Thermodynamic, counterion, and hydration effects for the incorporation of locked nucleic acid nucleotides into DNA duplexes

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA-modified oligonucleotides are known to exhibit enhanced hybridization affinity toward complementary DNA and RNA. In this work, we have evaluated the hybridization thermodynamics of a series of LNA-substituted DNA octamers, modified to various extents by one to three LNA substitutions, introduced at either adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) nucleotides. To understand the energetics, counterion effects, and the hydration contribution of the incorporation of LNA modification, a combination of spectroscopic and calorimetric techniques was used. The CD spectra of the corresponding duplexes showed that the modified duplexes adopt an A-type conformation. UV and DSC melting studies revealed that each type of duplex unfolds in a two-state transition. A complete thermodynamic profile at 5 degrees C indicated that the net effect of modification on thermodynamic parameters might be positional and that the neighboring bases flanking the modification might influence the favorable formation of the modified duplexes. Furthermore, relative to the formation of the unmodified reference duplexes, the formation of modified duplexes is accompanied by a higher uptake of counterions and a lower uptake of water molecules.     

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.     

Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers

Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 5′ end (LNA-5′), near the 3′ end (LNA-3′) and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, C_T) were characterized using two-way ANOVA. LNA-5′ increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5′ generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3′ and LNA-Even did not improve read lengths or C_T. ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence.     

Controlling nucleic acid secondary structure by intercalation: effects of DNA strand length on coralyne-driven duplex disproportionation

Small molecules that intercalate in DNA and RNA are powerful agents for controlling nucleic acid structural transitions. We recently demonstrated that coralyne, a small crescent-shaped molecule, can cause the complete and irreversible disproportionation of duplex poly(dA)·poly(dT) into triplex poly(dA)·poly(dT)·poly(dT) and a poly(dA) self- structure. Both DNA secondary structures that result from duplex disproportionation are stabilized by coralyne intercalation. In the present study, we show that the kinetics and thermodynamics of coralyne-driven duplex disproportionation strongly depend on oligonucleotide length. For example, disproportionation of duplex (dA)₁₆·(dT)₁₆ by coralyne reverts over the course of hours if the sample is maintained at 4°C. Coralyne-disproportioned (dA)₃₂· (dT)₃₂, on the other hand, only partially reverts to the duplex state over the course of days at the same temperature. Furthermore, the equilibrium state of a (dA)₁₆·(dT)₁₆ sample in the presence of coralyne at room temperature contains three different secondary structures [i.e. duplex, triplex and the (dA)₁₆ self-structure]. Even the well-studied process of triplex stabilization by coralyne binding is found to be a length-dependent phenomenon and more complicated than previously appreciated. Together these observations indicate that at least one secondary structure in our nucleic acid system [i.e. duplex, triplex or (dA)_n self-structure] binds coralyne in a length-dependent manner.     

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.     

Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’ for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by ‘hybrid’ phosphorothioate–LNA ODNs induces tumour necrosis factor-α production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.     

Effect of locked nucleic acid modifications on the thermal stability of noncanonical DNA structure

We studied the kinetic and thermodynamic effects of locked nucleic acid (LNA) modifications on parallel and antiparallel DNA duplexes. The LNA modifications were introduced at cytosine bases of the pyrimidine strand. Kinetic parameters evaluated from melting and annealing curves showed that the association and dissociation rate constants for the formation of the LNA-modified parallel duplex at 25.0 °C were 3 orders of magnitude larger and 6 orders of magnitude smaller, respectively, than that of the unmodified parallel duplex. The activation energy evaluated from the temperature-dependent rate constants was largely altered by the LNA modifications, suggesting that the LNA modifications affected a prenucleation event in the folding process. Moreover, thermodynamic parameters showed that the extent of stabilization by the LNA modification for parallel duplexes (3.6 kcal mol(-1) per one modification) was much more significant than that of antiparallel duplexes (1.6 kcal mol(-1)). This large stabilization was due to the decrease in ΔH° that was more favorable than the decrease in TΔS°. These quantitative parameters demonstrated that LNA modification specifically stabilized the noncanonical parallel duplex. On the basis of these observations, we succeeded to stabilize the parallel duplex by LNA modification at the physiological pH. These results can be useful in the rational design of functional molecules such as more effective antisense and antigene strands, more sensitive strands for detection of target DNA and RNA strands, and molecular switches responding to solution pH.     

Thermodynamics of single strand DNA base stacking

The thermodynamics of the stacking to unstacking transitions of 24 single-stranded DNA sequences (ssDNA), 10-12 bases in length, in sodium phosphate buffer were determined from 10 to 95 degrees C, using differential scanning calorimetry (DSC). An additional 22 ssDNA sequences did not exhibit an S<-->U transition in this temperature range. The transition properties of the ssDNA sequences with 相似文献   

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.     

