Template‐Directed Copying of RNA by Non‐enzymatic Ligation

The non‐enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA‐based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template‐directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′‐amino group at the 3′‐end of each oligonucleotide, in combination with an N‐alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.     

A study of fast and metastable dissociations of adenine-thymine binary-base oligonucleotides by using positive-ion MALDI-TOF mass spectrometry

In the present study, fast and metastable dissociations of a number of adenine-thymine binary-base oligonucleotides under the conditions of UV matrix-assisted laser desorption/ionization mass spectrometry were investigated. 2-Aminobenzoic acid/ammonium fluoride (ABA/NH4F) matrix system was used. The spectra obtained under metastable and fast dissociation conditions exhibit distinctive dissociation products. From the post-source-decay analysis, all oligonucleotides underwent predominantly metastable dissociations at the 3' C-O linkages to form [a(n)-B]+ and w(n)+ complimentary ion series. Based on the present results, the so-called "[wn+80]+" ions were postulated to be the complimentary [Z(8-n)AH]+ ions rather than the expected phosphate rearrangement products. In addition, these oligonucleotides were found to generate fast dissociation products of b(n)+, d(N)+, w(N)+ and y(N)+ ions through backbone cleavages at 5' C-O, 5' O-P, 3' C-O and 3' P-O linkages, respectively. Product ion series formed under PSD conditions were not observed. The implications of this mutually exclusive occurrence of the two sets of fragment ions under fast and metastable conditions using ABA/NH4F matrix would be discussed. A model of ion activation under UV-MALDI conditions was also proposed.     

Kinetics and mechanism of the general-acid-catalyzed ring-closure of the malondialdehyde-DNA adduct, N2-(3-oxo-1-propenyl)deoxyguanosine (N2OPdG-), to 3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin- 10(3H)-one (M1dG)

3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is the major product of the reaction of deoxyguanosine with malondialdehyde (MDA). M1dG blocks replication by DNA polymerases in vitro and is mutagenic in vivo. M1dG reacts with hydroxide to form the N2-(3-oxo-1-propenyl)deoxyguanosine anion (N2OPdG-). This reaction is pH-dependent and reverses under neutral and acidic conditions to form M1dG. Here we describe the kinetics and mechanism of the ring-closure reaction in both the nucleoside and oligonucleotides. Kinetic analysis of absorbance and fluorescence changes demonstrates that ring-closure is biphasic, leading to the rapid formation of an intermediate that slowly converts to M1dG in a general-acid-catalyzed reaction. The dependence of the rate of the rapid phase on pH reveals the pKa for protonated N2OPdG is 6.9. One-dimensional 1H NMR and DQF-COSY experiments identified two distinct intermediates, N2OPdG-H and 8-hydroxy-6,7-propenodeoxyguanosine (HO-Prene-dG), that are formed upon acidification of N2OPdG-. Characterization of ring-closure in single-stranded and in melted duplex oligonucleotides shows M1dG formation is also acid-catalyzed in single-stranded oligonucleotides and that the denaturation of an oligonucleotide duplex enhances ring-closure. This work details the complexity of ring-closure in the nucleoside and oligonucleotides and provides new insight into the role of duplex DNA in catalyzing ring-opening and ring-closing of M1dG and N2OPdG.     

pH-Independent triplex formation: hairpin DNA containing isoguanine or 9-deaza-9-propynylguanine in place of protonated cytosine

