Terminus Of Oligonucleotide Research Articles

A toolbox for enzymatic modification of nucleic acids with photosensitizers for photodynamic therapy.

Photodynamic therapy (PDT) is an approved cancer treatment modality. Despite its high efficiency, PDT is limited in terms of specificity and by the poor solubility of the rather lipophilic photosensitizers (PSs). In order to alleviate these limitations, PSs can be conjugated to oligonucleotides. However, most conjugation methods often involve complex organic synthesis and result in the appendage of single modifications at the 3'/5' termini of oligonucleotides. Here, we have investigated the possibility of bioconjugating a range of known PSs by polymerase-mediated synthesis. We have prepared a range of modified nucleoside triphosphates by different conjugation methods and investigated the substrate tolerance of these nucleotides for template-dependent and -independent DNA polymerases. This method represents a mild and versatile approach for the conjugation of single or multiple PSs onto oligonucleotides and can be useful to further improve the efficiency of the PDT treatment.

A Trisbenzimidazole Phosphoramidite Building Block Enables High-Yielding Syntheses of RNA-Cleaving Oligonucleotide Conjugates.

The RNA cleaving catalyst tris(2-aminobenzimidazole) when attached to the 5’ terminus of oligonucleotides cuts complementary RNA strands in a highly site-specific manner. Conjugation was previously achieved by the acylation of an amino linker by an active ester of the catalyst. However, this procedure was low yielding and not reliable. Here, a phosphoramidite building block is described that can be coupled to oligonucleotides by manual solid phase synthesis in total yields around 85%. Based on this chemistry, we have now studied the impact of LNA (locked nucleic acids) nucleotides on the rates and the site-specificities of RNA cleaving conjugates. The highest reaction rates and the most precise cuts can be expected when the catalyst is attached to a strong 5’ closing base pair and when the oligonucleotide contains several LNA units that are equally distributed in the strand. However, when placed in the 5’ position, LNA building blocks tend to diminish the specificity of RNA cleavage.

A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for Nanoscale Structural EPR Studies at Physiological Temperatures.

Triarylmethyl (trityl, TAM) radicals are a promising class of spin labels for nanometer-scale distance measurements in biomolecules at physiological temperatures. However, to date, existing approaches to site-directed TAM labeling of DNA have been limited to label attachment at the termini of oligonucleotides, thus hindering a majority of demanded applications. Herein, we report a new versatile strategy for TAM attachment at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides, which are then postsynthetically functionalized with TAM. We demonstrate a synthesis of a set of oligonucleotide complexes that are TAM-labeled at internal or terminal sites, as well as the possibility of measuring interspin distances up to ∼5-6 nm at 298 K using double quantum coherence electron paramagnetic resonance (EPR). Implementation of the developed approach strongly broadens the scope of nucleic acids and nucleoprotein complexes available for nanoscale structural EPR studies at room temperatures.

Synthesis and biological properties of triazole-linked locked nucleic acid.

We have synthesized and studied the biological and biophysical properties of triazole-linked ribo and xylo locked nucleic acid (LNA). The combination of LNA with the Isobe triazole linkage gave high binding affinity when incorporated at the 3' or 5' termini of oligonucleotides, but low binding affinity at internal positions. Antisense oligonucleotides (ASOs) and siRNAs containing triazole dimers were highly active and nuclease resistant. Surprisingly, the xyloLNA-modified siRNA was the most active of the series.

Enabling Multiple Conjugation to Oligonucleotides Using "Click Cycles".

An efficient method for the synthesis of multiply functionalized oligonucleotides (ONs) utilizing a novel H-phosphonate alkyne-based linker for multiple functionalization (LMF) is developed. The strategy allows for the conjugation of various active entities to oligonucleotide through the postsynthetic attachment of LMF at the 5'-terminus of ONs using H-phosphonate chemistry followed by conjugation of various entities via [3 + 2] copper(I) catalyzed cycloaddition in a stepwise manner. Each cycle is composed of attachment of the LMF followed by a click reaction with azido-containing units. Sequential solid-phase synthesis of oligonucleotide conjugates containing three attached entities was performed using an acetylated form of MIF peptide conjugated to azido linker, achieving high conversions at each unit addition. In addition, to show the versatility of the method, oligonucleotide conjugates with several different classes of compounds were synthesized. Each conjugate containing three different entities, whose structure and function varied (e.g., sugars, peptides, fluorescent labels, and m3G-Caps).

Phosphoramidate Ligation of Oligonucleotides in Nanoscale Structures.

