nucleoside-triphosphates-from-synthesis-to-biochemical

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

University of Bern

[structure: see text] A fluorinated OPA monomer containing the base thymine ((Z)-t-F-OPA) was synthesized in 12 steps, featuring a highly selective allylic over homoallylic Mitsunobu substitution for the introduction of the nucleobase. F-OPA modified PNA decamers were prepared by the MMTr/acyl protection strategy. The thermal stability of duplexes of PNA decamers containing (Z)-t-F-OPA units with antiparallel complementary DNA was measured. We found a strong dependence of stability from the sequential position of the (Z)-t-F-OPA units, ranging from DeltaT(m) of +2.4 to -8.1 degrees C/modification relative to unmodified PNA.

Synthesis and incorporation into PNA of fluorinated olefinic PNA (F-OPA) monomers.

The fluorinated olefinic peptide nucleic acid analogue (F-OPA) monomer containing the base thymine was synthesised in 13 steps. PNAs containing this unit were prepared and their pairing properties assessed by means of UV-melting experiments.

Fluorinated peptide nucleic acid.

The fluorinated olefinic peptide nucleic acid (F-OPA) system was designed as a peptide nucleic acid (PNA) analogue in which the base carrying amide moiety was replaced by an isostructural and isoelectrostatic fluorinated C-C double bond, locking the nucleobases in one of the two possible rotameric forms. By comparison of the base-pairing properties of this analogue with its nonfluorinated analogue OPA and PNA, we aimed at a closer understanding of the role of this amide function in complementary DNA recognition. Here we present the synthesis of the F-OPA monomer building blocks containing the nucleobases A, T, and G according to the MMTr/Acyl protecting group scheme. Key steps are a selective desymmetrization of the double bond in the monomer precursor via lactonization as well as a highly regioselective Mitsunobu reaction for the introduction of the bases. PNA decamers containing single F-OPA mutations and fully modified F-OPA decamers and pentadecamers containing the bases A and T were synthesized by solid-phase peptide chemistry, and their hybridization properties with complementary parallel and antiparallel DNA were assessed by UV melting curves and CD spectroscopic methods. The stability of the duplexes formed by the decamers containing single (Z)-F-OPA modifications with parallel and antiparallel DNA was found to be strongly dependent on their position in the sequence with T(m) values ranging from +2.4 to -8.1 degrees C/modification as compared to PNA. Fully modified F-OPA decamers and pentadecamers were found to form parallel duplexes with complementary DNA with reduced stability compared to PNA or OPA. An asymmetric F-OPA pentadecamer was found to form a stable self-complex (T(m) approximately 65 degrees C) of unknown structure. The generally reduced affinity to DNA may therefore be due to an increased propensity for self-aggregation.

Fluorinated olefinic peptide nucleic acid: synthesis and pairing properties with complementary DNA.

A highly selective DNAzyme sensor for mercuric ions.

Three modified nucleosides have been used in the in vitro selection of the self-cleaving DNAzyme 9-33. This DNAzyme operates in the absence of divalent metal cations with a first-order constant of approximately 0.05 min(-1).

In vitro selection of a DNAzyme with three modified nucleotides.

The selection of modified DNAzymes represents an important endeavor in expanding the chemical and catalytic properties of catalytic nucleic acids. Few examples of such exist and to date, there is no example where three different modified bases have been simultaneously incorporated for catalytic activity. Herein, dCTP, dATP and dUTP bearing, respectively, a cationic amine, an imidazole and a cationic guanidine, were enzymatically polymerized on a DNA template for the selection of a highly functionalized DNAzyme, called DNAzyme 9-86, that catalyzed (M(2+))-independent self-cleavage under physiological conditions at a single ribo(cytosine)phosphodiester linkage with a rate constant of (0.134 +/- 0.026) min(-1). A pH rate profile analysis revealed pK(a)'s of 7.4 and 8.1, consistent with both general acid and base catalysis. The presence of guanidinium cations permits cleavage at significantly higher temperatures than previously observed for DNAzymes with only amines and imidazoles. Qualitatively, DNAzyme 9-86 presents an unprecedented ensemble of synthetic functionalities while quantitatively it expresses one of the highest reported values for any self-cleaving nucleic acid when investigated under M(2+)-free conditions at 37 degrees C.

A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M2+).

A DNAzyme with three protein-like functional groups: enhancing catalytic efficiency of M2+-independent RNA cleavage.

