Unlocked Nucleic Acid Research Articles

Unlocked Nucleic Acids with a Pyrene‐Modified Uracil: Synthesis, Hybridization Studies, Fluorescent Properties and i‐Motif Stability

The synthesis of two new phosphoramidite building blocks for the incorporation of 5-(pyren-1-yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene-modified nucleobase component were found to destabilize an i-motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene-modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene-modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady-state fluorescence emission studies of oligonucleotides containing pyrene-modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene-modified UNA monomers was observed after formation of i-motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene-modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.

Inside Cover: A Chemical Perspective on Transcriptional Fidelity: Dominant Contributions of Sugar Integrity Revealed by Unlocked Nucleic Acids (Angew. Chem. Int. Ed. 47/2013)

Inside Cover: A Chemical Perspective on Transcriptional Fidelity: Dominant Contributions of Sugar Integrity Revealed by Unlocked Nucleic Acids (Angew. Chem. Int. Ed. 47/2013)

A Chemical Perspective on Transcriptional Fidelity: Dominant Contributions of Sugar Integrity Revealed by Unlocked Nucleic Acids

Unerlässlich für die Transkription: Ein biologischer Ansatz unter Anwendung von chemischer Synthese und geöffneten Nucleinsäuren unterstreicht die unerwartet große Bedeutung eines intakten Zuckerrückgrats für die Transkription durch RNA-Polymerase II (Pol II), was sowohl Effizienz als auch Genauigkeit betrifft. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A chemical perspective on transcriptional fidelity: dominant contributions of sugar integrity revealed by unlocked nucleic acids.

Transcription unlocked: A synthetic chemical biology approach involving unlocked nucleic acids was used to dissect the contribution of sugar backbone integrity to the RNA Polymerase II (Pol II) transcription process. An unexpected dominant role for sugar-ring integrity in Pol II transcriptional efficiency and fidelity was revealed.

G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K+ rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5′ with 3 nt and 3′ with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.

A quencher-free molecular beacon design based on pyrene excimer fluorescence using pyrene-labeled UNA (unlocked nucleic acid)

A quencher-free molecular beacon capable of generating pyrene excimer fluorescence has been constructed using strategically positioned pyrene-UNA monomers. Hybridization of a fully complementary RNA target was accompanied by a pyrene excimer emission increase of more than 900%, and detection of RNA in living cells was demonstrated.

5′ Unlocked Nucleic Acid Modification Improves siRNA Targeting

Optimization of small interfering RNAs (siRNAs) is important in RNA interference (RNAi)-based therapeutic development. Some specific chemical modifications can control which siRNA strand is selected by the RNA-induced silencing complex (RISC) for gene silencing. Intended strand selection will increase potency and reduce off-target effects from the unintended strand. Sometimes, blocking RISC loading of the unintended strand leads to improved intended strand-silencing potency, but the generality of this phenomenon is unclear. Specifically, unlocked nucleic acid (UNA) modification of the 5′ end of canonical (i.e., 19+2) siRNAs abrogates gene silencing of the modified strand, but the fate and potency of the unmodified strand has not been investigated. Here, we show that 5′ UNA-modified siRNAs show improved silencing potency of the unmodified strand. We harness this advantageous property in a therapeutic context, where a limited target region in a conserved HIV 5′ long terminal repeat U5 region would otherwise yield siRNAs with undesired strand selection properties and poor silencing. Applying 5′ UNA modification to the unintended sense (S) strand of these otherwise poorly targeted siRNAs dramatically improves on-target silencing by the intended antisense (AS) strand in pNL4-3.luciferase studies. This study highlights the utility of 5′ UNA siRNA modification in therapeutic contexts where siRNA sequence selection is constrained.