The folding of long DNA strands into designed nanostructures has evolved into an art. Being based on linear chains only, the resulting nanostructures cannot readily be transformed into covalently linked frameworks. Covalently linking strands in the context of folded DNA structures requires a robust method that avoids sterically demanding reagents or enzymes. Here we report chemical ligation of the 3'-amino termini of oligonucleotides and 5'-phosphorylated partner strands in templated reactions that produce phosphoramidate linkages. These reactions produce inter-nucleotide linkages that are isoelectronic and largely isosteric to phosphodiesters. Ligations were performed at three levels of complexity, including the extension of branched DNA hybrids and the ligation of six scaffold strands in a small origami.

Development of a 3′-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5′-termini of oligonucleotides; however, the ssH linker is not appropriate for 3′-terminal modification because it undergoes intramolecular trans-acylation under heat–aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat–alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3′-revH linker was efficiently linked with the 5′-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2′-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3′-terminal modification of oligonucleotides but also expands the utility range in combination with the 5′-ssH linker.

Synthesis and In Vivo PET Imaging of Hyaluronan Conjugates of Oligonucleotides.

Synthesis for (68)Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated oligonucleotide hyaluronan (HA) tetra- and hexasaccharide conjugates is described. A solid-supported technique is used to introduce NOTA-chelator into the 3'-terminus of oligonucleotides and a copper-free strain promoted azide alkyne cycloaddition (SPAAC) to HA/oligonucleotide conjugation. Protecting group manipulation, required for the HA-moieties, is carried out after the SPAAC-conjugation. Positron emission tomography (PET) is used (1) in the whole-body distribution kinetic studies of the conjugates in healthy rats and (2) to show the potential of hyaluronan-induced targeting of oligonucleotides into the infarcted area of rats with myocardial infarction.

Short-RNA selective binding of oligonucleotides modified using adenosine and guanosine derivatives that possess cyclohexylphosphates as substituents

We have developed new artificial oligonucleotides which distinguish short RNA targets from long ones. The modification of the 5' termini of oligonucleotides by using adenosine derivatives that possess a bulky cyclohexyl phosphate moiety at their base moiety and a phosphate group at the position of their 5'-hydroxyl group maximized their short RNA selectivity. The 2'-O-methyl-RNA (5'-XC(m)A(m)A(m)C(m)C(m)U(m)A(m)C(m)U(m)) having these modifications exhibits ca. 10 °C higher T(m) in the duplexes with the complementary short RNA (3'-GUUGGAUGA-5') than with the long RNA (3'-AUUAUAUGUUGGAUGAUGGUUA-5'). The oligodeoxynucleotides having the same modification exhibited similar selectivity. Such short-RNA selective binding of terminally modified oligonucleotides can be employed to distinguish between mature microRNAs and pre-microRNAs.

Synthesis of Oligonucleotides Carrying Amino Lipid Groups at the 3′-End for RNA Interference Studies

Novel lipid derivatives carrying amino and triazolyl groups were efficiently synthesized and covalently anchored at the 3'-termini of oligonucleotides. The desired amino-lipid conjugates were fully characterized by reversed-phase HPLC and MALDI-TOF mass spectrometry. The methodology was applied to the synthesis of lipid-siRNA designed to inhibit tumor necrosis factor (TNF-α) in order to obtain siRNAs with anti-inflammatory properties. The siRNA duplex carrying amino-lipids at the 3'-end of the passenger strand has inhibitory properties similar to those of unmodified RNA duplexes in HeLa cells, indicating that the new lipid derivatives are compatible with the RNA interference machinery.

ChemInform Abstract: Phosphorylation within DNA Duplexes. Synthesis of Modified Oligodeoxyribonucleotide.

Abstract An effective method was suggested for the activation of phos-phomonoester groups in nicks of a double-strand DNA (1,2). This approach allows to incorporate various sugar phosphate backbone modifications at a particular site when DNA duplexes are being assembled. A modifying group is first introduced at the 5′- or 3′-termini of oligonucleotides, then a duplex is formed and oligomers are coupled on the complementary template using water-soluble l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide or cyanogen bromide as the condensing agents. Various DNA duplexes containing not only natural phosphodiester but also phosphoramidate and pyrophospha-te internucleotide bonds, as well as phosphodiester bonds between nucleotide residues with modified sugar analogs (ribo-, arabino- and xylo-derivatives) were assembled by this method.

New, stronger nucleophiles for nucleic acid-templated chemistry: Synthesis and application in fluorescence detection of cellular RNA

Nucleic acid-templated chemistry is a promising strategy for imaging genetic sequences in living cells. Here we describe the synthesis of two new nucleophiles for use in templated nucleophilic displacements with DNA probes. The nucleophilic groups are phosphorodithioate and phosphorotrithioate; we report on synthetic methods for introducing these groups at the 3′-terminus of oligonucleotides. Both new nucleophiles are found to be more highly reactive than earlier phosphoromonothioates. This increased nucleophilicity is shown to result in more rapid templated reactions with electrophilic DNA probes. The new probes were demonstrated in detection of specific genetic sequences in solution, with clear signal over background being generated in as little as 20 min. The probes were also tested for imaging ribosomal RNA sequences in live Escherichia coli; useful signal was generated in 20 min to 1 h, approximately one quarter to one-half the time of earlier monothioate probes, and the signal-to-noise ratio was increased as well.

