Synthesis and Properties of Isobicyclo‐DNA

We present the synthesis of the isobicyclo‐DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo‐DNA synthesis. Dodecamers containing single isobicyclo‐thymidine incorporations, fully modified A‐ and T‐containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo‐DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self‐pairing. Isobicyclo‐DNA forms preferentially B‐DNA‐like duplexes with DNA and A‐like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self‐paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo‐DNA is stable in fetal bovine serum and does not elicit RNaseH activity.     

Synthesis and RNA-selective hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides

Three α-l-ribofuranosyl analogues of RNA nucleotides (α-l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′-C-hydroxymethyl-α-l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α-l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α-l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α-l-RNA and affinity enhancing α-l-LNA monomers (α-l-ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α-l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-hydroxymethyl-β-d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.     

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.     

Solution structure of a HNA-RNA hybrid

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.     

Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.     

Unlocked nucleic acid--an RNA modification with broad potential

The first unlocked nucleic acid (UNA) monomer was described more than a decade ago, but only recent reports have revealed the true potential applications of this acyclic RNA mimic. UNA monomers enable the modulation of the thermodynamic stability of various nucleic acid structures such as RNA and DNA duplexes, quadruplexes or i-motifs. Moreover, UNA monomers were found to be compatible with RNase H activity, a property which is important for single stranded antisense constructs. Notably, UNA monomers can be applied in the design of superior siRNAs, combining potent gene silencing and dramatically reduced off-target effects.     

Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.     

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

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Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA?RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D ‐threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross‐hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15‐mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.     

Phosphorylation Within DNA Duplexes. Synthesis of Modified Oligodeoxyribonucleotide

Abstract

An effective method was suggested for the activation of phos-phomonoester groups in nicks of a double-strand DNA (1,2). This approach allows to incorporate various sugar phosphate backbone modifications at a particular site when DNA duplexes are being assembled. A modifying group is first introduced at the 5′- or 3′-termini of oligonucleotides, then a duplex is formed and oligomers are coupled on the complementary template using water-soluble l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide or cyanogen bromide as the condensing agents. Various DNA duplexes containing not only natural phosphodiester but also phosphoramidate and pyrophospha-te internucleotide bonds, as well as phosphodiester bonds between nucleotide residues with modified sugar analogs (ribo-, arabino- and xylo-derivatives) were assembled by this method.     

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.     

Thermodynamic analysis of duplex formation of the heterochiral DNA with L-deoxyadenosine.

An L-DNA, the mirror-image isomer of natural DNA, has extraordinary nuclease resistance, and thus the molecules should be promising reagents for many applications, such as antisense technology. However, little is known about the structural and thermodynamic properties of DNAs with this modified nucleotide. In this study, we prepared the L-nucleotide (L-dA) and introduced it into oligodeoxyribonucleotides to assess the ability of the L-nucleotide as a functional molecule for many applications based on the DNA hybridization. Two decamers with an L-dA at the center were synthesized and duplexes with the complementary DNA strand were applied to structural and thermodynamic analyses. The structural study by CD spectra showed that the structures of both modified "L/D-D" duplexes were the typical B-form. This result suggests that the global structure of DNA was not collapsed by the introduction of an L-DNA. Thermodynamic parameters (deltaH degrees, deltaS degrees, and deltaG degrees 37) of the duplex formation, determined by UV melting experiments, indicated that the both duplexes were destabilized at about 2.5 to 3.0 kcal mol(-1) by the introduced L-dA, mainly due to an unfavorable enthalpic effect. In conjunction with information by other researchers, these results suggest that the L-DNA affect on the duplex structure and the stability vary locally; thus, the thermodynamic stability of modified L/D-D duplexes should be predictable by the nearest-neighbor thermodynamic parameters.     

Long RNA dangling end has large energetic contribution to duplex stability

Long terminal unpaired nucleotides known as dangling ends play interesting roles in biological systems. Previous studies, however, only dealt with the energy contributions of single dangling bases. The energy contributions of long dangling ends on the stability of duplexes have not been systematically studied. We now report a quantitative increase in stability of RNA-RNA and DNA-DNA duplexes containing a long dangling end. We found a larger enhancement of the stability by the long RNA dangling end of the RNA-RNA duplex than has been observed for the DNA duplexes. It is also found that structural stabilizations by long dangling ends seem to originate from the single-stranded stacking interactions of nucleotides. These results indicate that RNA stability can be achieved by increasing the length of the dangling end. The thermodynamic parameters of the long dangling ends are useful for designing ribozymes and antisense oligonucleotides, and for the prediction of the RNA secondary structure like the pseudoknot.     

