Control of DNA conformation using 3'-S-phosphorothiolate-modified linkages

An in-depth study into the incorporation of multiple 3-S-phosphorothiolate modifications into oligodeoxynucleotides (ODNs) and their subsequent effect on ODN/DNA and ODN/RNA duplex stability. 3-S-Phosphorothiolate linkages increase the stability of ODN/RNA duplexes and decrease the stability of ODN/DNA duplexes.     

CONTROL OF DNA CONFORMATION USING 3′-S-PHOSPHOROTHIOLATE-MODIFIED LINKAGES

An in-depth study into the incorporation of multiple 3′-S-phosphorothiolate modifications into oligodeoxynucleotides (ODNs) and their subsequent effect on ODN/DNA and ODN/RNA duplex stability. 3′-S-Phosphorothiolate linkages increase the stability of ODN/RNA duplexes and decrease the stability of ODN/DNA duplexes.     

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (T_m) of the peptide–RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as l-2-amino-4-guanidinobutyric acid (Agb) or l-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the l-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide–RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.     

The thermal stability of RNA duplexes containing modified base pairs placed at internal and terminal positions of the oligoribonucleotides

The presence of various modifications within oligomers changes their thermodynamic stability. To get more systematic data, we measured effects of 5- and 6-substituted uridine on thermal stability of (AUCU(Mod.)AGAU)2 and (AUCUAGAU(Mod.))2. Collected results lead to the following conclusions: (i) 5-halogenated and 5-alkylated substituents of the uridine affect thermal stability of the RNA duplexes differently. Moreover, the 5-fluorouridine changes stability of the RNA duplexes opposite to remaining 5-halogenouridines; (ii) for oligomers containing 5-chloro, 5-bromo or 5-iodouridine stronger hydrogen bond formed between oxygen-4 of the 5-halogenated uracil and 6-amino group of the adenine is presumably responsible for stabilizing effect; (iii) placing of A-U(5R) base pairs closer to the end of the duplex enhance thermal stability relatively to oligomer with central position of this base pair; (iv) the effects of 5-substituents are additive, particularly for substituents which stabilize RNA duplexes; (v) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine present at internal position of A-U(Mod.) inhibit duplexes formation; (vi) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine placed as terminal base pairs stabilize the duplexes mostly via 3'-dangling end effect.     

CD, absorption and thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes [d/r(AC)]12.[d/r(GT/U)]12 and the influence of phosphorothioate substitution.

Circular dichroism (CD) spectra and melting temperature (Tm) data for five duplexes containing phosphorothioate linkages were compared with data for four unmodified duplexes to assess the effect of phosphorothioate modification on the structure and stability of DNA. DNA and DNA.RNA duplexes. Nine duplexes were formed by mixing oligomers 24 nt long in 0.15 M K+(phosphate buffer), pH 7.0. Unmodified DNA.DNA and RNA.RNA duplexes were used as reference B-form and A-form structures. The CD spectra of the modified hybrids S-d(AC)12.r(GU)12 and r(AC)12.S-d(GT)12 differed from each other but were essentially the same as the spectra of the respective unmodified hybrids. They were more A-form than B-form in character. CD spectra of duplexes S-d(AC)12.d(GT)12 and d(AC)12.S-d(GT)12 were similar to that of d(AC)12.d(GT)12, except for a reduced long wavelength CD band. Sulfur modifications on both strands of the DNA duplex caused a pronounced effect on its CD spectrum. The order of thermal stability was: RNA.RNA > DNA.DNA > DNA.RNA > S-DNA.DNA > S-DNA. RNA > S-DNA.S-DNA. Phosphorothioation of one strand decreased the melting temperature by 7.8+/-0.6 degrees C, regardless of whether the substitution was in a hybrid or DNA duplex. Thermodynamic parameters were obtained from a multistate analysis of the thermal melting profiles. Interestingly, the destabilizing effect of the phosphorothioate substitution appears to arise from a difference in the entropy upon forming the DNA.DNA duplexes, while the destabilizing effect in the DNA.RNA hybrids appears to come from a difference in enthalpy.     

The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes

The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA.     

Thermodynamics of single mismatches in RNA duplexes

The thermodynamic properties and structures of single mismatches in short RNA duplexes were studied in optical melting and imino proton NMR experiments. The free energy increments at 37 degrees C measured for non-GU single mismatches range from -2.6 to 1.7 kcal/mol. These increments depend on the identity of the mismatch, adjacent base pairs, and the position in the helix. UU and AA mismatches are more stable close to a helix end, but GG mismatch stability is essentially unaffected by the position in the helix. Approximations are suggested for predicting stabilities of single mismatches in short RNA duplexes.     

