Additional Nucleobases Research Articles

Peptide Nucleic Acid with Double Face: Homothymine-Homocytosine Bimodal Cα-PNA (bm-Cα-PNA) Forms a Double Duplex of the bm-PNA2:DNA Triplex.

Cα-bimodal peptide nucleic acids (bm-Cα-PNA) are PNAs with two faces and are designed homologues of PNAs in which each aminoethylglycine (aeg) repeating unit in the standard PNA backbone hosts a second nucleobase at Cα through a spacer chain with a triazole linker. Such bm-Cα-PNA with mixed sequences can form double duplexes by simultaneous binding to two complementary DNAs, one to the base sequence on t-amide side and the other to the bases on the Cα side chain. The synthesis of bm-Cα-PNA with homothymine (T7) on the t-amide face and homocytosine (C5) on the Cα side chain through the triazole linker was achieved by solid phase synthesis with the global click reaction. In the presence of complementary DNAs dA8 and dG6 at neutral pH, bm-Cα-PNA 1 forms a higher order pentameric double duplex of a triplex composed of two bm-Cα-PNA-C5:dG5 duplexes built on a core (bm-Cα-PNA-T7)2:dA8 triplex. Circular dichroism studies showed that assembly can be achieved by either triplex first and duplex later or vice versa. Isothermal titration calorimetry data indicated that the assembly is driven by favorable enthalpy. These results validate concurrent multiple complex formation by bimodal PNAs with additional nucleobases at Cα or Cγ on the aeg-PNA backbone and open up ways to design programmed supramolecular assemblies.

Coupling of cyclo-l-Trp-l-Trp with Hypoxanthine Increases the Structure Diversity of Guanitrypmycins.

The cyclo-l-Trp-l-Trp (cWW, 1) tailoring P450 GutD2774 from Streptomyces lavendulae was characterized by expression in Streptomyces coelicolor, precursor feeding and enzyme assays. GutD2774 catalyzes mainly the transfer of hypoxanthine to C2 and C3 of the indole ring of 1. cWW adducts with guanine were detected as minor products. An orthologous cluster was identified in Streptomyces xanthophaeus. These results expand the spectrum of cyclodipeptide derivatives by involvement of an additional nucleobase and identification of new coupling patterns.

Further Probing of Cu2+-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes.

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu2+-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu2+-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2′-hydroxyl nucleophile with 2′-H or 2′-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu2+-dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target.

Covalent and non-covalent binding of platinated vitamin B12-derivatives to a B12 responsive riboswitch

The B12 responding btuB riboswitch is a short, non-coding RNA sequence involved in the gene-regulation of an outer membrane B12-transport protein in E. coli. This RNA is characterized by its selective high-affinity binding to coenzyme B12 and by the structural rearrangement it undergoes upon this interaction. Due to their involvement in (mostly) bacterial gene regulation, the btuB riboswitch as well as the further about twenty known classes of riboswitches received much attention in recent years. In case of the btuB riboswitch, the light sensitivity of its ligand, coenzyme B12, poses one of the greater challenges for its investigation. Vitamin B12 derivatives carrying a cyanide-bridged platinum(II) moiety offer an ideal strategy for the design of light stable coenzyme B12 analogs. Developed by Alberto and coworkers in 2005 these conjugates provide the possibility to coordinate an additional nucleobase making them potential coenzyme B12 mimics. However, although these derivatives show a high structural similarity to coenzyme B12, their functionality might differ and offers the opportunity to develop new classes of antibiotic agents. Especially the platinum(II)-complex could interact with RNA in an unexpected way, which is the main question addressed in the work presented here. We characterized the binding of three vitamin B12-platinum(II) complexes to two different RNAs: the B12-specific btuB riboswitch and the short RNA D1-45, which is a non-B12-binder. cisPt(II)VitB12+ (1) and enPt(II)VitB12+ (2) carry both a labile chloride ligand at the platinum(II) moiety whereas dienPt(II)VitB122+ (3), that carries no labile chloride ligand anymore, can be considered chemically inert under the applied conditions. Complexes 1 and 2 covalently bind to both RNAs as monitored by band-shift assays. In the case of the short D1-45 the shifted bands are well separated and were further analyzed by MALDI-MS proving the covalent interaction. Complex 3 is unable to covalently bind any of the two RNAs, which proves the general stability of the Pt(II) complex. However, the presence of this moiety has a dramatic influence on the binding property towards the B12-sensing riboswitch, as was observed in in-line probing assays by comparison to the natural ligand coenzyme B12.

Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non-Specificity, and Inhibition.

The single–strand–specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1–P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X–ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1–P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes–shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1–P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.

A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System.

Deoxynucleoside kinase from D.melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four additional nucleobase pairs to the genetic "alphabet". Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (respectively 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1'-β-d-2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one, dZ; 8-(1'β-d-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-β-d-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these nucleosides (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids containing AEGIS DNA.

