Translation of 2′-modified mRNAin vitroandin vivo

Abstract

2'-Fluoro- and 2'-amino-2'-deoxynucleoside triphosphates have been used for in vitro transcription of 2'-modified luciferase mRNA. The 2'-modified deoxynucleoside-containing transcripts were tested for the expression of luciferase in X.Laevis oocytes as well as in rabbit reticulocyte lysate. Only 2'-fluoro-2'-deoxy-adenosine-modified mRNA gave rise to luciferase as shown by SDS gel as well as by enzyme activity measurements in vivo as well as in vitro. 2'-Fluoro-2'-deoxy-pyrimidine nucleoside-modified mRNA did not give rise to luciferase activity. However, they directed incorporation of 35S-labeled methionine into peptide fragments in rabbit reticulocyte lysate indicating premature termination of translation. No or only extremely little of such incorporation could be detected with 2'-amino modified transcripts.

Nucleotides. Part LVII.. Synthesis of phosphoramidite building blocks of 2′‐amino‐2′‐deoxyribonucleosides: New compounds for oligonucleotide synthesis

“…Previous studies indicate that 2'-amino-2'-deoxynucleoside [48] and short oligonucleotides thereof [49] exist primarily in the 2'-endo-conformation which should favor DNA/ DNA duplex with a B-form geometry, whereas DNA/RNA duplex preferentially adopt an A-form geometry derived from 2'-exo-conformations of the basic nucleosides. These expectations could not be observed in the presented investigations, since we found in all cases of hybridizations decreased T, values due to destabilizing effects resulting probably from electrostatic interactions between the 2'-amino and the negatively charged phosphodiester function, and unknown secondary effects perturbing a normal duplex structure as also described by Shaharova and co-workers [24], Miller et al [49], and Eckstein and co-workers [50].…”

Antimycobacterial Agent Based on mRNA Encoding Human β-Defensin 2 Enables Primary Macrophages To Restrict Growth ofMycobacterium tuberculosis

“…Stability and translational efficiency may be improved by replacement of the native UTRs with those from ␤-globin, which is a very stable and efficiently translated mRNA in most cell types (17). The mRNA may also be further stabilized by alteration of the 2ЈOH groups (15) and by replacing some of the bridging phosphate groups with phosphorothioate groups (16) without abolishing translational activity (1).…”

Recognition of 2′‐hydroxyl groups by Escherichia coli ribonuclease HI

“…thermal stability of the duplex [36], whereas the 2'-fluoro-2'deoxynucleoside does not affect the duplex stability [37]. Therefore, the slightly larger Km value for the -2A substrate can be attributed to duplex destabilization caused by the amino substitution, although the experiments were performed at 30°C, at which the 9-base-pair duplexes should be formed.…”