NMR structure of an antisense DNA.RNA hybrid duplex containing a 3'-CH(2)N(CH(3))-O-5' or an MMI backbone linker.

The solution structure of an antisense DNA.RNA hybrid duplex, d(CGCGTT-MMI-TTGCGC).r(GCGCAAAACGCG) (designated R4), containing an MMI backbone linker [3'-CH(2)N(CH(3))-O5'], is elucidated. The structural details of the MMI linker, its structural effects on the neighboring residues, and the molecular basis of the MMI effects are examined. The lipophilic N-methyl group of MMI is peripheral to the helix, assuming a conformation that is most stable with regard to the N-O torsion angle. The MMI linker promotes a 3'-endo conformation for the sugar moieties at both 3'- and 5'-adjacent positions and a backbone kink involving distant residues along the 3'-direction. Comparison of R4 with other analogous hybrid duplexes previously studied in this laboratory reveals a new family of low-energy helical conformations that can be accommodated in stable duplexes and a common feature of C3'-modified sugars for adopting a C3'-endo pucker. The results of these studies emphasize the interplay of several factors that govern the formation of stable hybrid duplexes and provide a basis for the understanding of the biological role of the MMI modifications, which are important building blocks for a family of promising chimeric antisense oligonucleotides.     

Discrimination Between A-type and B-type Conformations of Double Helical Nucleic Acid Fragments in Solution by Means of Two-dimensional Nuclear Overhauser Experiments

Abstract

The solution structure of two double helical nucleic acid fragments, viz. r(CGCGCG) and d(CGCGCG), was probed by means of two-dimensional nuclear Overhauser effect spectroscopy. The two compounds were selected as models for the A-type and B-type double helical conformations, respectively, and it is shown that for each of the two model compounds the intensities of the NOE cross peaks between base- and H2′ (deoxy)ribose proteins are qualitatively in correspondence with the relative NOE intensities expected on basis of the supposed duplex conformations. Thus our results indicate that NOE-data can be used to differentiate between A- and B-type double helical conformations in solution.

Coupling constant data show that, except for G(6), all ribose rings in r(CGCGCG) adopt pure N (C3′-endo) conformations thereby manifesting that this molecule takes up a regular A-type double helical conformation in solution. In contrast, the deoxyribose rings in d(CGCGCG) retain conformational freedom in the duplex state, albeit that the N/S- equilibrium is biased towards the S (C2′-endo) sugar conformation. This finding indicates that in solution the B-DNA backbone is highly dynamic.     

The A and B conformations of DNA and RNA subunits. Potential energy calculations for dGpdC

In order to obtain a molecular picture of the A and B forms of a DNA subunit, potential energy calculations have been made for dGpdC with C(3′)-endo and C(2′)-endo [or C(3′)-exo] sugar puckerings. These are compared with results for GpC. The global minima for dGpdC and GpC are almost identical. They are like A-form duplex DNA and RNA, respectively, with bases anti, the ω′, ω angle pair near 300°, 280°, and sugar pucker C(3′)-endo. For dGpdC, a B-form helical conformer, with sugar pucker C(2′)-endo and ω′ = 257°, ω = 298°, is found only 0.4 kcal/mol above the global minimum. A second low-energy conformation (2.3 kcal/mol) has ω′ = 263°, ω = 158° and ψ near 180°. This has dihedral angles like the original Watson–Crick model of the double helix. In contrast, for GpC, the C(2′)-endo B form is 6.9 kcal/mol above the global minimum. These theoretical results are consistent with experimental studies on DNA and RNA fibers. DNA fibers exist in both A and B forms, while RNA fibers generally assume only the A form. A low-energy conformation unlike the A or B forms was found for both dGpdC and GpC when the sugars were C(3′)-endo. This conformation—ω′,ω near 20°,80°—was not observed for C(2′)-endo dGpdC. Energy surface maps in the ω′,ω plane showed that C(2′)-endo dGpdC has one low-energy valley. It is in the B-form helical region (ω′ ～ 260°, ω ～ 300). When the sugar pucker is C(3′)-endo, dGpdC has two low-energy regions: the A-form helical region and the region with the minimum at ω′ = 16°, ω = 85°.     

