A novel nucleic acid analogue called acyclic (S)-butyl nucleic acid (BuNA) composed of an acyclic backbone containing a phosphodiester linkage and bearing natural nucleobases was synthesized. Next, (S)-BuNA nucleotides were incorporated in DNA strands and their effect on duplex stability and changes in structural conformation were investigated. Circular dichroism (CD), UV-melting and non-denatured gel electrophoresis (native PAGE) studies revealed that (S)-BuNA is capable of making duplexes with its complementary strands and integration of (S)-BuNA nucleotides into DNA duplex does not alter the B-type-helical structure of the duplex. Furthermore, (S)-BuNA oligonucleotides and (S)-BuNA substituted DNA strands were studied as primer extensions by DNA polymerases. This study revealed that the acyclic scaffold is tolerated by enzymes and is therefore to some extent biocompatible.
Novel G-quadruplex structures are constructed by acyclic (L)-threninol nucleic acid and their synthesis and biophysical properties are described. Pyrene excimer fluorescence and circular dichroism (CD) data revealed that four strands of aTNA are oriented in antiparallel direction.
Acyclic (L)-threoninol nucleic acid (aTNA) containing thymine, cytosine and adenine nucleobases were synthesized and shown to form surprisingly stable triplexes with complementary single stranded homopurine DNA or RNA targets. The triplex structures consist of two (L)-aTNA strands and one DNA or RNA, and these triplexes are significantly stronger than the corresponding DNA or RNA duplexes as shown in competition experiments. As a unique property the (L)-aTNAs exclusively form triplex structures with DNA and RNA and no duplex structures are observed by gel electrophoresis. The results were compared to the known enantiomer (D)-aTNA, which forms much weaker triplexes depending upon temperature and time. It was demonstrated that (L)-aTNA triplexes are able to stop primer extension on a DNA template, showing the potential of (L)-aTNA for antisense applications.
Oligonucleotides are increasingly being used as a programmable connection material to assemble molecules and proteins in well‐defined structures. For the application of such assemblies for in vivo diagnostics or therapeutics it is crucial that the oligonucleotides form highly stable, non‐toxic, and non‐immunogenic structures. Only few oligonucleotide derivatives fulfil all of these requirements. Here we report on the application of acyclic l‐threoninol nucleic acid (aTNA) to form a four‐way junction (4WJ) that is highly stable and enables facile assembly of components for in vivo treatment and imaging. The aTNA 4WJ is serum‐stable, shows no non‐targeted uptake or cytotoxicity, and invokes no innate immune response. As a proof of concept, we modify the 4WJ with a cancer‐targeting and a serum half‐life extension moiety and show the effect of these functionalized 4WJs in vitro and in vivo, respectively.
Peptide nucleic acid (PNA) forms a triple helix with doublestranded RNA (dsRNA) stabilized by a hydrogen-bonding zipper formed by PNA's backbone amides (NÀ H) interacting with RNA phosphate oxygens. This hydrogen-bonding pattern is enabled by the matching~5.7 Å spacing (typical for A-form dsRNA) between PNA's backbone amides and RNA phosphate oxygens. We hypothesized that extending the PNA's backbone by one À CH 2 À group might bring the distance between PNA amide groups closer to 7 Å, which is favourable for hydrogen bonding to the B-form dsDNA phosphate oxygens. Extension of the PNA backbone was expected to selectively stabilize PNA-DNA triplexes compared to PNA-RNA. To test this hypothesis, we synthesized triplex-forming PNAs that had the pseudopeptide backbones extended by an additional À CH 2 À group in three different positions. Isothermal titration calorimetry measurements of the binding affinity of these extended PNA analogues for the matched dsDNA and dsRNA showed that, contrary to our structural reasoning, extending the PNA backbone at any position had a strong negative effect on triplex stability. Our results suggest that PNAs might have an inherent preference for A-form-like conformations when binding double-stranded nucleic acids. It appears that the original six-atom-long PNA backbone is an almost perfect fit for binding to A-form nucleic acids.
Triplex forming oligonucleotides are used as a tool for gene regulation and in DNA nanotechnology. By incorporating artificial nucleica cids, target affinity and biological stabilitys uperior to that of naturalD NA mayb e obtained. This work demonstrates how ac himeric clamp consisting of acyclic( L)-threoninol nucleic acid (aTNA) and DNA can bind DNA and RNA by the formationof ah ighly stable triplex structure. The (L)-aTNA clamp is released from the targeta gain by the addition of ar eleasing strand in as trand displacement type of reaction. It is shown that the clamp efficientlyi nhibits Bsu and T7 RNA polymerase activity and that polymerase activity is reactivated by displacing the clamp. The clamp was successfully appliedt o the regulation of luciferase expression by reversible binding to the mRNA. When targeting as equence in the double stranded plasmid, 40 %d ownregulation of protein expression is achieved.[a] Dr.
RNA folding is driven
by the formation of double-helical segments
interspaced by loops of unpaired nucleotides. Among the latter, bulges
formed by one or several unpaired nucleotides are one of the most
common structural motifs that play an important role in stabilizing
RNA–RNA, RNA–protein, and RNA–small molecule
interactions. Single-nucleotide bulges can fold in alternative structures
where the unpaired nucleobase is either looped-out (flexible) in a
solvent or stacked-in (intercalated) between the base pairs. In the
present study, we discovered that triplex-forming peptide nucleic
acids (PNAs) had unusually high affinity for single-purine-nucleotide
bulges in double-helical RNA. Depending on the PNA’s sequence,
the triplex formation shifted the equilibrium between looped-out and
stacked-in conformations. The ability to control the dynamic equilibria
of RNA’s structure will be an important tool for studying structure–function
relationships in RNA biology and may have potential in novel therapeutic
approaches targeting disease-related RNAs.
Acyclic (l)-threoninol nucleic acids ((l)-aTNA) containing poly-cytosines are prepared and investigated at various pH values, revealing the formation of a highly stable structure at lower pH that have the characteristics of an i-motif.
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