Hybridization energies of double strands composed of DNA, RNA, PNA and LNA

The electronic properties of double strands composed of trimeric LNA, PNA, DNA and RNA single strands were investigated by density-functional molecular orbital calculations. The computed hybridization energies for the double strands involving PNA or LNA are larger than those for DNA-DNA and RNA-RNA. The larger stability is attributed to the presence of a larger positive charge of the hydrogen atoms contributing to the hydrogen bonds in the PNA-DNA and LNA-DNA double-strands. These results are comparable to the experimental finding that PNA and LNA single strands display high affinity toward a complementary DNA or RNA single strand.     

Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids

Locked nucleic acids (LNAs) containing one or more 2'-O,4'-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. unprecedented helical thermostability when hybridized with cognate RNA and DNA. To acquire a detailed understanding of the structural features of LNA giving rise to its remarkable properties, we have conducted structural studies by use of NMR spectroscopy and now report high-resolution structures of two LNA:RNA hybrids, the LNA strands being d(5'-CTGAT(L)ATGC-3') and d(5'-CT(L)GAT(L)AT(L)GC-3'), respectively, T(L) denoting a modified LNA monomer with a thymine base, along with the unmodified DNA:RNA hybrid. In the structures, the LNA nucleotides are positioned as to partake in base stacking and Watson-Crick base pairing, and with the inclusion of LNA nucleotides, we observe a progressive change in duplex geometry toward an A-like duplex structure. As such, with the inclusion of three LNA nucleotides, the hybrid adopts an almost canonical A-type duplex geometry, and thus it appears that the number of modifications has reached a saturation level with respect to structural changes, and that further incorporations would furnish only minute changes in the duplex structure. We attempt to rationalize the conformational steering induced by the LNA nucleotides by suggesting that the change in electronic density at the brim of the minor groove, introduced by the LNA modification, is causing an alteration of the pseudorotational profile of the 3'-flanking nucleotide, thus shifting this sugar equilibrium toward N-type conformation.     

Determination of affinity constants of locked nucleic acid (LNA) and DNA duplex formation using label free sensor technology

Locked nucleic acid (LNA) is a nucleic acid analogue containing 2'-O,4'-C-methylene-beta-D-ribofuranosyl nucleotides, which have a bicyclic furanose unit locked in a RNA mimicking sugar conformation. Oligonucleotides containing LNA monomers show an enhanced thermal stability and robustness against nuclease mediated cleavage. Therefore special tailored LNA is a versatile tool for gene array analysis and single nucleotide polymorphism (SNP) analysis. The higher melting temperatures result from a higher affinity between the LNA and its complementary base. This was verified by the determination of the affinity constants of the duplex formation of 3 oligonucleotides: DNA, L-DNA, in which all thymidines are substituted by LNA, and a fully modified LNA, to their complementary DNA strand. Affinity constants were calculated to be 1.5 x 10(9), 4.0 x 10(9) and >10(12) L mol(-1). This was done using the label free and time resolved sensing technology reflectometric interference spectroscopy (RIfS), in an assay format similar to a titration called binding inhibition assay.     

LNA (locked nucleic acid) and analogs as triplex-forming oligonucleotides

The triplex-forming abilities of some conformationally restricted nucleotide analogs are disclosed and compared herein. 2'-Amino-LNA monomers proved to be less stabilising to triplexes than LNA monomers when incorporated into a triplex-forming third strand. N2'-functionalisation of 2'-amino-LNA monomers with a glycyl unit induced the formation of exceptionally stable triplexes. Nucleotide analogs containing a C2',C3'-oxymethylene linker (E-type furanose conformation) or a C2',C4'-propylene linker (N-type furanose conformation) had no significant effect on triplex stability proving that conformational restriction per se is insufficient to stabilise triplexes.     