Triplex-forming oligonucleotides (TFOs) containing 2'-deoxyisoguanosine (2), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) as well as the propynylated 9-deazaguanine N7-(2'-deoxyribonucleoside) were prepared. For this the phosphoramidites 9a, b of the nucleoside 1 and, the phosphoramidites 19, 20 of compound 3b were synthesized. They were employed in solid-phase oligonucleotide synthesis to yield the protected 31-mer oligonucleotides. The deblocking of the allyl-protected oligonucleotides containing 1 was carried out by Pd(0)[PPh3]4-PPh3 followed by 25% aq. NH3. Formation of the 31-mer single-stranded intramolecular triplexes was studied by UV-melting curve analysis. In the single-stranded 31-mer oligonucleotides the protonated dC in the dCH(+)-dG-dC base triad was replaced by 2'-deoxyisoguanosine (1), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) and, 9-deaza-9-propynylguanine N7-(2'-deoxyribonucleoside) (3b). The replacement of protonated dC by compounds 1 and 3b resulted in intramolecular triplexes which are formed pH-independently and are stable under neutral conditions. These triplexes contain "purine" nucleosides in the third pyrimidine rich strand of the oligonucleotide hairpin.     

Model transition states for methane diazonium ion methylation of guanine runs in oligomeric DNA

The DNA reaction pattern of the methane diazonium ion, which is the reactive intermediate formed from several carcinogenic methylating agents, was examined at N7 and O(6) sites in guanine runs occurring in oligonucleotides and model oligonucleotides. Density functional B3LYP/6-31G*, and SCF 3-21G and STO-3G energies of model transition states were calculated in the gas phase and in the CPCM reaction field. For nucleotides containing two, three, and four stacked guanines with counterions in the gas phase, O(6) reactivity is greater than N7 reactivity. In the reaction field, N7 reactivity is 9.0 to 9.8 times greater than O(6) reactivity. For a double-stranded oligonucleotide containing two stacked guanines with counterions in the reaction field, the N7 and O(6) reactivities of the 3'-guanine are 3.9 times greater than the corresponding sites in the 5'-guanine. For double-stranded oligonucleotides with three or four stacked guanines and counterions, the reactivities of the interior guanines are higher than corresponding reactivities of guanines at the ends. These reaction patterns agree with most of the available experimental data. Activation energy decomposition analysis for gas-phase reactions in a double-stranded dinucleotide containing two stacked guanines with counterions indicates that selectivity at O(6) is almost entirely due to electrostatic forces. Selectivity at N7 also has a large electrostatic interaction. However, the orbital interaction also contributes significantly to the gas-phase selectivity, accounting for 32% of the total interaction energy difference between the 3'- and 5'-guanine reactions. In aqueous solution, the relative orbital contribution to N7 selectivity is likely to be larger.     

Cleavage of oligonucleotides containing a P3'→N5' phosphoramidate linkage mediated by single-stranded oligonucleotide templates

Double-stranded DNA (dsDNA) templates can hybridize to and accelerate cleavage of oligonucleotides containing a P3'→N5' phosphoramidate (P-N) linkage. This dsDNA-templated cleavage of P-N linkages could be due to conformational strain placed on the linkage upon triplex formation. To determine whether duplex formation also induced conformational strain, we examined the reactivity of the oligonucleotides with a P-N linkage in the presence of single-stranded templates, and compared these reactions to those with dsDNA templates. P-N oligonucleotides that are cleaved upon duplex formation could be used as probes to detect single-stranded nucleic acids.     

Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages

[reaction: see text] H-Phosphonate monomers of 2'-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides containing either 2'-deoxy or 2'-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et(3)N in CCl(4)/Pyr/H(2)O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N(4)-acetyl deoxycytidine-3'-H-phosphonate monomers were used to avoid transamination during the oxidation step.     

XPS investigation of DNA binding to zirconium-phosphonate surfaces

The surface coverage of phosphorylated oligonucleotides immobilized on a zirconium-phosphonate surface was analyzed using X-ray photoelectron spectroscopy (XPS). By quantifying the intensity of the N 1s signal originating from the oligonucleotide and the Zr 3d peak from the metal-phosphonate surface, the surface coverage of the oligonucleotide could be calculated with a modified substrate-overlayer model. We found relatively low surface coverages indicating that once covalently bound via the terminal phosphate the polymer chain further physisorbs to the surface limiting the adsorption of additional molecules.     