Gel Retardation Assays for DNA-binding Proteins

MATERIALS 10x Tris-glycine buffer TBE buffer may be used in place of Tris-glycine. 6x Gel-loading buffer I 32P-labeled control DNA 32P-labeled target DNA of >20 bp (specific activity 2 x 107 cpm/μg [ Labeling of the DNA fragment can be accomplished by phosphorylation (Dephosphorylation of DNA Fragments with Alkaline Phosphataseor Phosphorylating the 5' Termini of Oligonucleotides), filling in of a 3'-recessed end using a DNA polymerase (Labeling 3' Termini of Double-stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase Ior Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I), or by using PCR to incorporate radiolabeled nucleotides into the body of the probe (The Basic Polymerase Chain Reaction). Control nuclear extract or protein fraction Ficoll 400 (20% w/v) Dissolve the Ficoll in sterile H2O and store the solution frozen in 100-μl aliquots at -20°C. Nuclear extract or Protein fraction(s) Prepare the extract by one of the methods described in Mapping Protein-binding Sites on DNA by DNase I Footprinting. Poly(dI-dC) (1 mg/ml) Gel Retardation Assays for DNA-binding Proteins -Sambrook and Rus... file:///C:/Users/MILI%20JOON/Documents/protocol/Electrophoresis,...

Cycle Sequencing: Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers

MATERIALS 5x Cycle-sequencing buffer AmpliTaq CS or other exonuclease-deficient versions of Taq DNA polymerase (5 units/μl) EDTA (0.05 M, pH 8.0) Formamide loading buffer Oligonucleotide Primers, radiolabeled at the 5' terminus with 33P or 32P To prepare end-labeled primers, please see Phosphorylating the 5' Termini of Oligonucleotides. Template DNAs Plasmids, cosmids, bacteriophage , and bacteriophage M13 DNAs, purified from largeor small-scale cultures can be used as templates. The table below shows the amounts of each type of template required.

Synthesis of Oligonucleotide Conjugates via Aqueous Diels‐Alder Cycloaddition

The conjugation of maleimide reporter groups to 5'-diene-modified oligonucleotides via aqueous Diels-Alder cycloaddition is described. Detailed basic protocols are provided for the preparation of a diene-amidite (5'-diene modifier), for the attachment of a diene group to the 5'-terminus of oligonucleotides, and for the conjugation of maleimide reporter groups to diene-modified oligonucleotides.

A new dark quencher for use in genetic analysis.

A novel, long-wavelength, non-fluorescent quencher (LQ), based on 1,4-diaminoanthraquinone, has been incorporated at the 3' and 5'-termini of oligonucleotides. The quencher has been used in Molecular beacons, efficiently quenching the long wavelength fluorophore, Cy5.

Controlled Detritylation of Antisense Oligonucleotides

The last chemical step in standard oligonucleotide synthesis, the acid-catalyzed removal of 4,4‘-dimethoxytrityl (DMTr) protecting groups from the 5‘-terminus of oligonucleotides is accompanied by hydrolysis of purine nucleoside glycosidic linkages (depurination). A mild procedure for controlled DMTr removal using NaOAc (10 mM, pH 3.0) under a fixed set of conditions has been developed. The calculation of the reaction time is based on a t1/2 versus pH profile, specifically determined for each individual oligonucleotide sequence. The effect of various solvent components on the reaction kinetics has been evaluated. As a result, oligonucleotides with improved impurity profile are obtained consistently from laboratory scale to production scale.

Attachment of Reporter and Conjugate Groups to the 3′ Termini of Oligonucleotides

Conjugation of oligonucleotides at the 3 terminus is less common because this site is used for covalent linkage to solid-phase oligonucleotide synthesis supports. However, 3-oligonucleotide conjugates have several valuable physicochemical properties, including their ability to stabilize nucleic acid hybridization complexes and to retard the activity of exonucleases. This unit discusses methods for preparing oligonucleotides conjugated at the 3 terminus.

Modification of the 5′ Terminus of Oligonucleotides for Attachment of Reporter and Conjugate Groups

Reporter and conjugate groups can be added directly to the 5' terminus of oligonucleotides by appropriate modification. Conjugate groups can be used to increase the affinity of complementary strands, induce irreversible modification of target sequences, or enable sequences to recognize and permeate target cell membranes. This overview discusses the 5' modifications that can be used and strategies for the covalent attachment of ligands to the modified oligonucleotides. Step-by-step protocols for attachment of conjugate groups are given elsewhere in the series.

Synthesis of metallointercalator-DNA conjugates on a solid support.

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.