Single-Stranded DNA and RNA Targeted Triplex-Formation: UV,CD and Molecular Modeling Studies of Foldback Triplexes Containing Different RNA, 2′-OMe-RNA and DNA Strand Combinations

Abstract

We studied the influence of different 2′-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D D:D, R* D:D, D D:R*, R* D:R*, D R:D, R* R:D, DR:R*, and R*-R:R*; where the first, middle, and last letters stand for the Hoogsteen Pyrimidine, Watson-Crick [WC] purine and WC pyrimidine strands, respectively, and D, R and R* stand for DNA, RNA and 2′-OMe-RNA strands, respectively) indicate more stable foldback triplex formation with a DNA purine strand than with an RNA purine strand. Of the four possible WC duplexes with RNA/DNA combinations, the duplex with a DNA purine strand and a 2′-O-Me-RNA pyrimidine strand forms the most thermally stable triplex, although its thermal stability is the lowest of all four duplexes. Irrespective of the duplex combination, a 2′-OMe-RNA Hoogsteen pyrimidine strand forms a stable foldback triplex over a DNA Hoogsteen pyrimidine strand confirming the earlier reports with conventional and circular triplexes. The CD studies suggest a B-type conformation for an all DNA homo-foldback triplex (D.D.D), while hetero-foldback triplex spectra suggest intermediate conformation to both Atype and B-type structures. A novel molecular modeling study has been carried out to understand the stereochemical feasibility of all the combinations of foldback triplexes using a geometric approach. The new approach allows use of different combinations of chain geometries depending on the nature of the chain (RNA vs. DNA).     

Remarkable stabilization of antiparallel DNA triplexes by strong stacking effects of consecutively modified nucleobases containing thiocarbonyl groups

The consecutive arrangement of 2′-deoxy-6-thioguanosines (s⁶Gs) and 4-thiothymidines (s⁴Ts) in antiparallel triplex-forming oligonucleotides (TFOs) considerably stabilized the resulting antiparallel triplexes with high base recognition ability by the strong stacking effects of thiocarbonyl groups. This result was remarkable because chemical modifications of the sugar moieties and nucleobases of antiparallel TFOs generally destabilize triplex structures. Moreover, in comparison with unmodified TFOs, it was found that TFOs containing s⁶Gs and s⁴Ts could selectively bind to the complementary DNA duplex but not to mismatched DNA duplexes or single-stranded RNA.     

SYNTHESIS AND HYBRIDIZATION PROPERTIES OF RNA CONTAINING 8-CHLOROADENOSINE

ABSTRACT

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3′- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by ～5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.     

Oligonucleotide analogues bearing an acyclonucleoside linked by an internucleotide amide bond

Oligonucleotide analogues bearing an acyclocytidine linked to thymidine with an amide (3′-O-CH₂-CO-N-5′) bond were synthesized. Melting curves of duplexes formed by modified oligonucleotides and complementary natural oligomers were obtained and thermodynamic parameters of their formation were measured. Replacement of dCpT by a modified dinucleotide only moderately decreased the melting temperature of these modified duplexes in comparison with unmodified duplexes containing complementary natural bases. CD spectra of modified duplexes were studied, and the duplex spatial structures are discussed.     

Conformations of cyclic 2,3-nucleotides and 2,3-Cyclic-5-diphosphates. Interrelation between the phase angle of pseudorotation of the sugar and the torsions about the glycosyl C(1)-N and the backbone C(4)-C(5) bonds

Semiempirical potential energy calculations have been carried out for cyclic 2′,3′-nucleotides and their 5′-phosphorylated derivatives, which are the intermediates in the hydrolysis of RNA. Calculations have been performed for both purine and pyrimidine bases for the observed O(1′)-endo, O(1′)-exo and the unpuckered planar sugar ring conformations. It is found that the mode of sugar pucker largely determines the preferred conformations of these molecules. For cyclic 2′,3′-nucleotides themselves, the O(1′)-endo sugars show a preference for the syn glycosyl conformation while the O(1′)-exo sugars exclusively favor the anti conformation regardless of whether the base is a purine or pyrimidine. For the unpuckered planar sugar, the syn conformation is favored for purines and anti for pyrimidines. Both the gauche (+) (60°) and trans (180°) conformations about the C(4′)? C(5′) bond are favored for O(1′)-endo sugars, while the gauche (?) (300°) and trans (180°) are favored for O(1′)-exo sugars. On the contrary, the 5′-phosphorylated cyclic 2′,3′-nucleotides of both purines and pyrimidines show a preference for the anti-gauche (+) conformational combination about the glycosyl and C(4′)? C(5′) bonds for the O(1′)-endo sugars and the anti-trans combination for the O(1′)-exo sugars. The correlation between the phase angle of the sugar ring and the favored torsions about the glycosyl and the backbone C(4′)? C(5′) bonds as one traverses along the pseudorotational pathway of the sugar ring is examined.     

