Solution structure of a dsDNA:LNA triplex

We have determined the NMR structure of an intramolecular dsDNA:LNA triplex, where the LNA strand is composed of alternating LNA and DNA nucleotides. The LNA oligonucleotide binds to the dsDNA duplex in the major groove by formation of Hoogsteen hydrogen bonds to the purine strand of the duplex. The structure of the dsDNA duplex is changed to accommodate the LNA strand, and it adopts a geometry intermediate between A- and B-type. There is a substantial propeller twist between base-paired nucleobases. This propeller twist and a concomitant large propeller twist between the purine and LNA strands allows the pyrimidines of the LNA strand to interact with the 5′-flanking duplex pyrimidines. Altogether, the triplex has a regular global geometry as shown by a straight helix axis. This shows that even though the third strand is composed of alternating DNA and LNA monomers with different sugar puckers, it forms a seamless triplex. The thermostability of the triplex is increased by 19°C relative to the unmodified DNA triplex at acidic pH. Using NMR spectroscopy, we show that the dsDNA:LNA triplex is stable at pH 8, and that the triplex structure is identical to the structure determined at pH 5.1.     

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.     

NMR structure of an alpha-L-LNA:RNA hybrid: structural implications for RNase H recognition

Alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) is a nucleotide analogue that raises the thermostability of nucleic acid duplexes by up to approximately 4 degrees C per inclusion. We have determined the NMR structure of a nonamer alpha-L-LNA:RNA hybrid with three alpha-L-LNA modifications. The geometry of this hybrid is intermediate between A- and B-type, all nucleobases partake in Watson-Crick base pairing and base stacking, and the global structure is very similar to that of the corresponding unmodified hybrid. The sugar-phosphate backbone is rearranged in the vicinity of the modified nucleotides. As a consequence, the phosphate groups following the modified nucleotides are rotated into the minor groove. It is interesting that the alpha-L-LNA:RNA hybrid, which has an elevation in melting temperature of 17 degrees C relative to the corresponding DNA:RNA hybrid, retains the global structure of this hybrid. To our knowledge, this is the first example of such a substantial increase in melting temperature of a nucleic acid analogue that does not act as an N-type (RNA) mimic. alpha-L-LNA:RNA hybrids are recognised by RNase H with subsequent cleavage of the RNA strand, albeit with slow rates. We attempt to rationalise this impaired enzyme activity from the rearrangement of the sugar-phosphate backbone of the alpha-L-LNA:RNA hybrid.     

The solution structure of a locked nucleic acid (LNA) hybridized to DNA

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations

Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–DNA and RNA–RNA duplexes, whereas the LNA–LNA duplex is found to have its helix partly unwound and does not resemble RNA–RNA duplex in a number of properties. Duplexes with an LNA strand have on average longer interstrand phosphate distances compared to RNA–DNA and RNA–RNA duplexes. Furthermore, intrastrand phosphate distances in LNA strands are found to be shorter than in DNA and slightly shorter than in RNA. In case of induced sugar puckering, LNA is found to tune the sugar puckers in partner DNA strand toward C3′-endo conformations more efficiently than RNA. The LNA–LNA duplex has lesser backbone flexibility compared to the RNA–RNA duplex. Finally, LNA is less hydrated compared to DNA or RNA but is found to have a well-organized water structure.     

The Solution Structure of a Locked Nucleic Acid (LNA) Hybridized to DNA

Abstract

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2′-0, 4′-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional'H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32Å. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3Å. The average twist over the sequence are 35.9° ± 0.3°. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by ¹H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5′-CT^LAG-3′ site than in the unmodified 5′-CT^LAG-3′ site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

Solution structure of an LNA hybridized to DNA: NMR study of the d(CT(L)GCT(L)T(L)CT(L)GC):d(GCAGAAGCAG) duplex containing four locked nucleotides