The structure of a mixed LNA/DNA:RNA duplex is driven by conformational coupling between LNA and deoxyribose residues as determined from 13C relaxation measurements

A study of the internal dynamics of an LNA/DNA:RNA duplex has been performed to further characterize the conformational changes associated with the incorporation of locked nucleic acid (LNA) nucleotides in a DNA:RNA duplex. In general, it was demonstrated that the LNA/DNA:RNA duplex has a very high degree of order compared to dsDNA and dsRNA duplexes. The order parameters of the aromatic carbon atoms in the LNA/DNA strand are uniformly high, whereas a sharp drop in the degree of order was seen in the RNA strand in the beginning of the AUAU stretch in the middle of the strand. This can be related to a return to normal dsRNA dynamics for the central A:U base pair. The high order of the heteroduplex is consistent with preorganization of the chimera strand for an A-form duplex conformation. These results partly explain the dramatic increase in T(m) of the chimeric heteroduplex over dsDNA and DNA:RNA hybrids of the same sequence.     

C5-functionalized DNA, LNA, and α-L-LNA: positional control of polarity-sensitive fluorophores leads to improved SNP-typing

Single nucleotide polymorphisms (SNPs) are important markers in disease genetics and pharmacogenomic studies. Oligodeoxyribonucleotides (ONs) modified with 5-[3-(1-pyrenecarboxamido)propynyl]-2'-deoxyuridine monomer X enable detection of SNPs at non-stringent conditions due to differential fluorescence emission of matched versus mismatched nucleic acid duplexes. Herein, the thermal denaturation and optical spectroscopic characteristics of monomer X are compared to the corresponding locked nucleic acid (LNA) and α-L-LNA monomers Y and Z. ONs modified with monomers Y or Z result in a) larger increases in fluorescence intensity upon hybridization to complementary DNA, b) formation of more brightly fluorescent duplexes due to markedly larger fluorescence emission quantum yields (Φ(F)=0.44-0.80) and pyrene extinction coefficients, and c) improved optical discrimination of SNPs in DNA targets. Optical spectroscopy studies suggest that the nucleobase moieties of monomers X-Z adopt anti and syn conformations upon hybridization with matched and mismatched targets, respectively. The polarity-sensitive 1-pyrenecarboxamido fluorophore is, thereby, either positioned in the polar major groove or in the hydrophobic duplex core close to quenching nucleobases. Calculations suggest that the bicyclic skeletons of LNA and α-L-LNA monomers Y and Z influence the glycosidic torsional angle profile leading to altered positional control and photophysical properties of the C5-fluorophore.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Comparison of the fragmentation behavior of DNA and LNA single strands and duplexes

DNA and locked nucleic acid (LNA) were characterized as single strands, as well as double stranded DNA‐DNA duplexes and DNA‐LNA hybrids using tandem mass spectrometry with collision‐induced dissociation. Additionally, ion mobility spectrometry was carried out on selected species. Oligonucleotide duplexes of different sequences — bearing mismatch positions and abasic sites of complementary DNA 15‐mers — were investigated to unravel general trends in their stability in the gas phase. Single‐stranded LNA oligonucleotides were also investigated with respect to their gas phase behavior and fragmentation upon collision‐induced dissociation. In contrast to the collision‐induced dissociation of DNA, almost no base loss was observed for LNAs. Here, backbone cleavages were the dominant dissociation pathways. This finding was further underlined by the need for higher activation energies. Base losses from the LNA strand were also absent in fragmentation experiments of the investigated DNA‐LNA hybrid duplexes. While DNA‐DNA duplexes dissociated easily into single stranded fragments, the high stability of DNA‐LNA hybrids resulted in predominant fragmentation of the DNA part rather than the LNA, while base losses were only observed from the DNA single strand of the hybrid.     

