the DNA core adopts the Z-conformation. The RNA/DNA hybrid at the ends of the oligomer adopt a conformation which is distinct from both B-DNA and A-RNA.     

Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.