We have used two-dimensional (1)H NMR spectroscopy at 750 MHz to determine a high-resolution solution structure of an oligonucleotide containing restricted nucleotides with a 2'-O, 4'-C-methylene bridge (LNA) hybridized to the complementary DNA strand. The LNA:DNA duplex examined contained four thymidine LNA modifications (T(L), d(C1T(L)2G3C4T(L)5T(L)6C7T(L)8G9C10):d( G11C12A13G14A15A16G17C 18A19G20). A total relaxation matrix approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Forty final structures were generated for the duplex from A-form and B-form DNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the 40 structures of the complex was 0.6 A. The sugar puckerings are averaged values of a dynamic interchange between N- and S-type conformation except in case of the locked nucleotides that were found to be fixed in the C3'-endo conformation. Among the other nucleotides in the modified strand, the furanose ring of C7 and G9 is predominantly in the N-type conformation whereas that of G3 is in a mixed conformation. The furanose rings of the nucleotides in the unmodified complementary strand are almost exclusively in the S-type conformation. Due to these different conformations of the sugars in the two strands, there is a structural strain between the A-type modified strand and the B-type unmodified complementary strand. This strain is relaxed by decreasing the value of rise and compensating with tip, buckle, and propeller twist. The values of twist vary along the strand but for a majority of the base pairs a value even lower than that of A-DNA is observed. The average twist over the sequence is 32+/-1 degrees. On the basis of the structure, we conclude that the high stability of LNA:DNA duplexes is caused by a local change of the phosphate backbone geometry that favors a higher degree of stacking.     

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.     

Chemistry of locked nucleic acids (LNA): Design, synthesis, and bio-physical properties

Preparation of LNA nucleosides requires a number of synthetic steps but very efficient procedures have been developed, as have protocols for synthesis of LNA oligonucleotides on automated DNA synthesizers. In all cases, LNA oligonucleotides have exhibited good aqueous solubility as would be expected from their close structural resemblance to the natural nucleic acids. The universality of LNA mediated high-affinity and specific hybridization has been demonstrated extensively with a large number of duplex forming LNA-oligonucleotides. Most importantly, most of the members of the LNA molecular family have been shown to exert their substantial affinity increase (i) in combination with standard DNA, RNA and contemporary analogues and (ii) whether inserted as single nucleosides in an oligonucleotide or as blocks of contiguous nucleotides, an important point. The works on TFO's is expanding the usefulness of LNA to double strand recognition and it has been demonstrated that LNA it is a promising structure for further base modifications in the pursuit of global sequence specific recognition of DNA.     

Chemistry of locked nucleic acids (LNA): Design, synthesis, and bio-physical properties

Summary Preparation of LNA nucleosides requires a number of synthetic steps but very efficient procedures have been developed, as have protocols for synthesis of LNA oligonucleotides on automated DNA synthesizers. In all cases, LNA oligonucleotides have exhibited good aqueous solubility as would be expected from their close structural resemblance to the natural nucleic acids. The universality of LNA mediated high-affinity and specific hybridization has been demonstrated extensively with a large number of duplex forming LNA-oligonucleotides. Most importantly, most of the members of the LNA molecular family have been shown to exert their substantial affinity increase (i) in combination with standard DNA, RNA and contemporary analogues and (ii) whether inserted as single nucleosides in an oligonucleotide or as blocks of contiguous nucleotides, an important point. The works on TFO's is expanding the usefulness of LNA to double strand recognition and it has been demonstrated that LNA it is a promising structure for further base modifications in the pursuit of global sequence specific recognition of DNA.     

LNA guanine and 2,6-diaminopurine. Synthesis, characterization and hybridization properties of LNA 2,6-diaminopurine containing oligonucleotides

LNA guanine and 2,6-diaminopurine (D) phosphoramidites have been synthesized as building blocks for antisense oligonucleotides (ON). The effects of incorporating LNA D into ON were investigated. As expected, LNA D containing ON showed increased affinity towards complementary DNA (Delta Tm +1.6 to +3.0 degrees C) and RNA (Delta Tm +2.6 to +4.6 degrees C) ON. To evaluate if LNA D containing ON have an enhanced mismatch sensitivity compared to their complementary LNA A containing ON thermal denaturation experiments towards singly mismatched DNA and RNA ON were undertaken. Replacing one LNA A residue with LNA D, in fully LNA modified ON, resulted in higher mismatch sensitivity towards DNA ON (Delta Delta Tm -4 to >-17 degrees C). The same trend was observed towards singly mismatched RNA ON (Delta Delta Tm D-a = -8.7 degrees C and D-g = -4.5 degrees C) however, the effect was less clearcut and LNA A showed a better mismatch sensitivity than LNA D towards cytosine (Delta Tm +5.5 degrees C).     

LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA

Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Structural studies have shown that LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.     

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 beta sheet and second beta sheet of RNase HI of Escherichia coli might participate in substrate binding.     

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.     

Proton NMR studies on the covalently linked RNA-DNA hybrid r(GCG)d(TATACGC). Assignment of proton resonances by application of the nuclear Overhauser effect.

Proton NMR spectra of a covalently linked self-complementary RNA X DNA hybrid, r(GCG)-d(TATACGC), are recorded in H2O and D2O. Imino proton resonances as well as the non-exchangeable base and H-1' resonances are unambiguously assigned by means of nuclear. Overhauser effect measurements. Additional information was obtained by 31P NMR and circular dichroism spectra. The RNA parts in the duplex attain full conformational purity and adopt the usual A-RNA conformation. The DNA residues opposite the RNA tract do not adopt an A-type structure completely. Their respective sugar rings still appear to possess a certain conformational freedom. The same holds true for the central d(-TATA-) sequence which forms a DNA X DNA duplex. There appears to be a structural break in this part: the first two residues, T(4) and A(5), are clearly influenced by the adjacent RNA structure, whereas residues T(6) and A(7) behave quite similar to what usually is found in DNA duplexes in aqueous solution.     

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.     

Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation

The design of modified nucleic acid probes, primers, and therapeutics is improved by considering their thermodynamics. Locked nucleic acid (LNA) is one of the most useful modified backbones, with incorporation of a single LNA providing a substantial increase in duplex stability. In this work, the hybridization DeltaH(o), DeltaS(o), and melting temperature (T(M)) were measured from absorbance melting curves for 100 duplex oligonucleotides with single internal LNA nucleotides on one strand, and the results provided DeltaDeltaH(o), DeltaDeltaS(o), DeltaDelta, and DeltaT(M) relative to reference DNA oligonucleotides. LNA pyrimidines contribute more stability than purines, especially A(L), but there is substantial context dependence for each LNA base. Both the 5' and 3' neighbors must be considered in predicting the effect of an LNA incorporation, with purine neighbors providing more stability. Enthalpy-entropy compensation in DeltaDeltaH(o) and DeltaDeltaS(o) is observed across the set of sequences, suggesting that LNA can stabilize the duplex by either preorganization or improved stacking, but not both simultaneously. Singular value decomposition analysis provides predictive sequence-dependent rules for hybridization of singly LNA-substituted DNA oligonucleotides to their all-DNA complements. The results are provided as sets of DeltaDeltaH(o), DeltaDeltaS(o), and DeltaDelta parameters for all 32 of the possible nearest neighbors for LNA+DNA:DNA hybridization (5' MX(L) and 5' X(L)N, where M, N, and X = A, C, G, or T and X(L) represents LNA). The parameters are applicable within the standard thermodynamic prediction algorithms. They provide T(M) estimates accurate to within 2 degrees C for LNA-containing oligonucleotides, which is significantly better accuracy than previously available.     

NMR solution structures of LNA (locked nucleic acid) modified quadruplexes

We have determined the NMR solution structures of the quadruplexes formed by d(TGLGLT) and d(TL₄T), where L denotes LNA (locked nucleic acid) modified G-residues. Both structures are tetrameric, parallel and right-handed and the native global fold of the corresponding DNA quadruplex is retained upon introduction of the LNA nucleotides. However, local structural alterations are observed owing to the locked LNA sugars. In particular, a distinct change in the sugar–phosphate backbone is observed at the G2pL3 and L2pL3 base steps and sequence dependent changes in the twist between tetrads are also seen. Both the LNA modified quadruplexes have raised thermostability as compared to the DNA quadruplex. The quadruplex-forming capability of d(TGLGLT) is of particular interest as it expands the design flexibility for stable parallel LNA quadruplexes and shows that LNA nucleotides can be mixed with DNA or other modified nucleic acids. As such, LNA-based quadruplexes can be decorated by a variety of chemical modifications. Such LNA quadruplex scaffolds might find applications in the developing field of nanobiotechnology.