Electrochemistry and in situscanning tunnelling microscopy of pure and redox-marked DNA- and UNA-based oligonucleotides on Au(111)-electrode surfaces

We have studied adsorption and electrochemical electron transfer of several 13- and 15-base DNA and UNA (unlocked nucleic acids) oligonucleotides (ONs) linked to Au(111)-electrode surfaces via a 5'-C6-SH group using cyclic voltammetry (CV) and scanning tunnelling microscopy in aqueous buffer under electrochemical potential control (in situ STM). 2,2',6',2''-Terpyridine (terpy) onto which the transition metal ions Fe(2+/3+), Os(2+/3+) and Ru(2+/3+) could be coordinated after UNA monolayer formation was attached to UNA via a flexible linker. The metal centres offer CV probes and in situ STM contrast markers, and the flexible UNA/linker a potential binder for intercalation. CV of pure and mercaptohexanol diluted ON monolayers displayed reductive desorption signals but also, presumably capacitive, signals at higher potentials. Distinct voltammetric signals arise on metal binding. Those from Ru-binding are by far the strongest and in accord with multiple site Ru-attachment. In situ STM disclosed molecular scale features in varying coverage on addition of the metal ions. The Ru-derivatives showed a bias voltage dependent broad maximum in the tunnelling current-overpotential correlation which could be correlated with theoretical frames for condensed matter conductivity of redox molecules. Together the data suggest that Ru-units are bound to both terpy and the UNA-DNA backbone.

Selective Biocatalytic Acylation Studies on 5′-O-(4,4′-Dimethoxytrityl)-2′,3′-Secouridine: An Efficient Synthesis of UNA Monomer

Lipozyme® TL IM (Theremomyces lanuginosus lipase immobilized on silica) in toluene catalyzes the acylation of the 2 ′-OH over the 3 ′-OH group in 5 ′-O-(4,4 ′-dimethoxytrityl)-2 ′,3 ′-secouridine (5 ′-O-DMT-2 ′,3 ′-secouridine) in a highly selective fashion in moderate to almost quantitative yields. The turn over during benzoyl transfer reactions mediated by vinyl benzoate or benzoic anhydride was faster than in acyl transfer reactions with vinyl acetate or C1 to C5 acid anhydrides; except in the case of butanoic anhydride. The 2 ′-O-benzoyl-5 ′-O-DMT-2 ′,3 ′-secouridine obtained by Lipozyme® TL IM catalyzed benzoylation of 5 ′-O-DMT-2 ′,3 ′-secouridine was successfully converted into its 3 ′-O-phosphoramidite derivative in satisfactory yield, which is a building block for the preparation of oligonucleotides containing the uracil monomer of UNA (unlocked nucleic acid).

Pyrene‐Modified Unlocked Nucleic Acids: Synthesis, Thermodynamic Studies, and Fluorescent Properties

Two pyrene-modified UNA monomers were synthesized and incorporated into 21-mer DNA oligonucleotides. Melting temperatures and thermodynamic properties of the modified duplexes were measured, and the fluorescence properties of single strands and duplexes containing one or more pyrene-UNA modifications were studied. It was found that incorporation of pyrene-UNA monomers increased duplex stability relative to UNA monomers, and thermodynamic studies revealed significant mismatch discriminative capabilities of the pyrene-UNA modified oligonucleotides. Furthermore, the steady-state fluorescence emission intensities of pyrene-UNA modified oligonucleotides were increased upon hybridization to DNA, which to the best of our knowledge is unprecedented for an acyclic pyrene modification in DNA. Interestingly, pyrene excimer emission was observed for single-stranded oligonucleotides containing three pyrene-UNA modifications, whereas this excimer emission disappeared after hybridization to DNA. In view of both the pyrene monomer and the excimer fluorescence emission, the triply modified oligonucleotides show intriguing properties relating to the development of new DNA/RNA detection tools.