The influence of various modified nucleotides placed as 3'-dangling end on thermal stability of RNA duplexes

The ribonucleic acids (RNA) form highly folded structures, which behind the helical fragments contain several secondary and tertiary structural motives. All of them have an influence on thermodynamic stability of the RNA. The 5'- and 3'-dangling ends are one of those structural motives, which effect stability of the adjacent helixes. In this paper, we described the influence of 14 different modified nucleotides, placed as 3'-dangling ends, on thermal stability of the RNA duplexes. Collected data demonstrate that: (i) 5-substituents of the uridine have an impact on the 3'-dangling end effect and the largest changes were observed for 5-chloro, bromo and methyl substituents; (ii) position of the methyl group within the uracil residue affect the thermal stability of the duplex; (iii) increasing a size of the heterocycle base placed as the 3'-terminal unpaired nucleotide enhances stabilization of duplexes.     

Different strategies for the synthesis of 2'-O-aminoethyl adenosine building blocks

The chemical modification of the 2'-O-position of nucleosides proved to be of great importance for the RNA stability. Greater stability of RNA duplexes allows a longer half life in the cell and, therefore, a better effect of RNA Interference. Here we investigated the synthesis of 2'-O-aminoethyl adenosine as a cationic modified building block.     

Different Strategies for the Synthesis of 2′- O -Aminoethyl Adenosine Building Blocks

The chemical modification of the 2′-O-position of nucleosides proved to be of great importance for the RNA stability. Greater stability of RNA duplexes allows a longer half life in the cell and, therefore, a better effect of RNA Interference. Here we investigated the synthesis of 2′-O-aminoethyl adenosine as a cationic modified building block.     

Simple molecular engineering of glycol nucleic acid: Progression from self-pairing to cross-pairing with cDNA and RNA

The acyclic chiral nucleic acid analogue, Glycol Nucleic Acid (GNA), displayed exceptional structural simplicity and atom economy while forming self-paired duplexes, using canonical Watson–Crick base pairing. We disclose here that the replacement of phosphodiester linker in GNA with somewhat rigid and shorter carbamate linker in Glycol Carbamate Nucleic Acid (GCNA) backbone allows unprecedented stability to the antiparallel self-paired duplexes. The R-GCNA oligomers were further found to form cross-paired antiparallel duplexes with cDNA and RNA following Watson–Crick base pairing. The stability of cross-paired GCNA:DNA and GCNA:RNA duplexes was higher than the corresponding DNA:DNA and DNA:RNA duplexes. The chiral (R) and (S) precursors were easily accessible from naturally occurring l-serine.     

Hybridization Properties of RNA Containing 8-Methoxyguanosine and 8-Benzyloxyguanosine

Modified nucleobase analogues can serve as powerful tools for changing physicochemical and biological properties of DNA or RNA. Guanosine derivatives containing bulky substituents at 8 position are known to adopt syn conformation of N-glycoside bond. On the contrary, in RNA the anti conformation is predominant in Watson-Crick base pairing. In this paper two 8-substituted guanosine derivatives, 8-methoxyguanosine and 8-benzyloxyguanosine, were synthesized and incorporated into oligoribonucleotides to investigate their influence on the thermodynamic stability of RNA duplexes. The methoxy and benzyloxy substituents are electron-donating groups, decreasing the rate of depurination in the monomers, as confirmed by N-glycoside bond stability assessments. Thermodynamic stability studies indicated that substitution of guanosine by 8-methoxy- or 8-benzyloxyguanosine significantly decreased the thermodynamic stability of RNA duplexes. Moreover, the presence of 8-substituted guanosine derivatives decreased mismatch discrimination. Circular dichroism spectra of modified RNA duplexes exhibited patterns typical for A-RNA geometry.     

The stability of intramolecular DNA G-quadruplexes compared with other macromolecules

DNA quadruplexes are often conceived as very stable structures. However, most of the free energy of stabilization derives from specific ion binding via inner sphere coordination of the GO6 of the guanine residues comprising the basic quartet. When compared with other nucleic acid structures such as DNA or RNA duplexes and hairpins, or proteins of the same number of atoms, metal-coordinated intramolecular quadruplexes are found to be of comparable or lower thermodynamic stability under similar solution conditions. Furthermore, intramolecular quadruplexes are actually less stable kinetically, than DNA duplexes or hairpins of the same size.     

The thermodynamics of 3'-terminal pyrene and guanosine for the design of isoenergetic 2'-O-methyl-RNA-LNA chimeric oligonucleotide probes of RNA structure

To facilitate design of short isoenergetic hybridization probes for RNA, we report the influence of adding 5'- or 3'-terminal 2'-O-methylguanosine (GM), LNA-guanosine (GL), or 3'-terminal pyrene pseudo-nucleotide (PPN) on the thermodynamic stability of 2'-O-methyl-RNA/RNA (2'-O-Me-RNA/RNA) duplexes with sequences 5'CMGMGMCMAM/3'AAXGCCGUXAA, where X is A, C, G, or U. A 3'-terminal GM or GL added to the 2'-O-Me-RNA strand to form a G-A, G-G or G-U mismatch enhances thermodynamic stability (DeltaDeltaG degrees 37) of the 2'-O-Me-RNA/RNA duplexes on average by 0.7 and 1.5 kcal/mol, respectively. A 3'-terminal GM or GL in a GM-C or GL-C pair stabilizes the 2'-O-Me-RNA/RNA duplex by 2.6 and 3.4 kcal/mol, respectively. A 5'-terminal GM or GL in a G-A or G-G mismatch provided less stabilization in comparison with a 3'-terminal G-A or G-G mismatch, but more stabilization in a G-C or G-U pair. In contrast to guanosine derivatives, pyrene residue (P) as PPN at the 3'-terminal position enhances thermodynamic stability of the 2'-O-Me-RNA/RNA duplexes on average by 2.3 +/- 0.1 kcal/mol, relatively independent of the type of ribonucleotide placed in the opposite strand. The thermodynamic data can be applied to design 2'-O-Me-RNA/RNA duplexes with enhanced thermodynamic stability that is also sequence independent. This is useful for design of hybridization probes to interrogate RNA structure and/or expression by microarray and other methods.     

