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Published online 26 March 2004

Nucleic Acids Research, 2004, Vol. 32, No. 6 1928-1936
© 2004 Oxford University Press

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

Nadezhda L. Mironova, Dmytryi V. Pyshnyi, Eugenya M. Ivanova, Marina A. Zenkova*, Hans J. Gross1 and Valentin V. Vlassov

Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Avenue 8, Novosibirsk, Russian Federation, 630090 and 1 Institute of Biochemistry, Biocenter, Am Hubland, D-97074 Würzburg, Germany

*To whom correspondence should be addressed. Tel: +7 3832 333761; Fax +7 3832 333677; Email: marzen{at}niboch.nsc.ru

Received December 23, 2003; Revised February 16, 2004; Accepted March 9, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In view of the important roles played by RNAs in recent years considerable effort has been expanded in designing compounds capable of cleaving RNA. These chemical constructs have been referred to as ‘artificial’ or ‘chemical’ ribonucleases (RNases). Artificial RNases are of great current interest because of their potential applications in molecular biology and drug design.

In the last few years a number of compounds capable of cleaving RNA under physiological conditions have been designed: metal complexes (1,2), organic compounds containing low basic amines (3) and imidazole groups (4–7), and small peptides (8). Some natural peptides belonging to the zinc-finger protein family are known to exhibit RNase-like activity (9,10). It was also found that synthetic polypeptides containing alternating hydrophobic (alanine, leucine) and basic (lysine, arginine) amino acids (11–13) could cleave RNA. Short oligopeptides built of alternating hydrophobic and basic amino acids displayed negligible RNase activity. However, conjugation of these peptides to oligodeoxyribonucleotides dramatically improves the activity of these catalysts towards RNA, but no DNA cleavage is observed (14–16). All the designed organic artificial RNases and some peptides display similar sequence-specificity: they cleave phosphodiester bonds in Pyr–A motifs, which are known to be particularly fragile (17,18). The reasons for the high sensitivity of Pyr–A motifs towards a wide range of catalysts are not clear and remain to be investigated (19,20). RNA cleavage by artificial RNases at phosphodiester bonds other than Pyr–A is not well documented.

In this paper we describe new artificial RNases, conjugates of different short oligodeoxyribonucleotides and peptides built of alternating arginine and leucine residues connected by a phosphamide bond. The oligonucleotides in the conjugates are not complementary to RNA substrates. The conjugates are able to cleave different RNAs at Pyr–A and G–X sequences and conjugate NH2-Gly-[ArgLeu]4-CCAAACA displayed pronounced G{downarrow}X cleavage specificity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
[{gamma}-32P]ATP (specific activity, >3000 Ci/mmol) and 5'-[32P]pCp (specific activity, 3000 Ci/mmol) were from Biosan Co. T4 RNA ligase and T4 polynucleotide kinase were purchased from Fermentas. The oligopeptide [LeuArg]4-Gly-amide was from the State Research Center of Virology and Biotechnology ‘Vector’. RNase T1 was from Boehringer Mannheim. FokI and Zsp2I restriction endonucleases were purchased from Sibenzyme. T7 RNA-polymerase was prepared by Dr V. Ankilova (this institute). All buffers were prepared using MilliQ water, contained 0.1 mM EDTA and were filtered through 0.22 µm Millipore filters.

Preparation of oligodeoxyribonucleotides
Oligodeoxyribonucleotides 16 [p(DEG)-CCCTGGACCCTCAGAT], 7 (p-CCAAACA) and 4 (p-TCAA) were synthesized by the standard solid phase phosphoramidite procedure on an automatic synthesizer ASM-700 (Biosset). Oligonucleotides were isolated by successive ion exchange and reverse phase HPLC on Polysil SA-500 columns (Russia) and LiChrosorb RP-18 columns (Merck), respectively, using standard protocols.