Deciphering nucleic acid modifications by chemical derivatization-mass spectrometry analysis

Chemical derivatization in combination with mass spectrometry (MS) analysis is a promising strategy for the sensitive and effective analysis of nucleic acid modifications. In this review, we summarize the recent advances for deciphering modifications in DNA and RNA by chemical derivatization-MS analysis.     

Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides

The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.     

Molecular dynamics simulation and binding energy calculation for estimation of oligonucleotide duplex thermostability in RNA-based therapeutics

For oligonucleotide-based therapeutics, a thorough understanding of the thermodynamic properties of duplex formation is critical to developing stable and potent drugs. For unmodified small interfering RNA (siRNA), DNA antisense oligonucleotide (AON) and locked nucleic acid (LNA), DNA/LNA modified oligonucleotides, nearest neighbor (NN) methods can be effectively used to quickly and accurately predict duplex thermodynamic properties such as melting point. Unfortunately, for chemically modified olignonucleotides, there has been no accurate prediction method available. Here we describe the potential of estimating melting temperature (T(m)) for nonstandard oligonucleotides by using the correlation of the experimental T(m) with the calculated duplex binding energy (BE) for oligonucleotides of a given length. This method has been automated into a standardized molecular dynamics (MD) protocol through Pipeline Pilot (PP) using the CHARMm component in Discovery Studio (DS). Results will be presented showing the correlation of the predicted data with experiment for both standard and chemically modified siRNA and AON.     

Highly fluorescent conjugated pyrenes in nucleic acid probes: (phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids

In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.     

Antisense oligonuclotides with oxetane-constrained cytidine enhance heteroduplex stability,and elicit satisfactory RNase H response as well as showing improved resistance to both exo and endonucleases

Antisense oligonucleotides (AONs) with single and double oxetane C modifications [1',2'-oxetane constrained cytidine, 1-(1',3'-O-anhydro-beta-D-psicofuranosyl)cytosine] have been evaluated, in comparison with the corresponding T-modified AONs, for their antisense potentials by targeting to a 15mer complementary RNA. Although the C modified mixmer AONs show approximately 3 degrees C drop per modification in melting temperature (Tm) of their hybrid AON-RNA duplexes, they are found to be good substrates for RNase H, in comparison with the native AON-RNA duplex. An AON with double C modifications along with 3'-DPPZ (dipyridophenazine) conjugation shows the Tm of the hybrid duplexes as high as that of the native, and the RNase H activity as good as its unconjugated counterpart. A detailed Michaelis-Menten kinetic analysis of RNase H cleavage showed that the single and double C modified AON-RNA duplexes as well as double C modifications along with 3'-DPPZ have catalytic activities (kcat) close to the native. However, the R Nase H binding affinity (1/Km) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (kcat/Km) for C modified AON-RNA duplexes. All oxetane modified AON-RNA hybrids showed a correlation of Tm with the 1/Km, Vmax, or Vmax/Km. The C modified AONs (with 3'-DPPZ), as in the T counterpart, showed an enhanced tolerance towards the endonuclease and exonuclease degradation compared to the native (the oxetane-sugar and the DPPZ based AONs are non-toxic to K562 cell growth, ref. 18). Thus a balance has been found between exo and endonuclease stability vis-a-vis thermostability of the heteroduplex and the R Nase H recruitment capability and cleavage with the oxetane-constrained cytidine incorporated AONs as potential antisense candidates with a fully phosphate backbone for further biological assessment.     

Recognition of chromosomal DNA by PNAs

The recognition of cellular nucleic acids by synthetic oligonucleotides is a versatile strategy for regulating biological processes. The vast majority of published studies have focused on antisense oligonucleotides that target mRNA, but it is also possible to design antigene oligonucleotides that are complementary to chromosomal DNA. Antigene oligomers could be used to inhibit the expression of any gene or analyze promoter structure and the mechanisms governing gene regulation. Other potential applications of antigene oligomers include activation of expression of chosen genes or the introduction of mutations to correct genetic disease. Peptide nucleic acid (PNA) is a nonionic DNA/RNA mimic that possesses outstanding potential for recognition of duplex DNA. Here we describe properties of PNAs and the challenges for their development as robust antigene agents.     

Spermine moiety attached to the C-5 position of deoxyuridine enhances the duplex stability of the phosphorothioate DNA/complementary DNA and shows the susceptibility of the substrate to RNase H

Novel phosphorothioate-modified oligodeoxynucleotides (S-ODNs) containing a deoxyuridine derivative bearing a spermine moiety at the C-5 position were synthesized. The study of the thermal stability and the thermodynamic stability showed that the modified S-ODNs have been able to form the stable duplexes with the complementary DNA. It was also found that the duplex composed of the modified S-ODN and its complementary RNA strand is the substrate for Escherichia coli RNase H, and the cleavage of the RNA strand by the enzyme was almost similar as in the case of the unmodified one.