Double-Headed Nucleotides: Building Blocks for New Nucleic Acid Architectures

Double-headed nucleotides are nucleotides with two nucleobases. These have been investigated recently with the purpose of using the recognition potential of the additional nucleobases in various nucleic acid constructs. Presented here is a review of the double-headed nucleotide monomers investigated so far and their effects in nucleic acid duplexes and other secondary structures.

Three new double-headed nucleotides with additional nucleobases connected to C-5 of pyrimidines; synthesis, duplex and triplex studies

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.

Double-coding nucleic acids: introduction of a nucleobase sequence in the major groove of the DNA duplex using double-headed nucleotides.

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.

Directed Evolution of Polymerases To Accept Nucleotides with Nonstandard Hydrogen Bond Patterns

Artificial genetic systems have been developed by synthetic biologists over the past two decades to include additional nucleotides that form additional nucleobase pairs independent of the standard T:A and C:G pairs. Their use in various tools to detect and analyze DNA and RNA requires polymerases that synthesize duplex DNA containing unnatural base pairs. This is especially true for nested polymerase chain reaction (PCR), which has been shown to dramatically lower noise in multiplexed nested PCR if nonstandard nucleotides are used in their external primers. We report here the results of a directed evolution experiment seeking variants of Taq DNA polymerase that can support the nested PCR amplification with external primers containing two particular nonstandard nucleotides, 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (trivially called P) that pairs with 6-amino-5-nitro-3-(1'-β-d-2'-deoxyribofuranosyl)-2(1H)-pyridone (trivially called Z). Variants emerging from the directed evolution experiments were shown to pause less when challenged in vitro to incorporate dZTP opposite P in a template. Interestingly, several sites involved in the adaptation of Taq polymerases in the laboratory were also found to have displayed "heterotachy" (different rates of change) in their natural history, suggesting that these sites were involved in an adaptive change in natural polymerase evolution. Also remarkably, the polymerases evolved to be less able to incorporate dPTP opposite Z in the template, something that was not selected. In addition to being useful in certain assay architectures, this result underscores the general rule in directed evolution that "you get what you select for".

Double-headed nucleotides in DNA-zipper structures; base–base interactions and UV-induced cross-coupling in the minor groove

Nucleic acids containing double-headed nucleotides with additional nucleobases attached to the 5′(S)-position of thymidine through a methylene linker are studied. The additional bases are oriented towards base-base interactions in the minor groove of the DNA-double helix. Two new examples with adenine or cytosine as the additional bases as well as an analogue with a 4N-methylpiperazine in the same position are introduced, and in a combined study with the original double-headed nucleotide containing two thymines, interactions between the additional nucleobases across the minor groove are detected. Finally, a duplex with two thymines in the minor groove is cross-linked using UV irradiation.

Functionalization of 2′-amino-LNA with additional nucleobases

Thymin-1-yl-acetic acid and adenin-9-yl-acetic acid have been coupled to the N2'-atom of a 2'-amino-LNA thymine nucleoside, and these "double-headed" LNA monomers have been incorporated into oligodeoxyribonucleotides via their corresponding phosphoramidite derivatives. Oligonucleotides containing these modified nucleotides show in most cases increased thermal stability when forming duplexes with complementary DNA, even allowing multiple incorporations. Incorporation of "double-headed" LNA monomers in both strands also led to stable duplexes, however, no indication of Watson-Crick base pairing between the extra nucleobases could be found.

Synthesis of double-headed nucleosides with the additional nucleobase in the 5'-C-position

Three analogues of double-headed nucleosides with the additional nucleobase in the 5'(S)-C-position have been synthesised. A thymine has been attached through a methylene or an ethylene linker and an adenine through a methylene linker. Thermal hybridisation studies indicate that this 5' (S)-C-position is ideal for placing the additional base in the minor groove and obtain specific interstrand stacking effects in a (-3)-zipper arrangement. However, this specific interaction is decreasing when elongating the linker from a methylene to an ethylene linker. The successful syntheses of the two nucleoside analogues with thymine in the 5'-C-position as well as different approaches towards a corresponding adenine analogue are reported.

Nucleic acid duplexes with zippers of additional nucleobases and aromatics in minor or major groove

Two series of thymidine derivatives with additional nucleobases/aromatics attached to either the 5'(S)-C- or the 5-position were prepared by epoxide opening and/or "click chemistry" cycloaddition protocols and introduced into DNA duplexes. Interstrand base-base communication in the minor groove and intrastrand stacking interactions in the major groove were detected.

ChemInform Abstract: Synthesis of Nucleosides with Additional Nucleobases

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Synthesis of Nucleosides with Additional Nucleobases

The syntheses of two nucleosides with additional nucleobases in the 2′-position are presented. The nucleosides have two- and one-carbon linkers to the additional nucleobase, respectively. The two nucleosides are synthesized from different strategies. The nucleoside with two carbons in the linker has been incorporated into oligonucleotides and showed stabilization of a tree-way junction.