Preparation of conjugates
Conjugates NH2-Gly-[ArgLeu]4-pDEG-CCCTGGACCCTCAGAT (pep-16), NH2-Gly-[ArgLeu]4-pCCAAACA (pep-7) and NH2-Gly-[ArgLeu]4-pTCAA (pep-4) were synthesized via phosphamide bond formation between the 5'-terminal phosphate of oligonucleotide and the N-terminal {alpha}-amino group of the oligopeptide [LeuArg]4-Gly-NH2 (21). The conjugates were isolated by reverse phase HPLC on LiChrosorb RP-18 columns. The homogeneity of oligonucleotides 16, 7 and 4 and the corresponding conjugates pep-16, pep-7 and pep-4 was tested by electrophoresis in 15% denaturing polyacrylamide gel containing 8 M urea followed by visualization of oligonucleotide material by ‘Stains-all’. The homogeneity of the oligonucleotides and the conjugates was 95–98%.

Preparation of substrate RNAs
RNA HIV-1 was prepared by in vitro transcription with T7 RNA polymerase using FokI-linearized plasmid pHIV-2 (22). The reaction was carried out in 300 µl of 40 mM Tris–HCl pH 7.5 containing 6 mM MgCl2, 2 mM spermidine, 10 mM sodium chloride, 10 mM DTT, 1 mM each of NTP, 30 µg of DNA template and 100 U T7 RNA polymerase for 2 h at 37°C. Reaction was quenched by 1:1 phenol–chloroform (v/v) extraction followed by ethanol precipitation. After centrifugation, RNA precipitate was rinsed twice with 80% ethanol and dissolved in water.

tRNA3Lys was prepared by in vitro transcription with T7 RNA polymerase using Zsp2I-linearized plasmid pHtk(wt) (22) under the same conditions.

The decarbonucleotide substrate was chemically synthesized by Dr M.N. Repkova (this institute).

Dephosphorylation
HIV-1 RNA transcript was dephosphorylated using bacterial alkaline phosphatase BAP (Fermentas) according to a described protocol (23). The reaction mixture, 50 µl of 50 mM Tris–HCl pH 8.5, containing 1 mM EDTA, 0.2% SDS, 2% formamide, 2.5 mM DTT, 0.1 A260 in vitro transcript of RNA HIV-1 and 2 U of bacterial alkaline phosphatase was incubated at 37°C for 1 h. BAP was added to the reaction mixture at 0 and 30 min incubation time. The reaction was quenched by 1:1 phenol–chloroform (v/v) extraction followed by extraction of water phase with ethyl ester and ethanol precipitation.

RNA 5'- and 3'-labeling
5'-End-labeling of decaribonucleotide and RNA HIV-1 was carried out using [{gamma}-32P]ATP and T4 polynucleotide kinase (Fermentas) according to a described protocol (23). 3'-End labeling of tRNA3Lys was carried out using 5'-[32P]cytidine-3',5'-bisphosphate and T4 RNA ligase (Fermentas) according to a published protocol (24). 32P-labeled RNAs were isolated by electrophoresis in 12% denaturing polyacrylamide gel with 8 M urea. RNAs were visualized by autoradiography on X-ray film. 32P-labeled RNAs were eluted from the gel with 300 µl of 0.3 M sodium acetate pH 5.5 and ethanol precipitated.

Cleavage of RNA by oligonucleotide–peptide conjugates
The reaction mixture (10 µl) contained 50 000 c.p.m. 32P-labeled RNA, one of the oligonucleotide–peptide conjugates at concentrations ranging from 5 to 50 µM, 50 mM Tris–HCl pH 7.0, 0.2 M KCl, 1 mM EDTA and 100 µg/ml total Escherichia coli tRNA as carrier. The mixtures were incubated at 37°C (for various times) and quenched by precipitation of RNA with 2% lithium perchlorate in acetone (150 µl). RNA was collected by centrifugation and dissolved in loading buffer (6 M urea, 0.025% bromophenol blue, 0.025% xylene cyanol). RNA and RNA cleavage products were resolved in 12% polyacrylamide–8 M urea gel using TBE (100 mM Tris–borate pH 8.3, 2 mM EDTA) as running buffer. To identify cleavage sites, an imidazole ladder (25) and a G-ladder produced by partial RNA cleavage with 2 M imidazole buffer (pH 7.0) and RNase T1 (26), respectively, were run in parallel. To obtain quantitative data, gels were dried, radioactive bands were cut out of the gel and their radioactivity was determined by Cherenkov counting. The total extent of RNA cleavage as well as the extent of RNA cleavage at any given site was determined as a ratio of radioactivity measured in the RNA fragment(s) to the total radioactivity applied on the gel.