C2'-pyrene-functionalized triazole-linked DNA: universal DNA/RNA hybridization probes

Development of universal hybridization probes, that is, oligonucleotides displaying identical affinity toward matched and mismatched DNA/RNA targets, has been a longstanding goal due to potential applications as degenerate PCR primers and microarray probes. The classic approach toward this end has been the use of "universal bases" that either are based on hydrogen-bonding purine derivatives or aromatic base analogues without hydrogen-bonding capabilities. However, development of probes that result in truly universal hybridization without compromising duplex thermostability has proven challenging. Here we have used the "click reaction" to synthesize four C2'-pyrene-functionalized triazole-linked 2'-deoxyuridine phosphoramidites. We demonstrate that oligodeoxyribonucleotides modified with the corresponding monomers display (a) minimally decreased thermal affinity toward DNA/RNA complements relative to reference strands, (b) highly robust universal hybridization characteristics (average differences in thermal denaturation temperatures of matched vs mismatched duplexes involving monomer W are <1.7 °C), and (c) exceptional affinity toward DNA targets containing abasic sites opposite of the modification site (ΔT(m) up to +25 °C). The latter observation, along with results from absorption and fluorescence spectroscopy, suggests that the pyrene moiety is intercalating into the duplex whereby the opposing nucleotide is pushed into an extrahelical position. These properties render C2'-pyrene-functionalized triazole-linked DNA as promising universal hybridization probes for applications in nucleic acid chemistry and biotechnology.     

The structure of LNA:DNA hybrids from molecular dynamics simulations: the effect of locked nucleotides

Locked nucleic acids (LNAs) exhibit a modified sugar fragment that is restrained to the C3'-endo conformation. LNA-containing duplexes are rather stable and have a more rigid structure than DNA duplexes, with a propensity for A-conformation of the double helix. To gain detailed insight into the local structure of LNA-modified DNA oligomers (as a foundation for subsequent exploration of the electron-transfer capabilities of such modified duplexes), we carried out molecular dynamics simulations on a set of LNA:DNA 9-mer duplexes and analyzed the resulting structures in terms of base step parameters and the conformations of the sugar residues. The perturbation introduced by a single locked nucleotide was found to be fairly localized, extending mostly to the first neighboring base pairs; such duplexes featured a B-type helix. With increasing degree of LNA modification the structure gradually changed; the duplex with one complete LNA strand assumed a typical A-DNA structure. The relative populations of the sugar conformations agreed very well with NMR data, lending credibility to the validity of the computational protocol.     

Electrochemical biosensor based on nanogold-modified poly-eriochrome black T film for BCR/ABL fusion gene assay by using hairpin LNA probe

A novel electrochemical biosensor is described for detection of breakpoint cluster region gene and a cellular abl (BCR/ABL) fusion gene in chronic myelogenous leukemia (CML) by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe. The hairpin LNA probe was immobilized on the nanogold (NG)/poly-eriochrome black T (EBT) film-modified glassy carbon electrode (GCE). The immobilized LNA probe could selectively hybridize with its target DNA on LNA/NG/EBT/GCE surface. The immobilization and hybridization of the LNA probe were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The hybridization of the immobilized LNA probe with the target DNA was detected by differential pulse voltammetry with the electroactive methylene blue as an indicator. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This novel electrochemical biosensor has been used for assay of PCR real sample with satisfactory result.     

Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world

Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.     

Synthesis of abasic locked nucleic acid and two seco-LNA derivatives and evaluation of their hybridization properties compared with their more flexible DNA counterparts

To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic acid) three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated with respect to duplex stabilities. The abasic LNA monomer (X(L), Figure 1) with the rigid carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the exceptional hybridization properties of LNA primarily originate from improved intrastrand nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives, obtained by cleavage of the C1'-O4' bond of an LNA monomer or complete removal of the O4'-furanose oxygen atom (Z(L) and dZ(L), respectively, Figure 1), were compared to their acyclic DNA counterpart (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives Z(L) and dZ(L) destabilize duplex formation even more than the flexible seco-DNA monomer Z.     

Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads

In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin–enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0–2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.     

Two-dimensional LNA/DNA arrays: estimating the helicity of LNA/DNA hybrid duplex

We measured the helical repeats of a non-natural nucleic acid, locked nucleic acid (LNA), by incorporating LNA strands into the outer arms of a DNA double crossover (DX) molecule; atomic force microscopy (AFM) imaging of the two-dimensional (2D) arrays self-assembled from these DX molecules allows us to derive the helical repeat of the LNA/DNA hetero-duplex to be 13.2 +/- 0.9 base pairs per turn.     