Covalent attachment and hybridization of DNA oligonucleotides on patterned single-walled carbon nanotube films

DNA oligonucleotides were covalently immobilized to prepatterned single-walled carbon nanotube (SWNT) multilayer films by amidation. SWNT multilayer films were constructed via consecutive condensation reactions creating stacks of functionalized SWNT layers linked together by 4,4'-oxydianiline. Aminated- or carboxylated-DNA oligonucleotides were covalently immobilized to the respective carboxylated or aminated SWNT multilayer films through amide bond formation using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. UV-vis-NIR spectroscopic analysis indicated that the SWNT film surface density increased uniformly according to the number of reaction cycles. Scanning electron microscopy and contact angle measurements of the SWNT multilayer film revealed a uniform coverage over the substrate surface. The covalent attachment of DNA oligonucleotides to the SWNT multilayer films and their subsequent hybridization with complementary oligonucleotides were verified using X-ray photoelectron spectroscopy and fluorescence-based measurements. This is the first report demonstrating that DNA oligonucleotides can be covalently attached to immobilized SWNT multilayer films. The anchored DNA oligonucleotides were shown to exhibit excellent specificity, realizing their potential in future biosensor applications.     

Oligonucleotide duplexes containing N8-glycosylated 8-aza-7-deazaguanine and self-assembly of 8-aza-7-deazapurines on the nucleoside and the oligomeric level

The 8-aza-7-deazaguanine N8-(2'-deoxy-beta-D-ribofuranoside) (1) was synthesized, converted into the phosphoramidite 4 and incorporated into oligonucleotides. Nucleoside 1 forms stable base pairs with 2'-deoxy-5-methylisocytidine in DNA with antiparallel chain orientation (aps) and with 2'-deoxycytidine in duplexes with parallel chains (ps). According to the CD spectra self-complementary oligonucleotides d(1-m5isoC)3 and d(1-C), form autonomous DNA-structures. Neither the nucleoside 1 nor the regularly linked 8-aza-7-deaza-2'-deoxyguanosine form G-like tetrads while the regularly linked 8-aza-7-deaza-2'-deoxyisoguanosine gives higher molecular assemblies which are destroyed by bulky 7-bromo substituents. This was verified on monomeric nucleosides by ESI-MS spectrometry and on oligonucleotides by HPLC analysis.     

Rapid characterization of oligonucleotides by capillary liquid chromatography-nano electrospray quadrupole time-of-flight mass spectrometry

A fast quality control method is developed allowing the desalting and characterization of oligonucleotides by capillary liquid chromatography and on-line nano-electrospray ionization quadrupole time-of-flight mass spectrometry using column switching. The influence of addition of ammonium acetate, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, formic acid or acetic acid to the sample, addition of ammonium acetate to the trapping solvent and variation of the trapping time on the further reduction of cation adduction was studied. Final conditions were the addition of 0.1 M ammonium acetate to the sample, the use of a trapping solvent consisting of 0.4 M aqueous 1,1,1,3,3,3-hexafluoro-2-propanol (HFLP) adjusted to pH 7.0 with triethylamine plus 10 mM ammonium acetate during 8 min and the elution of the oligonucleotides with 0.4 M HFIP in 50% methanol. The potential of the optimized procedure is demonstrated for different synthetic oligonucleotides.     

A phosphoramidate substrate analog is a competitive inhibitor of the Tetrahymena group I ribozyme

BACKGROUND: Phosphoramidate oligonucleotide analogs containing N3'-P5' linkages share many structural properties with natural nucleic acids and can be recognized by some RNA-binding proteins. Therefore, if the N-P bond is resistant to nucleolytic cleavage, these analogs may be effective substrate analog inhibitors of certain enzymes that hydrolyze RNA. We have explored the ability of the Tetrahymena group I intron ribozyme to bind and cleave DNA and RNA phosphoramidate analogs. RESULTS: The Tetrahymena group I ribozyme efficiently binds to phosphoramidate oligonucleotides but is unable to cleave the N3'-P5' bond. Although it adopts an A-form helical structure, the deoxyribo-phosphoramidate analog, like DNA, does not dock efficiently into the ribozyme catalytic core. In contrast, the ribo-phosphoramidate analog docks similarly to the native RNA substrate, and behaves as a competitive inhibitor of the group I intron 5' splicing reaction. CONCLUSIONS: Ribo-N3'-P5' phosphoramidate oligonucleotides are useful tools for structural and functional studies of ribozymes as well as protein-RNA interactions.     

