A 2'-acylamido cap that increases the stability of oligonucleotide duplexes.

[structure: see text]. Reported here is the synthesis of oligonucleotides containing a 2'-acylamido-2'-deoxyuridine residue at their 3'-terminus. Compared to control sequences bearing a thymidine residue, the quinolone-capped duplexes give higher UV melting points. In the case of (5'-ACGCGU-NA-2')2, where NA denotes a nalidixic acid residue, the melting point increase is up to 22 degrees C over that of (ACGCGT)2.

Solid-phase synthesis of cyclic peptide-DNA hybrids.

[reaction: see text] A methodology for preparing cyclic peptide-DNA hybrids on controlled pore glass in high yield is reported. This methodology employs Fmoc/Alloc-protected amino acid and nucleoside phosphoramidites on an omega-hydroxylauric acid-derivatized support and is suitable for library synthesis. A cyclic hybrid of the sequence Glu-Leu-TT-DP-Lys, where Glu and Lys are linked and T denotes a 5'-amino-5'-deoxynucleotide, exhibited high resistance to exo- and endonucleases.

Selection of modified oligonucleotides with increased target affinity via MALDI-monitored nuclease survival assays.

Reported here is how modified oligonucleotides with increased affinity for DNA or RNA target strands can be selected from small combinatorial libraries via spectrometrically monitored selection experiments (SMOSE). The extent to which target strands retard the degradation of 5'-acyl-, 5'-aminoacyl-, and 5'-dipeptidyl-oligodeoxyribonucleotides by phosphodiesterase I (EC 3.1.4.1) was measured via quantitative MALDI-TOF mass spectrometry. Oligonucleotide hybrids were prepared on solid support, and nuclease selections were performed with up to 10 modified oligonucleotides in one solution. The mass spectrometrically monitored experiments required between 120 and 300 pmol of each modified oligonucleotide, depending on whether HPLC-purified or crude compounds were employed. Data acquisition and analysis were optimized to proceed in semiautomated fashion, and functions correcting for incomplete degradation during the monitoring time were developed. Integration of the degradation kinetics provided "protection factors" that correlate well with melting points obtained with traditional UV melting curves employing single, pure compounds. Among the components of the five libraries tested, three were found to contain 5'-substituents that strongly stabilize Watson--Crick duplexes. Selecting and optimizing modified oligonucleotides via monitored nuclease assays may offer a more efficient way to search for new antisense agents, hybridization probes, and biochemical tools.

Monitoring the hybridization of the components of oligonucleotide mixtures to immobilized DNA via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Reported here is how the hybridization of individual components of oligonucleotide mixtures to solid-phase bound complementary strands can be monitored simultaneously by quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Three oligonucleotides, a DNA heptamer, a DNA octamer and a DNA octamer with a terminal cholic acid appendage were used as the test mixture. Upon cooling in the presence of a complementary undecamer on controlled pore glass, depletion of the components from the solution was observed. The resulting hybridization curves show the same relative affinities as traditional UV melting curves with single components and their complement. Assays of the kind described here may be used to select high affinity binders from combinatorial libraries of modified antisense oligonucleotides.