Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Mar 26;32(6):1928-36.
doi: 10.1093/nar/gkh514. Print 2004.

Covalently attached oligodeoxyribonucleotides induce RNase activity of a short peptide and modulate its base specificity

Nadezhda L Mironova  1 , Dmytryi V Pyshnyi, Eugenya M Ivanova, Marina A Zenkova, Hans J Gross, Valentin V Vlassov
Affiliations

Covalently attached oligodeoxyribonucleotides induce RNase activity of a short peptide and modulate its base specificity

Nadezhda L Mironova et al. Nucleic Acids Res. .

Abstract

New artificial ribonucleases, conjugates of short oligodeoxyribonucleotides with peptides containing alternating arginine and leucine, were synthesized and characterized in terms of their catalytic activity and specificity of RNA cleavage. The conjugates efficiently cleave different RNAs within single-stranded regions. Depending on the sequence and length of the oligonucleotide, the conjugates display either G-X>>Pyr-A or Pyr-A>>G-X cleavage specificity. Preferential RNA cleavage at G-X phosphodiester bonds was observed for conjugate NH2-Gly-[ArgLeu]4-CCAAACA. The conjugates function as true catalysts, exhibiting reaction turnover up to 175 for 24 h. Our data show that in the conjugate the oligonucleotide plays the role of a factor which provides an 'active' conformation of the peptide via intramolecular interactions, and that it is the peptide residue itself which is responsible for substrate affinity and catalysis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Oligonucleotide–peptide conjugates pep-16, pep-7 and pep-4, where 16, 7 and 4 indicate the respective oligonucleotides and pep corresponds to the peptide [ArgLeu]4-Gly-NH2. Deg, diethyleneglycol.
Figure 2
Figure 2
Cleavage of 5′-end-labeled decaribonucleotide 5′-[32P]U1UCA UGUAAA10-3′ by the conjugates pep-4 and pep-7. Autoradiograph of 12% polyacrylamide–8 M urea gel. Lanes L and T1, imidazole ladder and partial RNA digestion with RNase T1, respectively; lanes C1 and C2, oligonucleotide incubated without conjugates for 1 and 24 h, respectively. Oligonucleotide was incubated in the presence of conjugates pep-4 or pep-7 (1 µM) at 37°C for different times (shown at the top). Positions of RNA cleavage with the conjugates and with RNase T1 are shown on the right and the left, respectively.
Figure 3
Figure 3
(A) Cleavage of 5′-end-labeled in vitro transcript of HIV-1 RNA with conjugates pep-4, pep-7 and pep-16. Autoradiograph of 12% polyacrylamide–8 M urea gel. Lanes L and T1, imidazole ladder and partial RNA digestion with RNase T1, respectively; lanes C1 and C2, RNA incubated without conjugates for 1 and 24 h, respectively. 5′-[32P]RNA HIV-1 was incubated with the conjugates pep-4, pep-7 or pep-16 (10 µM) at 37°C for different times. The conjugates and the incubation times are indicated at the top. Positions of RNA cleavage with the conjugates and RNase T1 are shown on the right and left, respectively. (B) Secondary structure of the fragment of HIV-1 RNA and location of the cleavages by conjugates pep-4 (blue triangles), pep-7 (red squares) and pep-16 (green circles). Strong and weak cleavage sites are shown as full and open symbols, respectively. Parts 1–5 indicate elements of HIV-1 RNA secondary structure (31). The transcript has G instead of C in position 1.
Figure 3
Figure 3
(A) Cleavage of 5′-end-labeled in vitro transcript of HIV-1 RNA with conjugates pep-4, pep-7 and pep-16. Autoradiograph of 12% polyacrylamide–8 M urea gel. Lanes L and T1, imidazole ladder and partial RNA digestion with RNase T1, respectively; lanes C1 and C2, RNA incubated without conjugates for 1 and 24 h, respectively. 5′-[32P]RNA HIV-1 was incubated with the conjugates pep-4, pep-7 or pep-16 (10 µM) at 37°C for different times. The conjugates and the incubation times are indicated at the top. Positions of RNA cleavage with the conjugates and RNase T1 are shown on the right and left, respectively. (B) Secondary structure of the fragment of HIV-1 RNA and location of the cleavages by conjugates pep-4 (blue triangles), pep-7 (red squares) and pep-16 (green circles). Strong and weak cleavage sites are shown as full and open symbols, respectively. Parts 1–5 indicate elements of HIV-1 RNA secondary structure (31). The transcript has G instead of C in position 1.