Inhibition of RNase activity by diethylpyrocarbonate
One microlitre of 1% diethylpyrocarbonate (DEPC) (Sigma) was added to solutions (10 µl) containing RNase A (1 nmol) (Sigma) and conjugate pep-4 (10 µmol), and the mixtures were incubated for 1 h at 37°C. Then 1 µl of the reaction mixture was added to solutions (10 µl) containing 50 000 c.p.m. 3'-[32P]tRNA3Lys in 50 mM Tris–HCl pH 7.0, 0.2 M KCl and 1 mM EDTA, supplied with 100 µg/ml of RNA carrier. The solutions were incubated at 37°C for 10 min and 8 h with RNase A and conjugate pep-4, respectively. Then the solutions were mixed with 40 µl of 0.3 M sodium acetate pH 5.5 containing 1 µg of RNA carrier and 50 µl of water-saturated phenol. RNA was precipitated from the water phase with 150 µl ethanol and analyzed by electrophoresis.

Reaction turnover assay
Reaction mixtures (10 µl) contained 50 000 c.p.m. 5'-[32P]tRNA3Lys, 10 µM conjugate pep-4, 50 mM Tris–HCl pH 7.0, 0.2 M KCl, 1 mM EDTA and in vitro transcript of tRNA3Lys at concentrations ranging from 10 to 100 µM. The mixtures were incubated at 37°C and quenched by RNA precipitation with 100 µl of 2% lithium perchlorate solution in acetone followed by PAGE analysis as described above. The reaction turnover was calculated as follows:

n = [RNA] x N x Cex/[conjugate]

where n is the reaction turnover, i.e. the number of cleaved phosphodiester bonds per molecule of the conjugate for 24 h, [RNA] is the concentration of RNA substrate, N is the number of phosphodiester bonds cleaved by the conjugate within RNA substrate, Cex is the total cleavage extent after 24 h of reaction and [conjugate] is the concentration of the conjugate pep-4.

Effective rate constants of RNA cleavage
The cleavage reactions were performed under conditions of conjugate excess. Single reaction turnover conditions were as follows: 0.1 µM of [32P]RNA and 10 µM of conjugate. The reaction mixtures were prepared, incubated and analyzed as described above. The effective rate constants of RNA cleavage were calculated for individual phosphodiester bonds using a kinetic scheme of consecutive and parallel reactions (27):

k1k2kn

A0 -> P1 -> ... -> Pn

P1 -> P2 -> ... -> Pn

P2 -> P3 -> ... -> Pn

Pn–1 -> Pn1

where A0 is RNA and P1...Pn are products of cleavage at individual phosphodiester bonds.

Effective rate constants keff were obtained by minimizing the mean square deviation between the experimental data and theoretical curves of the system of equations

P1 = 100{exp[–(k2 +...+ kn)t] – exp[–(k1 + k2 +...+ kn)t]}

P2 = 100{exp[–(k3 +...+ kn)t] – exp[–(k2 + k3 +...+ kn)t]}

Pn = 100[1 – exp(–knt)]

P1 + P2 +...+ Pn = 100{1 – exp[–(k1 + k2 +...+ kn)t]}2

using Origin 6.0 software.

