Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo

Md Rowshon Alam, Alokes Majumdar, Arun Kalliat Thazhathveetil, Su Ting Liu, Ji Lan Liu, Nitin Puri, Bernard Cuenoud, Shigeki Sasaki, Paul S. Miller, Michael M. Seidman

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2′-O-methylribose (2′-OMe) and 2′-O-aminoethylribose (2′-AE) residues. Active TFOs contained four contiguous 2′-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2′-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2′-AE residues, the conformationally locked BNA/LNA ribose with a 2′-O,4′-C-methylene bridge, or the 2′-O,4′-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The T m value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences.

Original languageEnglish
Pages (from-to)10222-10233
Number of pages12
JournalBiochemistry
Volume46
Issue number35
DOIs
Publication statusPublished - Sep 4 2007

Fingerprint

Oligonucleotides
Sugars
Thermodynamic stability
Ribose
Bioassay
Hot Temperature
Bioactivity
Biological Assay
In Vitro Techniques
Association reactions
Kinetics
Gene Targeting
Chemical modification
Assays
Genes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Alam, M. R., Majumdar, A., Thazhathveetil, A. K., Liu, S. T., Liu, J. L., Puri, N., ... Seidman, M. M. (2007). Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo. Biochemistry, 46(35), 10222-10233. https://doi.org/10.1021/bi7003153

Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo. / Alam, Md Rowshon; Majumdar, Alokes; Thazhathveetil, Arun Kalliat; Liu, Su Ting; Liu, Ji Lan; Puri, Nitin; Cuenoud, Bernard; Sasaki, Shigeki; Miller, Paul S.; Seidman, Michael M.

In: Biochemistry, Vol. 46, No. 35, 04.09.2007, p. 10222-10233.

Research output: Contribution to journalArticle

Alam, MR, Majumdar, A, Thazhathveetil, AK, Liu, ST, Liu, JL, Puri, N, Cuenoud, B, Sasaki, S, Miller, PS & Seidman, MM 2007, 'Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo', Biochemistry, vol. 46, no. 35, pp. 10222-10233. https://doi.org/10.1021/bi7003153
Alam, Md Rowshon ; Majumdar, Alokes ; Thazhathveetil, Arun Kalliat ; Liu, Su Ting ; Liu, Ji Lan ; Puri, Nitin ; Cuenoud, Bernard ; Sasaki, Shigeki ; Miller, Paul S. ; Seidman, Michael M. / Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo. In: Biochemistry. 2007 ; Vol. 46, No. 35. pp. 10222-10233.
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