The conformations of locked nucleic acids (LNA)

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2'-O, 4'-C-methylene bridge. We have investigated a modified 9-mer single stranded oligonucleotide as well as three 9- and 10-mer modified oligonucleotides hybridized to unmodified DNA. The single-stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson-Crick base-pairing with all the nucleotides in anti-conformation. By use of selective DQF-COSY spectra we determined the ratio between the N-type (C3'-endo) and S-type (C2'-endo) sugar conformations of the nucleotides. In contrast to the corresponding single-stranded DNA (ssDNA), we found that the sugar conformations of the single-stranded LNA oligonucleotide (ssLNA) cannot be described by a major S-type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N-type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N-type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S-type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N-type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases.     

Lna (Locked Nucleic Acid)

Abstract

LNA (Locked Nucleic Acid) forms duplexes with complementary DNA, RNA or LNA with unprecedented thermal affinities. CD spectra show that duplexes involving fully modified LNA (especially LNA:RNA) structurally resemble an A-form RNA:RNA duplex. NMR examination of an LNA:DNA duplex confirm the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA is demonstrated suggesting strand invasion by LNA. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has been accomplished.     

Conformational studies on pyrimidine 5'-monophosphates and 3',5'-diphosphates. Effect of the phosphate groups on the backbone conformation of polynucleotides

In continuation of our studies on the effect of the base and the phosphate groups on the glycosyl and the sugar-phosphate backbone conformation, we have carried out semi-empirical potential energy calculations on the common 5′- and 3′5′-ribopyrimidine mono- and diphosphates by considering simultaneous rotations about the glycosyl (χ) and the C(4′)–C(5′) (ψ) bonds. This calculation provides an assessment of the nature and orientation of the base on the sugar–phosphate backbone conformation of nucleotides and polynucleotides. It is found that the attractive inetractions between the 5′-phosphate group and the base mutually stabilize the antiand the gauche-gauche (gg) conformations about χ and ψ, respectively, in 5′-ribopyrimidine nucleotides. The introduction of the 3′-phosphate group as in 3′,5′-ribopyrimidine diphosphates, still leaves the anti-gg as the most favored conformation with the important difference that the probability of occurrence of the anti, gauche-trans (gt) is how substantially increased. This is dependent to a large extent on the sugar conformation and to a lesser extent on the base. Uracil and thymine show a greater probability for the anti-gt than cytosine. The syn conformation is considerably less likely and its occurrence is also dependent on the base type, cytosine showing a lesser tendency than uracil and thymine. For the syn base, the most favourec conformation for ψ is gt, since gg is sterically disallowed and tg is destabilized by electrostatic repulsive interactions between the 3′ and 5′-phosphate groups. Thus, there is a striking correlation between the glycoysl and the backbone C(4′)–C(5′) bond conformations. The rest of the bonds of the backbone are considerable less dependent on the glycosyl conformation. These studies reveal that in poly-ribopyrimidine nucletides the majority of the nucleotide residues are expected to occur in the anti-gg conformation.     

Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: the structural basis of this difference

Through binding and fluorescence studies of oligonucleotides covalently attached to a pyrene group via one carbon linker at the sugar residue, we previously found that pyrene-modified RNA oligonucleotides do not emit well in the single-stranded form, yet the attached pyrene emits with a significantly high quantum yield upon binding to a complementary RNA strand. In sharp contrast, similarly modified pyrene–DNA probes exhibit very weak fluorescence both in the double-stranded and single-stranded forms. The pyrene-modified RNA oligonucleotides therefore provide a useful tool for monitoring RNA hybridization. The purpose of this paper is to present the structural basis for the different fluorescence properties of pyrene-modified RNA/RNA and pyrene-modified DNA/DNA duplexes. The results of absorption, fluorescence anisotropy and circular dichroism studies all consistently indicated that the pyrene attached to the RNA duplex is located outside of the duplex, whereas the pyrene incorporated into the DNA duplex intercalates into the double helix. ¹H NMR measurements unambiguously confirmed that the pyrene attached to the DNA duplex indeed intercalates between the base pairs of the duplex. Molecular dynamics simulations support these differences in the local structural elements around the pyrene between the pyrene–RNA/RNA and the pyrene–DNA/DNA duplexes.     

