Abstract
A novel approach to tritium-labeled antisense oligonucleotides (ASO) was established by conjugating N-succinimidyl propionate, as well as maleimide-derivatives, to the 3′-end of ASOs targeting metastasis-associated lung adenocarcinoma transcript 1 (Malat1) containing amino- or sulfhydryl-linkers. In vitro stability and Malat1 RNA reduction studies demonstrated that N-ethylmaleimide (NEM) could be used as a stable tag while maintaining the desired target interaction. The corresponding radioactive label conjugation using [3H]-NEM resulted in tritium-labeled ASOs with a high molar specific activity of up to 17 Ci/mmol. Single-dose in vivo studies in mice were carried out to compare [3H]-ASOs with their unlabeled counterpart ASOs, with and without conjugation to N-acetylgalactosamine (GalNAc), for tissue and plasma concentrations time profiles. Despite the structural modification of the labeled ASOs, in vitro target interaction and in vivo pharmacokinetic behaviors were similar to that of the unlabeled ASOs. In conclusion, this new method provides a powerful technique for fast and safe access to tritium-labeled oligonucleotides, e.g., for pharmacokinetic, mass balance, or autoradiography studies.Graphical abstract
Highlights
The transcription of DNA to RNA and subsequent translation to functional proteins is a central element in living cells
Standard locked nucleic acids (LNAs)-incorporating antisense oligonucleotides (ASO) are usually based on single-stranded 14–20-mers and contain three structural units: LNA nucleotides, DNA nucleotides, and a phosphorothioate backbone
Actual tritiated oligonucleotides used for this study were only prepared with [3H]-NEM as described in the section “Labeling procedure III: [3H]-N-ethylmaleimide ([3H]-3) conjugation on sulfhydryl-linker” (Route B)
Summary
The transcription of DNA to RNA and subsequent translation to functional proteins is a central element in living cells. Using viral RNA, Stephenson and Zamecnik (1978) demonstrated in 1978 that translation of proteins could be efficiently inhibited in vitro by competitive hybridization with antisense oligonucleotides (ASO). There is growing interest across the pharmaceutical industry to discover and develop new ASO-based therapies, employing various mechanisms to inhibit mRNA translation, e.g., formation of ASO/ RNA complexes that activate RNase H, which degrades the mRNA or prevents ribosome recruitment (Muntoni and Wood 2011). Carbon-14 is used to label small molecule drug candidates, since it can be introduced in a metabolically stable position. For larger molecules such as oligonucleotides, the resulting molar specific activity after carbon-14 (62.4 mCi/mmol) labeling might be insufficient for a quantitative determination by liquid scintillation counting (LSC). Tracing of carbon-14 is possible by accelerated mass spectrometry (AMS), but this technology requires special equipment
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