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Nucleotide monomers that hold two nucleobases each, i.e. double-headed nucleotides, have been shown to form two sets of functional Watson-Crick base pairs when incorporated into dsDNA, and they hereby behave as dinucleotides. To form the basis for fully modified double-headed nucleic acids (DhNA), we have prepared three new DhNA monomers and can now demonstrate that the molecular information of 10 Watson-Crick base pairs can be condensed to highly stable 5-mer DhNA duplexes.

A normal duplex holds as many Watson-Crick base pairs as the number of nucleotides in its constituent strands. Here we establish that single nucleotides can be designed to functionally imitate dinucleotides without compromising binding affinity. This effectively allows sequence information to be more compact and concentrated to fewer phosphates.

In the search for double-coding DNA-systems, three new pyrimidine nucleosides, each coded with an additional nucleobase anchored to the major groove face, are synthesized. Two of these building blocks carry a thymine at the 5-position of 2'-deoxyuridine through a methylene linker and a triazolomethylene linker, respectively. The third building block carries an adenine at the 6-position of pyrrolo-2'-deoxycytidine through a methylene linker. These double-headed nucleosides are introduced into oligonucleotides and their effects on the thermal stabilities of duplexes are studied. All studied double-headed nucleotide monomers reduce the thermal stability of the modified duplexes, which is partially compensated by using consecutive incorporations of the modified monomers or by flanking the new double-headed analogs with members of our former series containing propyne linkers. Also their potential in triplex-forming oligonucleotides is studied for two of the new double-headed nucleotides as well as the series of analogs with propyne linkers. The most stable triplexes are obtained with single incorporations of additional pyrimidine nucleobases connected via the propyne linker., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The formation of new nucleic acid motifs by using double-headed nucleotides is reported. Modified phosphoramidites carrying additional thymine or adenine attached to the 2'-position of arabinouridine through a methylene linker are conveniently prepared and incorporated into oligonucleotides to obtain the modified nucleotide monomers (a)U(T) and (a)U(A), respectively. The extension of a DNA double helix by one or two additional A:T base pairs is achieved by placing these modified monomers in the opposite strands in a so-called (+1)-zipper arrangement. Hence, 12 basepairs can be presented in an 11-mer or even a 10-mer duplex. The modified nucleotide monomers also behave as dinucleotides when base-paired with two complementary nucleotides from the opposite strand. A new nucleic acid motif is introduced when two (a)U(A) monomers recognize each other in the center of a duplex.

A series of double-headed nucleosides were synthesized using the Sonogashira cross-coupling reaction. In the reactions, additional nucleobases (thymine, cytosine, adenine, or guanine) were attached to the 5-position of 2'-deoxyuridine or 2'-deoxycytidine through a propyne linker. The modified nucleosides were incorporated into oligonucleotides, and these were combined in different duplexes that were analyzed by thermal denaturation studies. All of the monomers were well tolerated in the DNA duplexes and induced only small changes in the thermal stability. Consecutive incorporations of the monomers led to increases in duplex stability owing to increased stacking interactions. The modified nucleotide monomers maintained the Watson-Crick base pair fidelity. Stable duplexes were observed with heavily modified oligonucleotides featuring 14 consecutive incorporations of different double-headed nucleotide monomers. Thus, modified duplexes with an array of nucleobases on the exterior of the duplex were designed. Molecular dynamics simulations demonstrated that the additional nucleobases could expose their Watson-Crick and/or Hoogsteen faces for recognition in the major groove. This presentation of nucleobases may find applications in providing molecular information without unwinding the duplex.

Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers.

Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Triplex forming oligonucleotides (TFOs) are the most commonly used approach for site-specific targeting of double stranded DNA (dsDNA). Important parameters describing triplex formation include equilibrium binding constants (K(eq)) and association/dissociation rate constants (k(on) and k(off)). The 'fluorescent intercalator displacement replacement' (FIDR) assay is introduced herein as an operationally simple approach toward determination of these parameters for triplexes involving TC-motif TFOs. Briefly described, relative rate constants are determined from fluorescence intensity changes upon: (i) TFO-mediated displacement of pre-intercalated and fluorescent ethidium from dsDNA targets (triplex association) and (ii) Watson-Crick complement-mediated displacement of the TFO and replacement with ethidium (triplex dissociation). The assay is used to characterize triplexes between purine-rich dsDNA targets and TC-motif TFOs modified with six different locked nucleic acid (LNA) monomers, i.e. conventional and C5-alkynyl-functionalized LNA and α-L-LNA pyrimidine monomers. All of the studied monomers increase triplex stability by decreasing the triplex dissociation rate. LNA-modified TFOs form more stable triplexes than α-L-LNA-modified counterparts owing to slower triplex dissociation. Triplexes modified with C5-(3-aminopropyn-1-yl)-LNA-U monomer Z are particularly stable. The study demonstrates that three affinity-enhancing features can be combined into one high-affinity TFO monomer: conformational restriction of the sugar ring, expansion of the pyrimidine π-stacking surface and introduction of an exocyclic amine.

We have designed and synthesised a double-headed nucleotide that presents two nucleobases in the interior of a dsDNA duplex. This nucleotide recognises and forms Watson-Crick base pairs with two complementary adenosines in a Watson-Crick framework. Furthermore, with judicious positioning in complementary strands, the nucleotide recognises itself through the formation of a T:T base pair. Thus, two novel nucleic acid motifs can be defined by using our double-headed nucleotide. Both motifs were characterised by UV melting experiments, CD and NMR spectroscopy and molecular dynamics simulations. Both motifs leave the thermostability of the native dsDNA duplex largely unaltered. Molecular dynamics calculations showed that the double-headed nucleotides are accommodated in the dsDNA by entirely local perturbations and that the modified duplexes retain an overall B-type geometry with the dsDNA unwound by around 25 or 60°, respectively, in each of the modified motifs. Both motifs can be accommodated twice in a dsDNA duplex without incurring any loss of stability and extrapolating from this observation and the results of modelling, it is conceivable that both can be multiplied several times within a dsDNA duplex. These new motifs extend the DNA recognition repertoire and may form the basis for a complete series of double-headed nucleotides based on all 16 base combinations of the four natural nucleobases. In addition, both motifs can be used in the design of nanoscale DNA structures in which a specific duplex twist is required., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteen-mismatched targets, and high stability against 3?-exonucleases.

Abstract: A short synthetic route to the first β-l-ribo configured locked nucleic acid (LNA), that is, 2′-amino-β-l-LNA thymine phosphoramidite 6, has been developed from bicyclic nucleoside 1. Incorporation of 2′-amino-β-l-LNA thymine monomers into α-DNA strands results in probes forming stable duplexes with complementary RNA in parallel orientation. [Copyright &y& Elsevier]

Abstract: Three pyrimidine nucleosides with differently substituted phenyltriazoles attached to the 5-position were prepared by Cu(I)-assisted azide–alkyne cycloadditions (CuAAC) and incorporated into oligonucleotides. Efficient π–π-stacking between two or more phenyltriazoles in the major groove was found to increase the thermal stability of a DNA:RNA duplex significantly. The best stacking, and most stable duplex, was obtained by a sulfonamide substituted derivative. [Copyright &y& Elsevier]

Abstract: Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3–ON12 and ON15–ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage. [Copyright &y& Elsevier]