Optimizing processing parameters for signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy

Signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy was achieved by optimizing chip processing parameters. The parameters investigated were fabrication, blocking and guide dot concentration, probe concentration and modification, print buffer, humidity during arraying, slide agitation, spot volume and spotter compatibility. The optimum oligonucleotide concentration was 20 microM, while the optimum protein concentration was 0.05 mg/ml. Amino-modified oligonucleotides were best able to be bound to the resin's epoxy groups at pH 8, whereas thiol-modified oligonucleotides displayed an optimum coupling value of pH 7. So as to avoid background (BG) contamination of probes around bright guide dots, the concentration of fluorescent guide dots was set to 1 muM. The most suitable print buffers for oligonucleotide arrays using both piezo- and contact-printing systems proved to be 3 x SSC/1.5 M betaine and commercial ArrayLink. When 0.01% monochlortriazinyl-beta-cyclodextrin sodium salt (MCT) was added, the hybridization signal doubled in strength as compared to plain buffer. The optimum print buffer for proteins was 0.1 N phosphate buffer, pH 8/10% glycerine. The optimum humidity for arraying oligonucleotides was 60% and for proteins 40%. Initially agitating slides for 15 min was found just as effective as agitating slides over the total hybridization period (2.5 h), and this resulted in a three times stronger signal.     

Optimizing the stacking moiety and linker of 2'-acylamido caps of DNA duplexes with 3'-terminal adenine residues

Reported here is the synthesis of oligodeoxynucleotides with a 3'-terminal 2'-acylamido-2'-deoxyadenosine residue. The route to these oligonucleotides employs an N,O-Alloc-protected 5'-phosphoramidite of 2'-amino-2'-deoxyadenosine that was prepared in 11 steps from arabinoadenosine. Small combinatorial libraries of oligonucleotides were generated via acylation with a mixture of linker amino acids and subsequent acylation of their amino groups. Mass spectrometrically monitored nuclease selection assays led to oligonucleotides whose 2'-substituent increases the thermal stability of the DNA duplexes. A linker with three methylene groups between a perylene stacking moiety and the amido group gives a UV-melting point increase of up to 27.9 degrees C for the DNA sequence (TGCGCA*)2, where A* denotes the 2'-acylamidoadenosine residue. The same acylamido group improves mismatch discrimination at the terminal position with a melting point depression of >or=7 degrees C for any of the three mismatches in the target sequence of the octamer 5'-AGGTTGAA-3'. These results demonstrate how even a very weakly base-pairing nucleotide at the 3'-terminus of a DNA probe strand can be enforced to engage in strong and highly sequence-selective base-pairing interactions.     

Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis-electrospray ionization-quadrupole time of flight-mass spectrometry

A capillary zone electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometric method was developed for the characterization of oligonucleotides after synthesis, using model compounds. The major difficulty is the adduction of metal cations to the polyanionic backbone of the oligonucleotide sample, resulting in complex spectra and decreased sensitivity. Several approaches were investigated to circumvent this problem. Separation was performed in an ammonium carbonate buffer. During separation, the interfering metal ions were exchanged for ammonium ions, which are less tightly bound to the oligonucleotide when ionized. The influence of the addition of piperidine and imidazole or trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) to the running buffer for further reduction of cation adduction was investigated. Addition of CDTA to the buffer system resulted in a deconvoluted spectrum with very little adducts. On-line sample stacking proved vital to preconcentrate the samples. The pH and the concentration of the ammonium carbonate buffer as well as the electrophoresis voltage were optimized to achieve the best signal response for the oligonucleotides and a maximum reduction of the cation adducts as well as a short analysis time. Finally, the sheath liquid composition was examined for further improvement of the signal. The developed method was used to analyze different oligonucleotides (5000-9200 Da) in light of its use as a final quality control method for oligonucleotides in terms of purity and sequence homogeneity of the synthesized products. In all cases, very little adducts were observed in the deconvoluted spectra, and the relative errors of the measured molecular masses ranged from 3 to 35 ppm.     