The discovery of imidazole/amine-functionalized DNAzymes that efficiently cleave RNA independently of divalent metal cations (M(2+)) and cofactors underscores the importance of expanding the catalytic repertoire with modified nucleosides. Considerable effort has gone into defining polymerase tolerances of various modified dNTPs for synthesizing and amplifying modified DNA. While long linkers are generally found to enhance incorporation and therefore increase sequence space, shorter linkers may reduce the entropic penalty paid for orienting catalytic functionality. Catalytic enhancement ultimately depends on both the functional group and appropriate linkage to the nucleobase. Whether a shorter linker provides enough catalytic enhancement to outweigh the cost of reduced polymerizability can only be determined by the outcome of the selection. Herein, we report the selection of DNAzyme 20-49 (Dz20-49), which depends on amine, guanidine, and imidazole-modified dNTPs. In contrast to previous selections where we used dA(ime)TP (8-(4-imidazolyl)ethylamino-2'-dATP), here we used dA(imm)TP (8-(4-imidazolyl)methylamino-2'-dATP), in which the linker arm is shortened by one methylene group. Although the most active clone, Dz20-49, was absolutely dependent on the incorporation of either dA(imm)p or dA(ime)p, it catalyzed cofactor independent self-cleavage with a rate constant of 3.1 ± 0.3 × 10(-3) min(-1), a value not dissimilar from unmodified catalysts and strikingly inferior to modified catalysts selected with dA(ime)TP. These results demonstrate that very subtle differences in modified nucleotide composition may dramatically effect DNAzyme selection.

Protein-inspired modified DNAzymes: dramatic effects of shortening side-chain length of 8-imidazolyl modified deoxyadenosines in selecting RNaseA mimicking DNAzymes.

The enzymatic incorporation of a phenol-modified 2'-deoxyuridine triphosphate gave rise to a modified DNA library that was subsequently used in an in vitro selection for ribophosphodiester-cleaving DNAzymes in the presence of divalent zinc and magnesium cations. After 11 rounds of selection, cloning and sequencing resulted in 14 distinct sequences, the most active of which was Dz11-17PheO. Dz11-17PheO self-cleaved an embedded ribocytidine with an observed rate constant of 0.20 ± 0.02 min(-1) in the presence of 10 mM Mg(2+) and 1 mM Zn(2+) at room temperature. The activity was inhibited at low concentrations of Hg(2+) cations and somewhat higher concentrations of Eu(3+) cations.

A divalent metal-dependent self-cleaving DNAzyme with a tyrosine side chain.

Modified nucleoside triphosphates (dNTPs) are a versatile platform for the generation of high-density functionalized nucleic acids. The enzymatic polymerization of dNTPs allows the introduction of sensible functionalities that might not be compatible with the standard automated synthesis of oligonucleotides. Their application to in vitro selections, an elegant chemical approach to Darwinian evolution, delivers modified aptamers and catalytic nucleic acids with potentially enhanced properties. This review article highlights some recent synthetic examples of dNTPs bearing functionalities that are either found in the active site of protein enzymes or have been employed in organocatalysis and further underscores their usefulness in the development of some modified catalytic nucleic acids with special emphasis on M(2+)-independent RNA-cleaving DNAzymes.

Expanding the catalytic repertoire of DNAzymes by modified nucleosides.

Pyrene-deoxynucleoside triphosphates (dPTPs), varying by the positioning of the aromatic system, were synthesized. Their ability to function as substrates for the Klenow fragment of Escherichia coli DNA polymerase I and the TdT polymerase was assessed. The dPTPs are all equally well tolerated by the Klenow fragment, and lead to elongation of up to 5 extra nucleotides of a ssDNA primer in a TdT-mediated reaction. The tailing efficiency of the dPTPs compares favorably to other less drastically modified dNTPs.

Polymerase incorporation of pyrene-nucleoside triphosphates.

To expand the chemical array available for DNA sequences in the context of in vitro selection, I present herein the synthesis of five nucleoside triphosphate analogues containing side chains capable of organocatalysis. The synthesis involved the coupling of L-proline-containing residues (dU(tP)TP and dU(cP)TP), a dipeptide (dU(FP)TP), a urea derivative (dU(Bpu)TP), and a sulfamide residue (dU(Bs)TP) to a suitably protected common intermediate, followed by triphosphorylation. These modified dNTPs were shown to be excellent substrates for the Vent (exo(-)) and Pwo DNA polymerases, as well as the Klenow fragment of E. coli DNA polymerase I, although they were only acceptable substrates for the 9°N(m) polymerase. All of the modified dNTPs, with the exception of dU(Bpu)TP, were readily incorporated into DNA by the polymerase chain reaction (PCR). Modified oligonucleotides efficiently served as templates for PCR for the regeneration of unmodified DNA. Thermal denaturation experiments showed that these modifications are tolerated in the major groove. Overall, these heavily modified dNTPs are excellent candidates for SELEX.

Synthesis of deoxynucleoside triphosphates that include proline, urea, or sulfonamide groups and their polymerase incorporation into DNA.

Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate analogues represents a versatile platform for the facile and mild generation of (highly) functionalized nucleic acids. Numerous modified triphosphates have been utilized in a broad palette of applications spanning from DNA-tagging and -labeling to the generation of catalytic nucleic acids. This review will focus on the recent progress made in the synthesis of modified nucleoside triphosphates as well as on the understanding of the mechanisms underlying their polymerase acceptance. In addition, the usefulness of chemically altered dNTPs in SELEX and related methods of in vitro selection will be highlighted, with a particular emphasis on the generation of modified DNA enzymes (DNAzymes) and DNA-based aptamers.

Nucleoside triphosphates--building blocks for the modification of nucleic acids.

The convenient use of SELEX and related combinatorial methods of in vitro selection provides a formidable gateway for the generation of DNA enzymes, especially in the context of improving their potential as gene therapeutic agents. Here, we report on the selection of DNAzyme 12-91, a modified nucleic acid catalyst adorned with imidazole, ammonium, and guanidinium groups that provide for efficient M(2+)-independent cleavage of an all-RNA target sequence (kobs = 0.06 min(-1)). While Dz12-91 was selected for intramolecular cleavage of an all-RNA target, it surprisingly cleaves a target containing a lone ribocytosine unit with even greater efficiency (kobs = 0.27 min(-1)) than Dz9-86 (kobs = 0.13 min(-1)). The sequence composition of Dz12-91 bears a marked resemblance to that of Dz9-86 (kobs = 0.0014 min(-1) with an all-RNA substrate) that was selected from the same library to cleave a target containing a single ribonucleotide. However, small alterations in the sequence composition have a profound impact on the substrate preference and catalytic properties. Indeed, Dz12-91 displays the highest known rate enhancement for the M(2+)-independent cleavage of all-RNA targets. Hence, Dz12-91 represents a step toward the generation of potentially therapeutically active DNAzymes and further underscores the usefulness of modified triphosphates in selection experiments.

Toward the combinatorial selection of chemically modified DNAzyme RNase A mimics active against all-RNA substrates.

Modified nucleoside triphosphates (dA(Hs)TP, dU(POH)TP, and dC(Val)TP) bearing imidazole, hydroxyl, and carboxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared. These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reactions. Moreover, the combined use of terminal deoxynucleotidyl transferase (TdT) and the modified dNTPs led to efficient tailing reactions that rival those of natural counterparts. Finally, the triphosphates were tolerated by polymerases under PCR conditions, and the ensuing modified oligonucleotides served as templates for the regeneration of unmodified DNA. Thus, these modified dN*TPs are fully compatible with in vitro selection methods and can be used to develop artificial peptidases based on DNA.

Article	Year
Synthesis and incorporation into PNA of fluorinated olefinic PNA (F-OPA) monomers. Organic letters\| PubMed ID: 12762703	2003
Fluorinated peptide nucleic acid. Nucleosides, nucleotides & nucleic acids\| PubMed ID: 14565377	2003
Fluorinated olefinic peptide nucleic acid: synthesis and pairing properties with complementary DNA. The Journal of organic chemistry\| PubMed ID: 15822983	2005
A highly selective DNAzyme sensor for mercuric ions. Angewandte Chemie (International ed. in English)\| PubMed ID: 18435522	2008
In vitro selection of a DNAzyme with three modified nucleotides. Nucleic acids symposium series (2004)\| PubMed ID: 18776259	2008
A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M2+). Nucleic acids research\| PubMed ID: 19153138	2009
A DNAzyme with three protein-like functional groups: enhancing catalytic efficiency of M2+-independent RNA cleavage. Chembiochem : a European journal of chemical biology\| PubMed ID: 19591189	2009
Protein-inspired modified DNAzymes: dramatic effects of shortening side-chain length of 8-imidazolyl modified deoxyadenosines in selecting RNaseA mimicking DNAzymes. Organic & biomolecular chemistry\| PubMed ID: 21311779	2011
A divalent metal-dependent self-cleaving DNAzyme with a tyrosine side chain. Organic & biomolecular chemistry\| PubMed ID: 21670811	2011
Expanding the catalytic repertoire of DNAzymes by modified nucleosides. Chimia\| PubMed ID: 22054129	2011
Polymerase incorporation of pyrene-nucleoside triphosphates. Bioorganic & medicinal chemistry letters\| PubMed ID: 22633686	2012
Synthesis of deoxynucleoside triphosphates that include proline, urea, or sulfonamide groups and their polymerase incorporation into DNA. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 22996052	2012
Nucleoside triphosphates--building blocks for the modification of nucleic acids. Molecules (Basel, Switzerland)\| PubMed ID: 23154273	2012
Toward the combinatorial selection of chemically modified DNAzyme RNase A mimics active against all-RNA substrates. ACS combinatorial science\| PubMed ID: 23485334	2013
Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues--synthesis and biochemical characterization. Organic & biomolecular chemistry\| PubMed ID: 23817514	2013

Marcel Hollenstein

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