Examination by the molecular orbital method of the intrinsic conformation preferences of nucleic acid sequence]

This paper reports a systematic PCILO study of the conformation of the nucleic acid backbone. The authors principally studied the ω′ and ω phosphodiester torsion angles of the disugar triphosphate model as a simultaneous function of (1) the sugar nature, ribose or deoxyribose, (2) the different combinations of the sugar ring puckers C(2′)-endo-C(2′)-endo, C(3′)-endo-C(3′)-endo, C(3′)-endo-C(2′)-endo, and C(2′)-endo-C(3′)-endo, and (3) the different conformations around the ψ(C4′–C5′) exocyclic bond. The dependence of the (ω′,ω) conformational energy maps upon these different factors, is discussed. The results are in very good agreement with the observed structures of ribonucleic (RNA10, RNA11, A′-RNA12, tRNA^Phe) and deoxyribonucleic acids (D-DNA, C-DNA 9.3, B-DNA 10, A-DNA 11). Thus the validity of this model, the disugar triphosphate unit, is ensured. The main conclusions that can be drawn from this systematic study are the following:

• 1 The torsion around P-05′ (angle ω) is, as a general rule, more flexible than the torsion around P-03′ (angle ω′).
• 2 There is no notable difference between the ribose–triphosphate units and the deoxyribose–triphosphate units for the C(3′)-endo–C(3′)-endo and C(3′)-endo–C(2′)-endo sugar puckers.
• 3 The deoxyribose–triphosphate units with C(2′)-endo–C(2′)-endo and C(2′)-endo–C(3′)-endo sugar puckers show much more ω′ flexibility than the ribose–triphosphate units with the same sugar puckers and cis position for the 2′hydroxyl group.
• 4 The preferred values of ω′ are independent of the sugar nature (ribose or deoxyribose) and of ψ values; they are correlated with the sugar pucker of the first sugar-phosphate unit:
  • C(3′)-endo-C(3′)-endo and C(3′)-endo-C(2′)-endo puckers ? ω′ ? 240° (g^? region)
  • C(2′)-endo-C(2′)-endo and C(2′)-endo-C(3′)-endo puckers ? ω′ 180° (t region)
• 5 The preferred values of ω are independent of the nature and the puckering of the sugars; they are correlated with the rotational state of the torsion angle ψ(C4′–C5′): ψ ? 60° (gg) ? ω ? 300° (g^?), ψ ? 180° (gt) or 300° (tg) ? ω ? 60° (g⁺)

     

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2′-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2′-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2′-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first β sheet and second β sheet of RNase HI of Escherichia coli might participate in substrate binding.     

Interaction of molecules with nucleic acids. II. Two pairs of families of intercalation sites, unwinding angles, and the neighbor-exclusion principle