High affinity nucleic acid aptamers for streptavidin incorporated into bi-specific capture ligands

We have isolated 2′-Fluoro-substituted RNA aptamers that bind to streptavidin (SA) with an affinity around 7 ± 1.8 nM, comparable with that of recently described peptide aptamers. Binding to SA was not prevented by prior saturation with biotin, enabling nucleic acid aptamers to form useful ternary complexes. Mutagenesis, secondary structure analysis, ribonuclease footprinting and deletion analysis provided evidence for the essential structural features of SA-binding aptamers. In order to provide a general method for the exploitation of these aptamers, we produced derivatives in which they were fused to the naturally structured RNA elements, CopT or CopA. In parallel, we produced derivatives of CD4-binding aptamers fused to the complementary CopA or CopT elements. When mixed, these two chimeric aptamers rapidly hybridized, by virtue of CopA–CopT complementarity, to form stable, bi-functional aptamers that we called ‘adaptamers’. We show that a CD4–SA-binding adaptamer can be used to capture CD4 onto a SA-derivatized surface, illustrating their general utility as indirect affinity ligands.     

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.     

Identification of template strand in heteroduplex DNA directing cell-free enzyme synthesis

Summary Cell-free synthesis of galactose enzymes was directed by heteroduplex DNA carrying an amber mutation in thegal T gene on one DNA strand, the wildtype configuration on the complementary strand. Comparison with the corresponding homoduplex DNA preparations shows that the presence of the wildtype base sequence in the l-strand is necessary and sufficient for the synthesis of active enzyme. Single stranded DNA of either configuration is inactive in this system.     

Evidence that the nucleic acid base queuine is incorporated intact into tRNA by animal cells

Queuine (the base of queuosine, Q) catalytically reduced with tritium or deuterium yields a derivative in which the proton at C-8 (purine numbering system) has been exchanged and the cyclopentene ring has been reduced to a cyclopentane ring. Mouse fibroblast tRNA has been labeled by culturing the cells in medium supplemented with [3H]- and [2H]dihydroqueuine. Such tRNA yields, upon hydrolysis, the nucleoside dihydroqueuosine and a saccharide derivative of dihydroqueuosine. Each product has been identified unambiguously by mass spectrometry and chromatography. Both the 3H- and 2H-labeled material coeluted, and no unlabeled Q nucleoside was found. Therefore, dihydroqueuine is incorporated intact into tRNA in mammalian cells. Furthermore, fractionation of the labeled tRNA on concanavalin A-agarose, which specifically binds the mannosyl-Q-containing tRNAAsp, has shown that the dihydroqueuosine-containing tRNAAsp is mannosylated. This is the first direct evidence that queuine is incorporated intact into mammalian tRNA in vivo.     

Synthesis of locked pyranosyl nucleic acid (LpNA)

A new locked pyranosyl nucleoside was synthesized by phenylsulfinyl-assisted chemistry. The novel building block was inserted into oligonucleotides and provides new insight on conformational restricted pyranosyl nucleosides on duplex formation     

Methylphosphonate LNA: a locked nucleic acid with a methylphosphonate linkage

Synthesis of an oligonucleotide containing one methylphosphonate locked nucleic acid (LNA) thymine monomer using the phosphoramidite approach is described. The binding affinity of this 9-mer methylphosphonate LNA towards complementary DNA and RNA oligonucleotides was increased compared to the reference DNA, but decreased compared to the reference LNA. In the 9-mer sequence context studied, introduction of a single methylphosphonate LNA monomer, contrary to a single LNA monomer, efficiently inhibits 3'-exonucleolytic degradation.     

Effects of hypoxanthine substitution on bleomycin-mediated DNA strand degradation in DNA-RNA hybrids.

We have reported on the differences in site-specific cleavage between DNA and DNA-RNA hybrids by various prototypic DNA cleavers (accompanying paper). In the case of bleomycin (BLM), degradation at 5'-GC-3'sites was suppressed relative to the same sequence in double-stranded DNA, while 5'-GT-3' damage remained constant. We now present results of our further investigation on the chemical and conformational factors that contribute to BLM-mediated DNA strand cleavage of DNA-RNA hybrids. Substitution of guanine by hypoxanthine on the RNA strand of hybrids resulted in a significant enhancement of 5'-GC-3' site damage on the DNA strand relative to double-stranded DNA, thus reversing the suppression noted at these sites. Additionally, 5'-AT-3' sites, which are damaged significantly more in the hybrid than in DNA, exhibit decreased product formation when hypoxanthine is present on the RNA strand of hybrids. However, when hypoxanthine is substituted for guanine on the DNA strand (a GC cleavage site becomes IC), 5'-IT-3' and 5'-IC-3' site cleavage is almost completely suppressed, whereas AT site cleavage is dramatically enhanced. The priority in metallobleomycin site-specific cleavage of hybrids changes with hypoxanthine substitution: the cleavage priority is AT > GT > GC in native hybrid; GC > GT > AT in hybrids substituted with hypoxanthine in the RNA strand; AT >> GT approximately GC in hybrids substituted with hypoxanthine in the DNA strand. The results of kinetic isotope effect studies on BLM cleavage are presented and, in most cases, the values are larger for the hypoxanthine-substituted hybrid. The results suggest that the 2-amino groups of guanine residues on both strands of the nucleic acid play an important role in modulation of the binding and cleavage specificity of BLM.