Monitored selection of DNA-hybrids forming duplexes with capped terminal C:G base pairs.

Reported here are the results of a search for modified oligodeoxynucleotides with a 5'-terminal cytidine residue whose affinity for target strands is enhanced by 5'-acylamido groups. These acylamido groups were envisioned to act as molecular caps that bind to the exposed terminal base pair of the duplex with the target strand. A total of 52 capped oligonucleotides of the sequence R-CGGTTGAC, where R denotes the 5'-appendage and C a 5'-amino-2',5'-dideoxycytidine residue, were tested. Among the building blocks employed to modify the 5'-amino group of the DNA strand were carboxylic acid residues, either appended directly or via an amino acid residue, and aromatic aldehydes, coupled via reductive amination. The carboxylic acids employed ranged from Fmoc-glycine to (Fmoc)(2)-vancomycin and included a number of aromatic acids and bile acids. Small libraries were subjected to MALDI-monitored nuclease selection experiments, and selected compounds were tested in UV-melting assays with target strands. Cholic acid appendages stabilized terminal C:G base pairs to the greatest extent, with melting point increases of up to 10 degrees C. Further, the cholic acid residue enhanced base pairing fidelity at the terminus, as determined in melting analyses with target strands containing a mismatched nucleobase at the 3'-terminus.

Rapid genotyping by MALDI-monitored nuclease selection from probe libraries.

Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with individual genes. Efficient technologies for rapidly detecting SNPs will therefore facilitate the mining of genomic information. Known methods for SNP analysis include restriction-fragment-length polymorphism polymerase chain reaction (PCR), allele-specific oligomer hybridization, oligomer-specific ligation assays, minisequencing, direct sequencing, fluorescence-detected 5'-exonuclease assays, and hybridization with PNA probes. Detection by mass spectrometry (MS) offers speed and high resolution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products, mass-tagged oligonucleotides, DNA created by restriction endonuclease cleavage, and genomic DNA. We have previously reported MALDI-TOF-monitored nuclease selections of modified oligonucleotides with increased affinity for targets. Here we use nuclease selections for genotyping by treating DNA to be analyzed with oligonucleotide probes representing known genotypes and digesting probes that are not complementary to the DNA. With phosphodiesterase I, the target-bound, complementary probe is largely refractory to nuclease attack and its peak persists in mass spectra (Fig. 1A). In optimized assays, both alleles of a heterozygote were genotyped with six nonamer DNA probes (> or = 125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).

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.

5'-Peptidyl substituents allow a tuning of the affinity of oligodeoxyribonucleotides for RNA.

The affinity of amide-linked 5'-aminoacyl and 5'-dipeptidyl DNA octamers for two RNA undecamers with 3'-overhangs was measured via UV melting analysis. A sequence-dependent increase in melting points was observed. At low ionic strength, two appended lysine residues elevate melting points more than two additional A:U base pairs.