Intercalation-site geometries are generated for a tetramer duplex extracted from B-DNA. Glycosidic angles and puckers of the deoxyribose sugar groups bonded to base pairs BP₁ and BP₄, namely, those at either end of the tetramer duplex, are assumed to be those of B-DNA to insure continuity. All possible geometrical conformations for combinations of C(2′)-endo, C(3′)-endo, C(2′)-exo, and C(3′)-exo sugar puckers are determined for the tetranucleotide backbone. Those with minimum energy are selected as candidates for intercalation sites. Calculations reveal two pairs of physically meaningful families of intercalation sites which occur in two distinct regions, I and II, of helical angles which orient BP₂ relative to BP₃ and with the helical axis disjointed between these base pairs. For each site I and II within BP₂ and BP₃, there are two distinct backbone conformations, A and B, connecting BP₃ to BP₄ or BP₁ to BP₂ which do not disrupt backbone conformations connecting BP₂ to BP₃. Hence two pairs, IA and IB, and IIA and IIB, of intercalation sites exist in which the sugar puckers along the backbone of the tetramer alternate from C(2′)-endo to C(3′)-endo on the backbone (5′p3′) connecting BP₂ to BP₃. The glycosidic angles of the C(3′)-endo sugar χ₃^γ are, coincidentally, 80° ± 2° for both conformations γ = A and B connecting BP₃ to BP₄ along the phosphate backbone (5′p3′). Consistent with the theoretical results, the experimental unwinding angles can be grouped into two categories with absolute values of 18° and 26°. The theoretical unwinding angles for sites IA and IB of 16° and for sites IIA and IIB of 20° occur for a displacement of -0.8 Å in the helical axes of BP₂ and BP₃ and for a 100% G·C composition, with a decrease depending on the amount of A·T base pairs present. Ratios of theoretical unwinding angles of sites I and II, which range from 0.75 to 0.84 for the two principal sites, compare well with the experimental value of 0.71. The theoretical results, in agreement with experimental observation, provide a new interpretation of the nature and conformation of the possible binding sites. Conformations obtained from these studies of intercalation sites in a tetramer duplex are used to rationalize the well-known neighbor-exclusion principle. The possibility of violation of this principle is demonstrated by the existence of two families of physically meaningful conformations. Conformations of unconstrained dimer duplexes are also obtained, one of which corresponds to the experimental crystal structure of ethidium–dinucleoside complexes, but these cannot be joined to the B-DNA structure. Backbone conformations of the tetramer duplex can be constructed until the base-pair separation reaches 8.25 Å, which may limit the molecules that can intercalate.     

Enhancement and Inhibition by 2′-O-Hydroxyethyl Residues of Gene Targeting Mediated by Triple Helix Forming Oligonucleotides

Abstract

Reagents that recognize and bind specific genomic sequences in living mammalian cells would have great potential for genetic manipulation, including gene knockout, strain construction, and gene therapy. Triple helix forming oligonucleotides (TFOs) bind specific sequences via the major groove, but pyrimidine motif TFOs are limited by their poor activity under physiological conditions. Base and sugar analogues that overcome many of these limitations have been described. In particular, 2′-O-modifications influence sugar pucker and third strand conformation, and have been important to the development of bioactive TFOs. Here we have analyzed the impact of 2′-O-hydroxyethyl (2′-HE) substitutions, in combination with other 2′ modifications. We prepared modified TFOs conjugated to psoralen and measured targeting activity in a gene knockout assay in cultured hamster cells. We find that 2′-HE residues enhance the bioactivity of TFOs containing 2′-O-methyl (2′-OMe) modifications, but reduce the bioactivity of TFOs containing, in addition, 2′-O-aminoethyl (2′-AE) residues.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

STRUCTURAL,DYNAMIC, AND ENZYMATIC PROPERTIES OF MIXED α/β-OLIGONUCLEOTIDES CONTAINING POLARITY REVERSALS

We employ NMR structure determination, thermodynamics, and enzymatics to uncover the structural, thermodynamic and enzymatic properties of α/β-ODNs containing 3′-3′ and 5′-5′ linkages. RNase H studies show that α/β-gapmers that are designed to target erbB-2 efficiently elicit RNase H activity. NMR structures of DNA · DNA and DNA · RNA duplexes reveal that single α-anomeric residues fit well into either duplex, but alter the dynamic properties of the backbone and deoxyriboses as well as the topology of the minor groove in the DNA · RNA hybrid.     

The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes.

In an effort to discover novel oligonucleotide modifications for antisense therapeutics, we have prepared oligodeoxyribonucleotides containing more than 200 different modifications and measured their affinities for complementary RNA. These include modifications to the heterocyclic bases, the deoxy-ribose sugar and the phosphodiester linkage. From these results, we have been able to determine structure-activity relationships that correlate hybridization affinity with changes in oligonucleotide structure. Data for oligonucleotides containing modified pyrimidine nucleotides are presented. In general, modifications that resulted in the most stable duplexes contained a heteroatom at the 2'-position of the sugar. Other sugar modifications usually led to diminished hybrid stability. Most backbone modifications that led to improved hybridization restricted backbone mobility and resulted in an A-type sugar pucker for the residue 5'to the modified internucleotide linkage. Among the heterocycles, C-5-substituted pyrimidines stood out as substantially increasing duplex stability.