Determination of Km
The initial cleavage rates V0 for conjugate pep-4 and HIV-1 RNA were determined under conditions of substrate excess. The cleavage reactions (10 µl) contained 10 µM conjugate, 100 µM HIV-1 RNA, 50 000 c.p.m. 5'-32P-labeled HIV-1 RNA, 50 mM Tris–HCl pH 7.0, 200 mM KCl and 1 mM EDTA. The reactions were incubated, quenched and analyzed as described above. The concentration of substrate converted to cleavage products was plotted as a function of time. The initial cleavage rates V0 for each substrate concentration were obtained from the slope of the best-fit line derived from five data points within the linear portion of the plot. Further, the initial cleavage rates were plotted as a function of substrate concentration and the data were analyzed using the Michaelis–Menten equation. Km was obtained from the Lineweaver–Burk plot of 1/V0 versus 1/[S0] (28).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Design of oligonucleotide–peptide conjugates and their RNase activity
In the present work we investigated RNase activity of the conjugates of peptide [LeuArg]4-Gly-NH2 with different short oligonucleotides in experiments with RNA which did not contain sequences complementary to the oligonucleotide domains of the conjugates longer than dinucleotides: the synthetic decaribonucleotide 5'-UUCAUGUAAA-3', the in vitro transcripts of human tRNA3Lys and HIV-1 RNA (123–218 nt), comprising the primer binding site (PBS). Figure 1 shows the oligonucleotide–peptide conjugates pep-16, pep-7 and pep-4 designed for the present study. The conjugates were synthesized as described (21). In the conjugate pep-16 a diethyleneglycol residue was introduced between the 5'-phosphate and the N-terminal leucine in order to provide additional flexibility to the peptide. The conjugates pep-4, pep-7 and pep-16 were homogeneous and did not contain non-conjugated peptide or oligonucleotide.



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  		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.

 
Ribonuclease activity and sequence specificity of the conjugates were assayed in 50 mM Tris–HCl buffer pH 7.0 containing 0.2 M KCl, 1 mM EDTA and physiological temperature 37°C (referred to subsequently as standard conditions). Mg2+ was omitted from the reaction mixtures because natural enzymes RNase A and RNase T1 do not require Mg2+ for their activity. The reaction mixtures contained 5'-32P-labeled decaribonucleotide (1 x 10–6 M) (pep-4, pep-7) or 3'-32P-labeled tRNA3Lys (1 x 10–7 M) (pep-4, pep-7) or a 96 nt long fragment of HIV-1 RNA (1 x 10–7 M) (pep-4, pep-7, pep-16) supplemented with total tRNA E.coli (100 µg/ml) as RNA carrier (see Materials and Methods).

Cleavage of decaribonucleotide by conjugates pep-4 and pep-7 (Fig. 2)
Conjugates pep-4 and pep-7 cleave RNA in a non-random manner: the major cuts occur at phosphodiester bonds C3–A4, G6–U7 and U7–A8. Conjugate pep-4 cleaves the decaribonucleotide at C3–A4 and U7–A8 motifs more efficiently than at G6–U7. Conjugate pep-7 cleaves phosphodiester bond G6–U7 more efficiently than the bonds in sequences C3–A4 and U7–A8; with the increase of pep-7 concentration (from 1 to 5 µM) the ratio of cleavage extents at C–A, G–U and U–A sites is kept constant (data not shown).

The kinetics presented in Figure 2 indicate that cleavages at each of the sites C3–A4, U7–A8 and G6–U7 occur as parallel independent reactions. At longer incubation times formation of the shortest cleavage product bearing the 32P label and corresponding to cleavage at the C3–A4 bond is observed, which suggests complete cleavage at each sensitive phosphodiester bond (primary data not shown). The decaribonucleotide was not expected to form any stable secondary structure under the experimental conditions; therefore differences in the cleavage specificity displayed by pep-4 and pep-7 can be attributed to the features of the conjugate itself.



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  		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.

 
Cleavage of HIV-1 RNA by conjugates pep-4, pep-7 and pep-16
Cleavage specificity of the conjugates was also investigated in experiments with the HIV-1 RNA fragment (Fig. 3A). Conjugates pep-4 and pep-16 (10 µM) cleaved RNA at phosphodiester bonds located in Pyr–A and G–X sequences in hairpin 1, in the junction between stem 2 and hairpin 3, in the loops of hairpins 3, 4 and 5, and in the junction between hairpin 5 and stem 2 (Fig. 3B, triangles and circles for pep-4 and pep-16, respectively). Conjugate pep-7 under the same conditions cleaved the RNA at these sites except for C91–A92 and additionally phosphodiester bonds within 8 G–X sequences (Fig. 3A, squares). Among the 21 phosphodiester bonds cleaved by conjugate pep-7, seven sites are located in the C–A and U–A sequences and the other in the G–X sequences. Very strong cleavage at phosphodiester bond G1–U2 is observed in the case of both RNase T1 and pep-7 (Fig. 3A, lanes T1 and pep-7); the G1–U2 site is adjacent to the 5'-32P label and turns out to be very sensitive towards cleavage under various conditions.