Modulating Anti-MicroRNA-21 Activity and Specificity Using Oligonucleotide Derivatives and Length Optimization

MicroRNAs are short, endogenous RNAs that direct posttranscriptional regulation of gene expression vital for many developmental and cellular functions. Implicated in the pathogenesis of several human diseases, this group of RNAs provides interesting targets for therapeutic intervention. Anti-microRNA oligonucleotides constitute a class of synthetic antisense oligonucleotides used to interfere with microRNAs. In this study, we investigate the effects of chemical modifications and truncations on activity and specificity of anti-microRNA oligonucleotides targeting microRNA-21. We observed an increased activity but reduced specificity when incorporating locked nucleic acid monomers, whereas the opposite was observed when introducing unlocked nucleic acid monomers. Our data suggest that phosphorothioate anti-microRNA oligonucleotides yield a greater activity than their phosphodiester counterparts and that a moderate truncation of the anti-microRNA oligonucleotide improves specificity without significantly losing activity. These results provide useful insights for design of anti-microRNA oligonucleotides to achieve both high activity as well as efficient mismatch discrimination.

SiRNA-optimized Modifications for Enhanced In Vivo Activity

Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo. 2'-O-benzyl and 2'-O-CH2Py(4) modifications were tested at each position individually along the guide strand in five sequences to determine positions that tolerated the modifications. The positions were combined together and found to increase potency and duration of siRNAs in vivo compared to their unmodified counterparts when delivered using lipid nanoparticles. For 2'-O-benzyl, four incorporations were tolerated with similar activity to the unmodified siRNA in vivo, while for 2'-O-CH2Py(4) six incorporations were tolerated. Increased in vivo activity was observed when the modifications were combined at positions 8 and 15 on the guide strand. Understanding the optimal placement of siRNA-optimized modifications needed for maximal in vivo activity is necessary for development of RNA-based therapeutics.

Stepping towards highly flexible aptamers: enzymatic recognition studies of unlocked nucleic acid nucleotides

Enzymatic recognition of unlocked nucleic acid (UNA) nucleotides was successfully accomplished. Therminator DNA polymerase was found to be an efficient enzyme in primer extension reactions. Polymerase chain reaction (PCR) amplification of a 81 mer UNA-modified DNA library was efficiently achieved by KOD DNA polymerase.

Non-nucleosidic analogs of nucleic acids

This paper presents several analogs of nucleic acids, including their structure and selected physico-chemical properties, reported recently as being obtained from non-nucleosidic units connected via phosphorodiester bonds. These analogs are built from units that instead of sugar residues carry different diol structures functionalized either with natural nucleobases or with other ring systems of an aromatic character. Some interesting nonnucleosidic oligonucleotide analogs and their applications are described. Unlocked (UNA) and glycerol nucleic acids (GNA) are also described.

ChemInform Abstract: Unlocked Nucleic Acid — An RNA Modification with Broad Potential

AbstractReview: 62 refs.

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2′-C-piperazino-UNA monomer

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2′-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2′-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28–0.44kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG37°=−1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.

Unlocking G-quadruplex: Effect of unlocked nucleic acid on G-quadruplex stability

G-quadruplexes are common structural motifs in aptamers. UNA or unlocked nucleic acid is the latest nucleic acid modification. We have attempted to evaluate the impact of UNA modification on the structure and stability of G-quadruplex oligonucleotides for application in aptamer design. We show using CD spectroscopy that UNA modifications can cause structural transitions in some cases although they retain the inherent G- quadruplex signature. From UV melting studies we showed a position dependent effect of UNA modifications such that quadruplexes with UNA modified loops are further stabilized whereas UNA modifications in stem of the G-quadruplex significantly destabilize the structure. The impact of UNA modification on different nucleobases is also investigated. From the analysis of UV melting results, thermodynamic profile was computed and it was concluded that all the sequences are stable at 37 °C. Finally, a greater serum stability of the modified oligonucleotides in comparison with unmodified ones is also demonstrated. Overall, the position dependent effect of single UNA substitutions was observed and analysed.

Locked and unlocked nucleosides in functional nucleic acids.

Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes.

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.

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.