Figure 4
Figure 4
(A) Cleavage of 3′-end-labeled in vitro transcript of tRNA3Lys with conjugates pep-4 and pep-7. Autoradiograph of 12% polyacrylamide–8 M urea gel. Lanes L and T1, imidazole ladder and partial RNA digestion with RNase T1, respectively; lanes C1 and C2, RNA incubated without conjugates for 1 and 24 h, respectively. 3′-[32P]tRNA3Lys was incubated with the conjugates pep-4 or pep-7 (50 µM) at 37°C for different times. The conjugates and incubation times are indicated at the top. Positions of RNA cleavage with the conjugates and RNase T1 are shown on the right and left, respectively. (B) Secondary structure of in vitro transcript of human tRNA3Lys. Phosphodiester bonds cleaved by conjugates pep-4 and pep-7 are indicated by blue triangles and red squares, respectively. Strong and weak cleavage sites are shown as full and open symbols, respectively.
Figure 4
Figure 4
(A) Cleavage of 3′-end-labeled in vitro transcript of tRNA3Lys with conjugates pep-4 and pep-7. Autoradiograph of 12% polyacrylamide–8 M urea gel. Lanes L and T1, imidazole ladder and partial RNA digestion with RNase T1, respectively; lanes C1 and C2, RNA incubated without conjugates for 1 and 24 h, respectively. 3′-[32P]tRNA3Lys was incubated with the conjugates pep-4 or pep-7 (50 µM) at 37°C for different times. The conjugates and incubation times are indicated at the top. Positions of RNA cleavage with the conjugates and RNase T1 are shown on the right and left, respectively. (B) Secondary structure of in vitro transcript of human tRNA3Lys. Phosphodiester bonds cleaved by conjugates pep-4 and pep-7 are indicated by blue triangles and red squares, respectively. Strong and weak cleavage sites are shown as full and open symbols, respectively.
Figure 5
Figure 5
Cleavage of 3′-32P-labeled tRNA3Lys with RNase A and conjugate pep-4 treated with DEPC. Autoradiograph of 12% polyacrylamide–M urea gel. Lanes L and T1, imidazole ladder and partial tRNA3Lys digestion with RNase T1, respectively. (A) Controls: lane C1, tRNA3Lys, incubation control; lane C2, tRNA3Lys incubated with 1 µM peptide [ArgLeu]4-Gly-amide; lanes C3 and C4, tRNA3Lys incubated with 10 µM oligonucleotides TCAA and CCAAACA, respectively; lanes C5 and C6, tRNA3Lys incubated in the presence of an equimolar mixture of each oligonucleotide at concentration 10 µM with the peptide. (B) Lanes C7 and C8, tRNA3Lys incubated without conjugate pep-4 and RNase A for 10 min (control for RNase A) and 24 h (control for conjugate), respectively; lanes ‘RNase A, –DEPC’ and ‘RNase A, +DEPC’, tRNA3Lys incubated in the presence of RNase A (0.1 nM) and RNase A treated with 0.1% DEPC, respectively; lanes ‘conjugate pep-4, –DEPC’ and ‘conjugate pep-4, +DEPC’, tRNA3Lys incubated in the presence of conjugate pep-4 (10 µM) and conjugate pep-4 treated with 0.1% DEPC, respectively.
Figure 6
Figure 6
Effect of concentration on cleavage of HIV-1 RNA by conjugates pep-4, pep-7 and pep-16. Assay conditions: 5′-[32P]RNA HIV-1 was incubated with the conjugates under standard conditions at 37°C for 8 h.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Trawick B.N., Daniher,A.T. and Bashkin,J.K. (1998) Inorganic mimics of ribonucleases and ribozymes: from random cleavage to sequence-specific chemistry to catalytic antisense drugs. Chem. Rev., 98, 939–960. - PubMed
    1. Cowan J.A. (2001) Chemical nucleases. Curr. Opin. Chem. Biol., 5, 634–642. - PubMed
    1. Bashkin J.K, Frolova,E.I. and Sampath,U.S. (1994) Sequence-specific cleavage of HIV mRNA by a ribozyme mimic. J. Am. Chem. Soc., 116, 5981–5982.
    1. Ushijima K. and Takaku,H. (1998) Site-specific cleavage of tRNA by imidazole and/or primary amine groups bound at the 5′-end of oligodeoxyribonucleotides. Biochim. Biophys. Acta, 1379, 217–223. - PubMed
    1. Hovinen J., Guzaev,A., Azhayev,A. and Lonnberg,H. (1995) Imidazole tethered oligodeoxyribonucletides: synthesis and RNA cleaving activity. J. Org. Chem., 60, 2205–2209.

Publication types