Book of Abstracts: Albany 2007
June 19-23 2007
Small RNA cleaving molecules artificial ribonucleases (aRNases) can find applications in biotechnology for manipulating RNA and may provide new opportunities for design of RNA-targeted therapeutics. We designed three different groups of artificial ribonucleases. One group of the compounds was prepared by conjugation of cationic organic molecules to different structures containing the groups known to be involved in catalysis in the active sites of natural ribonucleases: imidazole, carboxylic, and amino groups. The highest ribonuclease activity was displayed by the compounds built of catalytic domain containing imidazole and carboxylic groups, and RNA binding domain built of cationic 1,4-diazabicyclo[2.2.2]octane (DABCO) residues bearing an aliphatic fragment (Fig. 1A). The compounds cleave linkages in single-stranded regions of RNA yielding 2?,3?-cyclophosphate and 5?-OH groups at the cleavage site. The cleavage specificity of these aRNases is similar to that of ribonuclease A (CAUACG). Under conditions of excess of RNA substrate, each molecule of the ribonuclease mimic can cleave up to 150 phosphodiester bonds within 24h.
The second group of aRNases are amphifilic molecules discovered in experiments with combinatorial libraries of conjugates of (1,4-diazabicyclo[2.2.2]-octane) and hydrophobic molecules (Fig. 1B). These molecules lacking traditional catalytic groups cleave RNA displaying the specificity Y-A > Y-C > C-N (were Y is pyrimidine and N ? any base), which is characteristic of spontaneous cleavage of different phosphodiester bonds in water solution: UA > CA > YC > YG > YU.
The third family of the aRNases is represented by conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine. Some of these compounds designed by rational screening, display T1 ribonuclease-like activity. The conjugate built of a nanodeoxyribonucletide and peptide [Leu-Arg]4-Gly-amide connected by a linker of three abasic deoxyribonucleotides (conjugate pep-9) (Fig. 1C) cleaves RNA exclusively at G-X linkages. Physico-chemical studies revealed that the conjugated oligonucleotide provides specific conformation of the peptide via a set of intramolecular interactions, and that it is the peptide residue itself, which is responsible for affinity of the compound to RNA and catalysis.
Potential of the DABCO based aRNases as probes for investigation of RNA structure in solution was studied in experiments with different RNAs. The compounds displayed high structure and sequence specificity: they cleaved RNA under physiological conditions within single-stranded regions and mismatches after pyrimidine residues with a marked preference to CA and UA sequences.
The aRNases were tested as potential tools for production of antiviral vaccines. The compound ABL3C3 (Fig. 1A) was shown to efficiently inactivate influenza A virus. Mice immunized intranasally with the aRNase inactivated virus were protected against challenge with lethal doses of homologous influenza virus.
Valentin V. Vlassov*
Institute of Chemical Biology and Fundamental Medicine
Figure 1: Artificial ribonucleases. (A) ABLkCm series: conjugates of 1,4-diazabicyclo[2.2.2]-octane and imidazole; (B) Compound Dp12 ; (C) Oligonucleotide-peptide conjugate pep-9
This research was supported by RFBR 05-04-48985, RFBR 04-04-48566, FCSTP RI-112/001/254 and RAS programs ?Molecular and Cellular Biology,? ?Science to Medicine,? "Origin of life and evolution of the biosphere."
References and Footnotes