Enzymatic Incorporation of LNA Nucleotides into DNA Strands

  title={Enzymatic Incorporation of LNA Nucleotides into DNA Strands},
  author={Rakesh N. Veedu and Birte Vester and Jesper Wengel},
Unlocking uses of locked nucleic acids: LNA nucleoside 5′-triphosphates have been synthesized, and their ability to serve as substrates for polymerases have been investigated. Phusion high-fidelity DNA polymerase was found to be an efficient enzyme for incorporating LNA nucleoside 5′-triphosphates into DNA strands. 

Polymerase-directed synthesis of C5-ethynyl locked nucleic acids.

Polymerase directed incorporation studies of LNA-G nucleoside 5′-triphosphate and primer extension involving all four LNA nucleotides

An extension experiment showed that KOD DNA polymerase can even incorporate 21 LNA nucleotides successively to a full-length extension product and revealed that LNA-containing extension products were much more resistant towards the 3′→5′ exonucleolytic cleavage activity of the enzyme than DNA extension products.

Enzymatic polymerisation involving 2'-amino-LNA nucleotides.

Highly efficient enzymatic synthesis of 3'-deoxyapionucleic acid (apioNA) having the four natural nucleobases.

Therminator DNA polymerase was shown to incorporate these apioNTPs effectively giving 43mer DNA-apioNA chimera.

Structures of KOD and 9°N DNA Polymerases Complexed with Primer Template Duplex

Replicate it: Structures of KOD and 9°N DNA polymerases, two enzymes that are widely used to replicate DNA with highly modified nucleotides, were solved at high resolution in complex with

Locked and Unlocked Nucleosides in Functional Nucleic Acids

This review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic Acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes.

Locked nucleic acid nucleoside triphosphates and polymerases: on the way towards evolution of LNA aptamers.

Recent developments in LNA-based aptamers for diagnostic and therapeutic applications remain largely unexplored, but scientific breakthroughs relating to enzymatic polymerization using LNA nucleoside triphosphates as substrates are highlighted.

Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids

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).

Enzymatic incorporation of emissive pyrimidine ribonucleotides.

The enzymatic incorporation of a series of emissive pyrimidine analogues into RNA oligonucleotides is explored and the 5-modified non-natural UTP is found to be the most "polymerase-friendly" and well accommodated by T7 RNA polymerase.

Enzymatic Synthesis of 7',5'-Bicyclo-DNA Oligonucleotides.

The synthesis of a bicyclo-DNA triphosphate (i.e., 7',5'-bc-TTP) is described and its potential to serve for the generation of new xenonucleic acids (XNAs) based on this scaffold is shown.



LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition

A novel class of nucleic acid analogues, termed LNA (locked nucleic acids), is introduced. Following the Watson–Crick base pairing rules, LNA forms duplexes with complementary DNA and RNA with

Recognition of threosyl nucleotides by DNA and RNA polymerases.

It is found that for both the Vent (exo-) DNA polymerase and HIV reverse transcriptase K(m) values were increased and kcat values decreased for the incorporation of tTTP in comparison to their natural counterparts.

Enzymatic incorporation in DNA of 1,5-anhydrohexitol nucleotides.

Kinetic parameters for incorporation of one anhydrohexitol adenine nucleoside were similar to those of its natural analogue and a selective incorporation proved only feasible under experimental conditions where no more than two analogues could be inserted.

LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA.

The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.

Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization

CeNA is suggested as an ideal candidate to use in directed evolution methods for the development of a polymerase capable of replicating CeNA.

High fidelity TNA synthesis by Therminator polymerase

Therminator DNA polymerase is an efficient DNA-dependent TNA polymerase capable of polymerizing TNA oligomers of at least 80 nt in length. In order for Therminator to be useful for the in vitro

Analysis and suppression of DNA polymerase pauses associated with a trinucleotide consensus.

A novel class of DNA sequences that cause DNA polymerases to pause are studied, which have the central consensus Py-G-C and are not necessarily adjacent to hairpins in the DNA template.