Synthesis and Binding Properties of Oligonucleotides Carrying Nuclear Localization Sequences

Abstract

The synthesis of oligonucleotides carrying nuclear localization peptide sequences is described using two strategies: first, oligonucleotides carrying a thiol group at the 5' end were reacted with maleimido peptides; second, peptide and oligonucleotide were prepared stepwise on the same support, yielding oligonucleotide-3'-peptide conjugates. This second approach was thoroughly studied. Using amino acids and small peptides as model compounds, some side reactions were analyzed, detected, and minimized. Oligonucleotides complementary to Ha-ras gene and carrying nuclear localization peptides at the 3' and 5' ends were prepared. Melting temperature studies showed that duplexes containing nuclear localization peptides were more stable than duplexes with unmodified oligonucleotides. Moreover, oligonucleotide-peptide conjugates maintain a good mismatch discrimination when they bind to their target RNA.

Synthesis of OligonucleotidePeptide Conjugates Carrying the c‐myc Peptide Epitope as Recognition System

“…Previously, we observed phosphoramidite coupling reactions to be more efficient when phosphoramidites were dissolved in dry CH 2 Cl 2 [21,22] but in this case coupling efficiencies were equally high (>98%) using both solvents. A similar effect was observed during the synthesis of oligonucleotides carrying trifunctional amino acids at the 3'-end [23] and we believe that differences on coupling yields are due to the influence of the amino acids on the swelling of the PEG-PS support.…”

“…Oligonucleotide-peptide sequences 1-3 ( Table 1) were prepared essentially as described for the synthesis of oligonucleotides carrying nuclear localization peptide (NLS) sequences [21,22]. First, the peptide sequence was assembled using Boc-amino acids on a sarcosyl-polyethyleneglycol-polystyrene (PEG-PS) support carrying the 5-aminopentyl linker [23].…”

Stepwise synthesis of RNA conjugates carrying peptide sequences for RNA interference studies

“…The melting temperature (Tm) of the duplex containing nucleoplasmine was 83.8 ºC while the Tm of the unmodified duplex was 83.5 ºC. A slighty higher stabilization of the duplex has been described for this peptide on DNA duplexes [32] and it has been attributed to a possible interaction between lysine side chain and DNA phosphates. In the present study two unpaired thymidines separated the peptide from the DNA backbone, therefore it is reasonable to expect no differences between modified and unmodified duplexes.…”

“…Polyethylenglycol-polystyrene (PEG-PS) was selected as solid support since it gave the best results for the coupling of amino acids and nucleoside phosphoramidites [20,32,33]. Scheme 1 shows the outline of the synthesis of the oligonucleotide-peptide conjugates on the same support [20,32,33].…”

“…Scheme 1 shows the outline of the synthesis of the oligonucleotide-peptide conjugates on the same support [20,32,33]. To avoid the use of strong acids in the presence of the oligonucleotide, the peptide part was first synthesized using the acidlabile Boc group to protect the a-amino function.…”