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  		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.

 
The electrophoretic mobility of fragments formed upon RNA cleavage with the conjugates corresponds to mobility of fragments formed by RNA cleavage with RNase T1 and imidazole (Figs 2 and 3A), which suggests similar product formation upon cleavage with the synthetic compounds and RNase T1. It is known that under conditions of partial cleavage RNase T1 produces fragments bearing 5'-hydroxyl and 2',3'-cyclophosphate.

Prolonged incubation (1–24 h) results in a higher yield of the cleavage products, but the relative intensities of cleavage at G–X and Pyr–A sequences remained constant (see Supplementary Table 1). The extent of RNA cleavage with conjugate pep-7 at G–X sequences is 2.3 times higher than at Pyr–A sequences. For conjugates pep-4 and pep-16, the extent of RNA cleavage at Pyr–A sequences is 2.6 times higher than at G–X motifs. Among the tested compounds conjugate pep-7 displayed the highest RNase activity (68.5% of total cleavage extent in 24 h), whereas pep-16 displayed somewhat lower (55%) and conjugate pep-4 displayed 2-fold lower (34%) activity under the same conditions.

Similar results were obtained in experiments with tRNA3Lys (Fig. 4). Conjugate pep-4 cleaves tRNA3Lys preferentially at C–A and U–A sequences in single-stranded regions (loops, junction), whereas pep-7 additionally cleaves the major part of the G–X motifs presented in this tRNA.




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  		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.

 
Analysis of structural specificity of RNA cleavage
In the case of HIV-1 RNA, all Pyr–A sequences located within the loops and junctions and one Pyr–A sequence (U11–A12) within the stem of hairpin 1 are cleaved by the conjugates (Fig. 3B and Supplementary Table 2). Conjugate pep-7 cleaves all G–X phosphodiester bonds in loops. Among 14 G–X sequences located in stem regions of HIV-1 RNA, six are also sensitive to cleavage with pep-7. Phosphodiester bonds G5 and G13 are located within stem 1, which is known to possess an unstable secondary structure (29), so that in the absence of magnesium ions these sites can be considered as single-stranded. Phosphodiester bonds after G34 and G38 in stem 3 flanked by AU and UA base pairs, and G37 located near the bulge region are also cleaved by pep-7, although with decreased efficiency. An abnormally strong cut is observed after G87 in stem 5. Probably the high sensitivity of this site (G87–U88) is explained by simultaneous cleavage of adjacent U88–A89 phosphodiester bond located in the junction, which leads to unfolding of the adjacent G87·C74 base pair. It should be noted that no cleavage occurs within highly stable stem 2 and 4, and part of stem 3 adjacent to the junction containing several neighboring GC pairs.

Similar sequence and structure specificity was observed for conjugates pep-4 and pep-7 in experiments with tRNA3Lys (Fig. 4). Conjugates pep-4 and pep-7 cleave three of four Pyr–A sequences within loops and the C28–A29 phosphodiester bond in the anticodon stem which is known to be cleaved spontaneously under various conditions (17,29). Conjugate pep-4 did not cleave tRNA3Lys at G–X sequences. Conjugate pep-7 cleaves all G–X sequences located within loops and nine of 13 sites within stems (Fig. 4B and Supplementary Table 2). For the conjugate pep-7 increasing of cleavages within the acceptor stem (positions 65–70) is observed with increasing cleavage at the T loop; thus cleavages within the stem can be considered as secondary cuts appearing upon changing of tRNA structure after cleavage within the loop. Weak cleavages within stems are secondary cuts and can be explained by the fragile structure of the transcript in the absence of magnesium ions (30). These data clearly show that the conjugates are ‘single-stranded’ RNases capable of cleaving RNA within loops and junctions: the conjugate pep-4 and pep-16 are Pyr–A>>G–X RNases, and pep-7 displays pronounced G–X specificity.

Activity controls
A series of control experiments with oligonucleotides, peptide and conjugates were performed (Fig. 5A). Neither the oligonucleotides nor the peptide nor equimolar mixtures of oligonucleotides with the peptide display any RNase activity under the assay conditions.