Synthesis and modelling of DNA junction and minor groove zipper motifs incorporating the double-headed nucleoside 5′(S)–C-(thymine-1-ylmethyl)thymidine

A nucleoside with two nucleobases, a so-called double-headed nucleoside, 5'(S)-C-(thymine-1-ylmethyl)thymidine 3, is synthesised and incorporated into oligonucleotides. The additional nucleobase is hereby positioned in the minor groove of the duplexes, which are formed with complementary DNA and RNA-sequences. Slight thermal destabilisation of these duplexes as compared to unmodified duplexes is observed. With other target sequences forming bulged duplexes or three-way junctions, no additional influence of the additional base on the thermal stability is observed. On the other hand, a base-base stacking interaction and subsequent stabilisation is observed when two double-headed nucleotide moieties are positioned in two complementary DNA-sequences forming a DNA-zipper motif.

Stabilisation of nucleic acid secondary structures by oligonucleotides with an additional nucleobase; synthesis and incorporation of 2′-deoxy-2′-C-(2-(thymine-1-yl)ethyl)uridine

A nucleoside with two nucleobases is incorporated into oligonucleotides. The synthetic building block, 2'-deoxy-2'-C-(2-(thymine-1-yl)ethyl)uridine, 2, is prepared from uridine via 5',3'-TIPDS-protected 2'-deoxy-2'-C-allyluridine by an oxidative cleavage of the allyl group, a Mitsunobu reaction for the introduction of thymine and appropriate deprotection reactions. This compound is converted into a DMT-protected phosphoramidite and incorporated once into a 13-mer oligodeoxynucleotide sequence, once in an isosequential LNA-modified oligodeoxynucleotide and four times in the middle of a 12-mer oligodeoxynucleotide. These sequences are mixed with different complementary DNA and RNA sequences in order to study the effect of the additional nucleobase in duplexes, in bulged duplexes and in three-way junctions. The first additional thymine is found to be well-accommodated in a DNA-RNA duplex, whereas a DNA-DNA duplex was slightly destabilised. A three-way junction with the additional thymine in the branching point is found to be stabilised in both a DNA-DNA and a DNA-RNA context but destabilised where the modified LNA-sequence is used. In a Mg2+-containing buffer, however, the relative stability of the three-way junctions is found to be opposite with especially the LNA-modified DNA-DNA complex being significantly stabilised by the additional nucleobase.

Complex Formation of the Antiviral 9‐[2‐(Phosphonomethoxy)Ethyl]Adenine (PMEA) and of Its N 1, N 3, and N 7 Deaza Derivatives with Copper(II) in Aqueous Solution

AbstractThe stability constants of the 1:1 complexes formed between Cu2+ and the anions of the N 1, N 3, and N 7 deaza derivatives of 9‐[2‐(phosphonomethoxy)‐ethyl]adenine (PA2‐), Cu(H;PA)+ and Cu(PA), were determined by potentiometric pH titration in aqueous solution (25 °C; I = 0.1 M, NaNO3) and compared with previous results for 9‐[2‐(phosphonomethoxy)ethyl]adenine (PMEA2‐) and (phosphonomethoxy)ethane (PME2‐). A microconstant scheme reveals that in Cu(H;PA)+ Cu2+ is coordinated to the nucleobase, H+ being at the phosphonate group, in about 90% of the Cu(H;PMEA)+ and Cu(H;1‐deaza‐PMEA)+ species, but only in about 37% and 12% of the corresponding complexes with H(3‐deaza‐PMEA)‐ and H(7‐deaza‐PMEA)‐, respectively. Straight‐line plots of log KCuCu(R–PO3) versus pKHH(R–PO3) for simple phosph(on)ate ligands show that all the Cu(PA) complexes, including those with PMEA2‐ and PME2‐, are more stable than expected simply from the basicity of the ‐PO2‐ group; to some extent five‐membered chelates (Cu(PA)cl/O) involving the ether oxygen of the ‐CH2–O–CH2–PO2‐3 chain are formed, and in all complexes an additional nucleobase–metal‐ion interaction occurs. Based on 1H NMR line‐broadening measurements and structural considerations it is concluded that in Cu(3‐deaza‐PMEA) the interaction occurs with N7 whereas in Cu(7‐deaza‐PMEA), Cu(1‐deaza‐PMEA), and Cu(PMEA) it occurs with N3. The proof of a metal ion–N3 interaction is important (and also of relevance regarding DNA) because so far this interaction has received little attention. In all Cu(PA) systems three major isomeric species are in equilibrium; for example, 17(∓3)% of Cu(PMEA) exists as an isomer with a sole Cu2+–phosphonate coordination, 34(∓10)% as Cu(PMEA)cl/O, and in 49(∓10)% the Cu2+ is bound to the phosphonate group, the ether O, and N3. In contrast, 54(∓8)% of Cu(5′‐AMP) occurs as an isomer with sole Cu2+–phosphate coordination and 46(∓8)% as a macrochelate involving N7 too.