Christopher J. Nulf

Peptide Nucleic Acids Conjugated to Short Basic Peptides Show Improved Pharmacokinetics and Antisense Activity in Adipose Tissue

A peptide nucleic acid (PNA) targeting a splice junction of the murine PTEN primary transcript was covalently conjugated to various basic peptides. When systemically administered to healthy mice, the conjugates displayed sequence-specific alteration of PTEN mRNA splicing as well as inhibition of full length PTEN protein expression. Correlating activity with drug concentration in various tissues indicated strong tissue-dependence, with highest levels of activity observed in adipose tissue. While the presence of a peptide carrier was found to be crucial for efficient delivery to tissue, little difference was observed between the various peptides evaluated. A second PNA-conjugate targeting the murine insulin receptor primary transcript showed a similar activity profile, suggesting that short basic peptides can generally be used to effectively deliver peptide nucleic acids to adipose tissue.

Novel HLA-B35 subtypes putative gene conversion events with donor sequences from alleles common in native americans (HLA-B*4002 or B*4801)

In a study of 523 normal subjects of differing ethnic groups, including 189 South American Indians, we have described novel hybridization pattern corresponding to 22 potentially new HLA-B locus alleles. Three of these alleles were subtypes of B35. The locally, assigned alleles, B-3504v, B-3505v, and B-3508v have been sequenced and were officially designated as B*3512, B*3517, and B*3518, respectively. In addition, we determined the nucleotide sequence of another new variant, locally designated B-3509.2. B*3517, was found in 3 individuals (2 Hispanic, 1 Caucasian), it differs from B*3505 by 3 nucleotide substitutions that lead to changes in residues 94, 95, and 103. B*3517 differs from B*3501 in residues 97 and 103. B*3518 was found in 7 South American Indian individuals (6 of 124 Toba Indians, 1 of 18 Pilaga Indians). It differs from B*3509 by 2 silent nucleotide substitutions and by one nonsynonymous substitution in codon 156 (Arg-->Leu). B*3512 differs from B*3504 by 3 nucleotides, one of them leading to a substitution in residue 103 (Val-->Leu). B*3509 was observed in 3 individuals from the Wichi tribe. The nucleotide sequence of one of these was determined and was found to differ from B*35091 by two synonymous nucleotide substitutions. The distinguishing amino acid substitutions in residues 95, 97, and 156 contribute to the structure of specificity pockets F, C, and E, and D and E respectively, therefore, it is possible that some of the new alleles may have different peptide binding profiles. It has been shown that differences at residue 156 may elicit different allorecognition and mediate graft-versus-host disease and rejection in bone marrow transplantation. The mechanisms for the generation of these novel alleles may involve gene conversion events in which short exon-3 segments from the common Native American alleles B*4002 or B*4801 were inserted in HLA-B35 backbone structures. The novel allele B*3518 is closely related to B*35092 and to B*3508. Two alternative hypotheses for its generation can be suggested, the most plausible one would involve B*35092, the putative progenitor of B*3518, since both alleles are prevalent in the same Indian tribes.

Synthesis and Purification of Peptide Nucleic Acids

Peptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2‐aminoethyl glycine linkages. Hybridization of PNAs with RNA or DNA follows normal rules for Watson‐Crick base pairing and occurs with high affinity. Thus, PNAs are a promising choice for applications that benefit from high‐affinity hybridization. They are assembled using techniques adapted from peptide chemistry. Protocols are given for both automated and manual synthesis of PNAs as well as their purification. The advantages of each method are discussed, as are the different monomers and reagents that are required. Additionally, protocols are given for adding peptides to PNAs (which can enhance hybridization or cell uptake of the PNA) and for adding a biotin label.

DNA assembly using bis-peptide nucleic acids (bisPNAs)

DNA nanostructures are ordered oligonucleotide arrangements that have applications for DNA computers, crystallography, diagnostics and material sciences. Peptide nucleic acid (PNA) is a DNA/RNA mimic that offers many advantages for hybridization, but its potential for application in the field of DNA nanotechnology has yet to be thoroughly examined. We report the synthesis and characterization of tethered PNA molecules (bisPNAs) designed to assemble two individual DNA molecules through Watson-Crick base pairing. The spacer regions linking the PNAs were varied in length and contained amino acids with different electrostatic properties. We observed that bisPNAs effectively assembled oligonucleotides that were either the exact length of the PNA or that contained overhanging regions that projected outwards. In contrast, DNA assembly was much less efficient if the oligonucleotides contained overhanging regions that projected inwards. Surprisingly, the length of the spacer region between the PNA sequences did not greatly affect the efficiency of DNA assembly. Reasons for inefficient assembly of inward projecting DNA oligonucleotides include non-sequence-specific intramolecular interactions between the overhanging region of the bisPNA and steric conflicts that complicate simultaneous binding of two inward projecting strands. These results suggest that bisPNA molecules can be used for self-assembling DNA nanostructures provided that the arrangement of the hybridizing DNA oligonucleotides does not interfere with simultaneous hybridization to the bisPNA molecule.