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  		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.

 
To exclude the possibility of RNA hydrolysis by contamination of conjugate preparations with traces of RNases, the experiments with oligonucleotide–peptide conjugates treated with DEPC were performed. DEPC is known to react with imidazole residues in catalytic centers of RNases, thus resulting in inactivation of enzymes. Conjugate pep-4 was treated with 0.1% DEPC at 37°C for 1 h and then incubated with 3'-[32P]tRNA3Lys. A similar experiment was carried out with RNase A. In parallel, tRNA3Lys was incubated in the presence of conjugate pep-4 and RNase A without DEPC treatment (Fig. 5B). Treatment of RNase A with DEPC completely inhibited the enzyme (lanes RNase A, +DEPC) whereas the activity of conjugate pep-4 was not affected (lanes pep-4, +DEPC).

Kinetic parameters of RNA cleavage by conjugates pep-4, pep-7 and pep-16
Kinetic parameters of RNA cleavage by the conjugates were measured at a conjugate concentration much higher (10 µM) than the concentration of RNA substrate (0.1 µM) corresponding to the conditions of a single-turnover reaction ([pep-ON]>>[S0], where ON stands for oligodeoxyribonucleotide). We studied the cleavage activity, the dependence of RNA cleavage on conjugate concentration and the evaluated effective rate constants of RNA cleavage at individual phosphodiester bond. The conditions of multiple-turnover reaction were used to determine the catalytic activity (reaction turnover) and affinity of the conjugates to RNA (Michaelis constant).

Concentration dependence of RNA cleavage by the conjugates
Figure 6 displays the dependences of RNA cleavage on the concentration of the conjugates. In these experiments 5'-[32P]RNA HIV-1 was incubated in the presence of one of the conjugates at concentrations ranging from 1 to 100 µM under standard conditions for 8 h. It is seen that the curves for conjugates pep-4, pep-7 and pep-16 are similar: they reach a plateau at a conjugate concentration of 3 x 10–5 M and differ only in plateau values. This curve shape (curve with saturation) characterizes reactions proceeding via complex formation (28). However, no stable complementary complexes between RNA and the conjugates were detected by gel-shift analysis and enzymatic probing under assay conditions (see Supplementary Fig. 1). The similarity of the curves, which reach a plateau at the same conjugate concentration, suggests that the reactions occur via formation of complexes of similar type. The nature of these complexes can be rationalized assuming that the interaction occurs due to the peptide, which was identical in all the conjugates.



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  		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.

 
Affinity of oligonucleotide–peptide conjugates to RNA
Cleavage of phosphodiester bonds in RNA by oligonucleotide–peptide conjugates occurs via formation of specific transient complexes. We estimated the Km value using RNA HIV-1 as a substrate and the reaction turnover using tRNA3Lys. 5'-[32P]RNA HIV-1 at concentrations of 1–10 µM, which correspond to concentrations of phosphate groups of 20–200 µM, was incubated (under standard conditions) in the presence of 10 µM conjugate pep-4 (multiple-turnover reaction conditions). Km was calculated as described in Materials and Methods. The Michaelis constant Km for conjugate pep-4 was estimated to be (1.5 ± 0.7) x 10–4 M. It is interesting to note that the Km for RNase T1 in the case of cleavage of minimal substrate GpC, to which the enzyme displays the lowest affinity, is (1.31 ± 0.07) x 10–4 M (31).

The reaction turnover of conjugate pep-4 was evaluated using tRNA3Lys as substrate. One molecule of the conjugate can cleave up to 175 phosphodiester bonds in 24 h. Thus the oligonucleotide–peptide conjugates are true catalysts of RNA cleavage and exhibit some features comparable to those of natural RNases: specific complex formation, reaction turnover and cleavage products.

Effective rate constants of RNA cleavage by the conjugates
Kinetic parameters of RNA cleavage by conjugates pep-4, pep-7 and pep-16 were determined using conditions of single-turnover reactions. The reaction was interpreted using a kinetic scheme for a consecutive parallel reaction (see Materials and Methods) in which formation of individual cleavage products is assumed to occur as a result of parallel and independent reactions (32).