Improved Utility of Photolabile Solid Phase Synthesis Supports for the Synthesis of Oligonucleotides Containing 3'-Hydroxyl Termini

Oligonucleotides are synthesized on, and cleaved from, a solid phase support (6) using the o-nitrobenzyl intramolecular photochemical redox reaction. The yields of isolated oligonucleotides relative to yields obtained using conventional hydrolytic cleavage vary between 67% and 82.5%. Synthesis of oligonucleotides using phosphoramidites that do not contain N-benzoyl protecting groups enables one to photolytically cleave the biopolymers in good yields using a commonly available UV irradiation source. Tritium labeling indicates that less than 3% thymidine.thymidine photodimers are formed during photolytic cleavage of polythymidylates from 6 using a transilluminator. No UV-induced damage is detected via HPLC analysis of enzymatically digested oligonucleotides that were obtained following photolytic cleavage from 6.     

Template-Directed Copying of RNA by Non-enzymatic Ligation

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′-amino group at the 3′-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.     

Synthesis of oligonucleotides containing Fapy.dG (N(6)-(2-deoxy-alpha,beta-D-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) using a 5'-dimethoxytrityl dinucleotide phosphoramidite

Fapy.dG (N(6)()-(2-deoxy-alpha,beta-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) is a modified purine lesion produced by a variety of DNA-damaging agents, which shows interesting biochemical properties. The previous method for synthesizing oligonucleotides containing Fapy.dG utilized a reverse dinucleotide phosphoramidite, which also required the synthesis of the appropriate reverse phosphoramidites. An improved method for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites, is described. Fapy.dG containing dinucleotide phosphoramidites containing 5'-thymidine (11a) or 5'-deoxycytidine (15) are prepared and employed in oligonucleotide synthesis. Oligonucleotide purity is assayed using the DNA repair enzyme formamidopyrimidine DNA glycosylase and by ESI-MS.     

Analysis of oligonucleotides by hydrophilic interaction liquid chromatography coupled to negative ion electrospray ionization mass spectrometry

Hydrophilic interaction liquid chromatography (HILIC) is here successfully coupled to negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of synthetic and chemically modified oligonucleotides. Separation was performed on a 2.1 mm × 100 mm PEEK ZIC^® HILIC column packed with hydrophilic stationary phase with a permanent zwitterionic functional group and a particle size of 3.5 μm with an average pore diameter of 200 Å. A method was developed to separate homogeneous and heterogeneous oligonucleotides as well as methylated oligonucleotides using a quaternary pumping system containing ammonium acetate and water with an acetonitrile gradient. Analyses of oligonucleotides were performed by LC/MS with a detection limit of 2.5 picomole (20 mer) with signal to noise ratio (S/N) of 4.12. The influence of the eluent composition, type of buffer and its concentration, and organic modifier were also evaluated. The HILIC LC/MS method presented in this paper used common, ‘MS friendly’, mobile phases achieving sensitive and selective oligonucleotide analysis.     

New reagent for the preparation of oligonucleotides involving a 5′-thiophosphate or a 5′-phosphate group

We report here a novel, simple reagent enabling the chemical incorporation of a thiophosphate or a phosphate group at the 5′-end of oligonucleotides using very mild basic deprotection conditions. This method can be useful in the case of alkali sensitive modified oligonucleotides. This reagent also gives access to the preparation of bifunctional oligonucleotides with either a thiophosphate group at the 5′-end and a phosphate at the 3′-end, or two thiophosphate groups at both the 5′- and the 3′-ends, or a 5′-thiophosphate group and a 3′-amino-containing linker.