An efficient method for producing the covalent closure of oligonucleotides on complementary templates by the action of BrCN was developed. A rational design of linear precursor oligonucleotides was studied, and the effect of factors such as oligonucleotide concentration and oligomer-template length ratio was evaluated. The efficiency of circularization was shown to correlate well with the secondary structure of the precursor oligomer (as predicted by a simple computer analysis), hairpin-like structures bearing free termini clearly favouring the circularization reaction. A novel idea, consisting of the incorporation of non-nucleotide insertions in the precursor oligomer (namely, 1,2-dideoxy-D-ribofuranose residues), may render this method universal and highly effective. An original set of assays was developed to confirm the circular structure of the covalently closed oligonucleotides.

No abstract

A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2'-deoxynucleoside 5' to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37-72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37-72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38-39 had reacted specifically with Lys51 in the basic region of Tat peptide (37-72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.