Structure and stability of the consecutive stereoregulated chiral phosphorothioate DNA duplex

The duplex structures of the stereoregulated phosphorothioate DNAs, [R(p),R(p)]- and [S(p),S(p)]-[d(GC(ps)T(ps)ACG)] (ps, phosphorothioate; PS-DNA), with their complementary RNA have been investigated by combined use of (1)H NMR and restrained molecular dynamics calculation. Compared to those obtained for the unmodified duplex structures (PO-DNA.RNA), the NOE cross-peak intensities are virtually identical for the PS-DNA.RNA hybrid duplexes. The structural analysis on the basis of the NOE restraints reveals that all of the three DNA.RNA duplexes take a A-form conformation and that there is no significant difference in the base stacking for the DNA.RNA hybrid duplexes. On the other hand, the NOE cross-peak intensities of the protons around the central T(ps)A step of the PS-DNA.DNA duplexes are apparently different from those of PO-DNA. DNA. The chemical shifts of H8/6 and H1' at the T(ps)A step are also largely different among PS-DNA.DNAs and PO-DNA.DNA, suggesting that the DNA.DNA structure is readily changed by the introduction of the phosphorothioate groups to the central T(p)A step. The structure calculations indicate that all of these DNA.DNA duplexes are B-form although there exist some small differences in helical parameters between the [R(p),R(p)]- and [S(p),S(p)]PS-DNA.DNA duplexes. The melting temperatures (T(m)) were determined for all of the duplexes by plotting the chemical shift change of isolated peaks as a function of temperature. For the PS-DNA.RNA hybrid duplexes, the [S(p),S(p)] isomer is less stable than the [R(p),R(p)] isomer while this trend is reversed for the PS-DNA.DNA duplexes. Consequently, although the PS-DNA.RNA duplexes take the similar A-form structure, the duplex stability is different between PS-DNA.RNA duplexes. The stability of the DNA.RNA duplexes may not be governed by the A-form structure itself but by some other factors such as the hydration around the phosphorothioate backbone, although the T(m) difference of the DNA.DNA duplexes could be explained by the structural factor.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.     

Thermodynamics of RNA duplexes modified with unlocked nucleic acid nucleotides

Thermodynamics provides insights into the influence of modified nucleotide residues on stability of nucleic acids and is crucial for designing duplexes with given properties. In this article, we introduce detailed thermodynamic analysis of RNA duplexes modified with unlocked nucleic acid (UNA) nucleotide residues. We investigate UNA single substitutions as well as model mismatch and dangling end effects. UNA residues placed in a central position makes RNA duplex structure less favourable by 4.0–6.6 kcal/mol. Slight destabilization, by ∼0.5–1.5 kcal/mol, is observed for 5′- or 3′-terminal UNA residues. Furthermore, thermodynamic effects caused by UNA residues are extremely additive with ΔG°₃₇ conformity up to 98%. Direct mismatches involving UNA residues decrease the thermodynamic stability less than unmodified mismatches in RNA duplexes. Additionally, the presence of UNA residues adjacent to unpaired RNA residues reduces mismatch discrimination. Thermodynamic analysis of UNA 5′- and 3′-dangling ends revealed that stacking interactions of UNA residues are always less favourable than that of RNA residues. Finally, circular dichroism spectra imply no changes in overall A-form structure of UNA–RNA/RNA duplexes relative to the unmodified RNA duplexes.     

Serologic and immunochromatographic detection of oxygenated polyenoic acids in Euglena gracilis var. bacillaris

The extent of evolutionary conservation of DNA complimentary to RNA stored in the mature oocyte of the sea urchin S. purpuratus has been assessed. To do this, such DNA was hybridized with total genomic DNA of S. purpuratus and S. franciscanus and the thermal stability of the resultant duplexes was measured by two methods. In the first method, the duplexes were bound to hydroxylapatite and thermally eluted; the difference in thermal stability between homologous and heterologous duplexes averaged 6.9 degrees C in duplicate determinations. In the second experiment, the same hybrids were thermally melted in 2.4M tetraethylammonium chloride, then assayed with S1 nuclease; the difference in thermal stability of homologous and heterologous duplexes was 4.8 degrees C. Either value is significantly lower than the divergence of total single-copy DNA among these species as measured by the same techniques. This demonstrates that DNA sequences complimentary to maternal RNA are conserved during evolution, and thus that a high fraction of them are likely to be physiologically functional.