Base-modified Oligonucleotides Research Articles

N-Methoxy-1,3-oxazinane nucleic acids (MOANAs) - a configurationally flexible backbone modification allows post-synthetic incorporation of base moieties.

(2R,3S)-4-(Methoxyamino)butane-1,2,3-triol was converted into a protected phosphoramidite building block and incorporated into the middle of a short DNA oligonucleotide. O1 and O3 of the (2R,3S)-4-(methoxyamino)butane-1,2,3-triol were engaged in phosphodiester linkages, leaving O2 and the methoxyamino function available to form an N-methoxy-1,3-oxazinane ring through reaction with an aldehyde. In modified oligonucleotides thus obtained, the oxazinane ring formally replaces the furanose ring and the aldehyde, the base moiety of natural nucleosides. The feasibility of synthesizing base-modified oligonucleotides by this approach was demonstrated with several aromatic and aliphatic aldehydes featuring various functional groups.

Additions of Thiols to 7-Vinyl-7-deazaadenine Nucleosides and Nucleotides. Synthesis of Hydrophobic Derivatives of 2'-Deoxyadenosine, dATP and DNA.

Additions of alkyl- or arylthiols to 7-vinyl-7-deaza-2'-deoxyadenosine gave a series of 7-[2-(alkyl- or arylsulfanyl)ethyl]-7-deaza-2'-deoxyadenosines in 45-85% yields. The nucleosides were converted to 5'-O-mono-(dASRMP) or triphosphates (dASRTP) by phosphorylation. The modified triphosphates were also prepared by thiol addition to 7-vinyl-7-deaza-dATP. The triphosphates dASRTP were good substrates for DNA polymerases useful in the enzymatic synthesis of base-modified oligonucleotides (ONs) or DNA containing flexibly linked hydrophobic substituents in the major groove. Primer extension was used for the synthesis of ONs with one or several modifications, PCR was used for the synthesis of heavily modified DNA, whereas terminal deoxynucleotidyl transferase was used for a single-nucleotide labeling of the 3'-end.

Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates.

A simple and elegant method for inhibition of non-templated nucleotide addition by DNA polymerases and for following DNA 3'-heterogeneity in enzymatic DNA synthesis by primer extension (PEX) is described. When template bearing ortho-twisted intercalating nucleic acid (ortho-TINA) at the 5'-end is used, non-templated nucleotide addition is reduced in both the A- and B-family DNA polymerases (KOD XL, KOD (exo-), Bst 2.0, Therminator, Deep Vent (exo-) and Taq). Formation of a single oligonucleotide product was observed with ortho-TINA modified template and KOD XL, KOD (exo-), Bst 2.0, Deep Vent (exo-) and Taq DNA polymerases. This approach can be applied to the synthesis of both unmodified and base-modified oligonucleotides.

Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR

Enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.

ChemInform Abstract: 3-Deaza-2′-deoxyadenosine: Synthesis via 4-(Methylthio)-1H-imidazo(4,5-c)pyridine 2′-Deoxyribonucleosides and Properties of Oligonucleotides.

The synthesis of 4-(methylthio)-1H-imidazo[4,5-c]pyridine 2′-deoxy-β-D-ribonucleosides 2 and 9 and the conversion of the N1-isomer 2 into the 2′,3′-didehydro-2′,3′-dideoxyribonucleoside 3a or (via7) 3-deaza-2′-deoxyadenosine (1) is described. Phosphonate building blocks of 1 were employed in solid-phase synthesis of self-complementary base-modified oligonucleotides. Their properties were studied with regard to duplex stability and hydrolysis by the restriction enzyme Eco RI.

Cross‐Coupling Modification of Nucleoside Triphosphates, PEX, and PCR Construction of Base‐Modified DNA

A novel, efficient, two-step methodology is presented for construction of base-modified oligonucleotides or DNA, involving aqueous cross-coupling reactions of halogenated nucleoside triphosphates (dNTPs) with terminal acetylenes or arylboronic acids, followed by polymerase incorporation of the modified dNTPs either using primer extension (PEX) or polymerase chain reaction (PCR). Curr. Protoc. Chem Biol. 2:1-14. © 2010 by John Wiley & Sons, Inc.

Heterocyclic modifications of oligonucleotides and antisense technology.

Modification of the heterocyclic moiety of oligonucleotides has led to the discovery of potent antisense compounds. This review describes the physicochemical factors that are responsible for duplex stabilization through base modification. A summary is given of the different heterocyclic modifications that can be used to beneficially influence this duplex stability. The biologic activity of base-modified oligonucleotides is described, and the different factors that are important for obtaining in vivo antisense activity with heterocyclic-modified oligonucleotides are summarized.

Hybridization properties of base-modified oligonucleotides within the double and triple helix motif

In this review we describe the influence of modified nucleobases in order to understand the changes they introduce in double and triple helix forming oligomers with the aim to study their potential therapeutic use in the antisense/antigene/ribozyme field. The hybridization properties of the studied modified oligomers depend on the structure of the modified nucleoside, the number of modifications, the selected sequence and the location of the modified nucleoside along the sequence. Unfortunately, most available information is based on melting point determination, eventually CD experiments and only in a few cases on X-ray and NMR studies which makes it difficult to put forward general rules for predicting modified nucleic acid structures. On the other hand, several of the described base modifications allow us to select new oligonucleotides with improved properties in the different application fields.

Synthesis and properties of base-modified oligonucleotides containing the lumazine moiety

Synthesis and properties of base-modified oligonucleotides containing the lumazine moiety