Conformationally controlled high-affinity targeting of RNA or DNA by novel 2'-amino-DNA/LNA mixmers and pyrenyl-functionalized 2'-amino-DNA

9-Mer DNA sequences containing 2'-N-methyl-2'-N-(pyren-1-ylmethyl)-2'-amino-DNA monomers display significantly increased affinity towards DNA complements whereas the corresponding 2'-amino-DNA monomer has a detrimental effect on duplex stability. These effects are efficiently reversed by incorporation of four LNA nucleotides inducing a B-DNA to A-DNA conformational change.     

An exocyclic methylene group acts as a bioisostere of the 2'-oxygen atom in LNA

We show for the first time that it is possible to obtain LNA-like (Locked Nucleic Acid 1) binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2'-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2'-position and a propynyl group at the C-4' position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2'-deoxyribose residue of the 5'-terminal G of a neighboring duplex, resulting in the formation of a CH...O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2'-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA antisense oligonucleotides (ASOs) showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moieties in nucleic acid structure and emphasizes greater use of this bioisostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.     

Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals

Oligonucleotide chemistry has been developed greatly over the past three decades, with many advances in increasing nuclease resistance, enhancing duplex stability and assisting with cellular uptake. Locked nucleic acid (LNA) is a structurally rigid modification that increases the binding affinity of a modified-oligonucleotide. In contrast, unlocked nucleic acid (UNA) is a highly flexible modification, which can be used to modulate duplex characteristics. In this tutorial review, we will compare the synthetic routes to both of these modifications, contrast the structural features, examine the hybridization properties of LNA and UNA modified duplexes, and discuss how they have been applied within biotechnology and drug research. LNA has found widespread use in antisense oligonucleotide technology, where it can stabilize interactions with target RNA and protect from cellular nucleases. The newly emerging field of siRNAs has made use of LNA and, recently, also UNA. These modifications are able to increase double-stranded RNA stability in serum and decrease off-target effects seen with conventional siRNAs. LNA and UNA are also emerging as versatile modifications for aptamers. Their application to known aptamer structures has opened up the possibility of future selection of LNA-modified aptamers. Each of these oligonucleotide technologies has the potential to become a new type of therapy to treat a wide variety of diseases, and LNA and UNA will no doubt play a part in future developments of therapeutic and diagnostic oligonucleotides.     

Efficient Discrimination of Single Nucleotide Polymorphisms (SNPs) Using Oligonucleotides Modified with C5‐Pyrene‐Functionalized DNA and Flanking Locked Nucleic Acid (LNA) Monomers

Oligodeoxyribonucleotides modified with 5‐[3‐(1‐pyrenecarboxamido)propynyl]‐2′‐deoxyuridine monomer X and proximal LNA monomers display higher affinity for complementary DNA, more pronounced increases in fluorescence emission upon DNA binding, and improved discrimination of SNPs at non‐stringent conditions, relative to the corresponding LNA‐free probes across a range of sequence contexts. The results reported herein suggest that the introduction of LNA monomers influences the position of nearby fluorophores via indirect conformational restriction, a characteristic that can be utilized to develop optimized fluorophore‐labeled probes for SNP‐discrimination studies.     

NUCLEIC ACID HYBRIDIZATION ACCOMPANIED WITH EXCIMER FORMATION FROM TWO PYRENE-LABELED PROBES

Abstract— We developed a novel nucleic acid hybridization method based on excimer formation. We used two different 16-mer oligonucleotide probes that had a combined continuous-sequence run that was complementary to a target 32-mer. Prior to hybridization, the adjacent terminal ends (i.e. the 3'-terminal of one probe and the 5'-terminal of the other probe) were each labeled with one pyrene residue. When these probes simultaneously hybridized to the target, a 495 nm broad fluorescence band was produced. The intensity of this band increased as the intensity of the pyrene monomer bands decreased, indicating that the 495 nm band was attributed to the pyrene excimer. The excimer fluorescence, easily differentiated from the monomer bands for emission wavelength, opens up a new way to perform homogeneous hybridization assays and in vivo imaging of nucleic acids.