Values of keff for the bonds C3–A4 and U7–A8 are approximately equal for both conjugates in the case of decaribonucleotide. The value of keff (G6–U7) for conjugate pep-7 is 10-fold higher than that for conjugate pep-4 (see Supplementary Table 3).

In HIV-1 RNA, conjugates pep-4 and pep-16 cleave 14 phosphodiester bonds and conjugate pep-7 cleaves 21. Since conjugates pep-16 and pep-7 display similar total cleavage activities but different sequence specificities, the kinetic parameters were derived for these two conjugates (Table 1).


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  		Table 1. Effective rate constants of the cleavage of phosphodiester bonds in fragments of HIV-1 RNA by conjugates pep-7 and pep-16
 
Comparison of effective rate constants reveals that conjugate pep-16 cleaves C–A and U–A motifs 1.5–3 times faster than conjugate pep-7 (Table 1). For both conjugates cleavages of phosphodiester bonds at G–X motifs are characterized by similar values of reaction rate constants except for phosphodiester bonds G5–G6 and G6–G7. Conjugate pep-16 seems to have a low ability to cleave G–X phosphodiester bonds; eight G–X sequences (among 24 existing in HIV-1 RNA) cleaved by this conjugate are the most sensitive to cleavage by RNase T1 (29).

The efficiency (keff) of RNA cleavage at G–X motifs by conjugate pep-7 strongly depends on the phosphodiester bond location: single strand, loop, junction or bulge. G–X sequences located in single-stranded regions are characterized by similar keff values (0.6 x 10–6 s–1), except for G1–U2 which is characterized by abnormal efficiency (keff = 3.98 x 10–6 s–1); G–X sequences in double-stranded regions are cleaved at least 1.5 times slower (0.38 x 10–6 s–1). The phosphodiester bonds G5–G6 and G6–U7 displayed the highest sensitivity (1.1–1.2) x 10–6 s–1, thus indicating the lability of RNA structure in this region. Thus differences in cleavage rates of G–X phosphodiester bonds correlate with their availability towards cleavage but not with the preferential cleavage of certain types of phosphodiester bonds (G–U, G–A, G–G etc.) by the conjugate pep-7.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY MATERIAL
 REFERENCES
 
The synthetic conjugates of short oligonucleotides and peptide [ArgLeu]4-Gly-NH2 cleave phosphodiester bonds in RNA, displaying no apparent contribution of the oligonucleotide fragment to the sequence specificity of the cleavage. However, the oligonucleotide domain is essential for activity of the compounds: the peptide alone does not cleave RNA under the same conditions. Conjugate pep-7 displayed pronounced G–X specificity, whereas the other conjugates cleaved RNA preferentially at Pyr–A positions. Cleavage at Pyr–A sequences is a typical specificity of synthetic RNases; however, cleavage at G–X motifs is observed for the first time.

Conjugates pep-4, pep-7 and pep-16 built from oligonucleotides TCAA, CCAAACA and CCCTGGACCCTCA GAT and an identical peptide display different cleavage specificity and activity. Previously efficient cleavages of RNA within non-complementary sequences by oligonucleotide–peptide conjugates targeting specific RNA regions were observed (15,16). It could be supposed that in the conjugates tested in this work oligonucleotides provide for binding with RNA; however, a search for possible interactions did not reveal any complementarity. Additionally no changes of reactivities of phosphodiester bonds or RNA structure were observed upon probing the RNA structure in the presence of the conjugates. Therefore an oligonucleotide linked to a peptide via a covalent bond does not support binding with RNA by base-pairing but could change peptide conformation and properties.

Affinity of conjugates to RNA is characterized by a Michaelis constant Km = 1.5x10–4 M. Therefore the cleavage reaction proceeds via formation of some specific complex between the RNA substrate and the conjugate. The dependences of RNA cleavage on conjugate concentration (Fig. 6) provide evidence for similar affinities of conjugates towards RNA substrate. The peptide [LeuArg]4-Gly-NH2 is the only common element of the conjugates: thus it seems reasonable to assume that the peptide is responsible for affinity of conjugates to RNA. Arginine-rich peptide motifs or a single arginine are common elements of different RNA and DNA binding proteins (33–35) and display high affinity to guanine residues, forming a network of hydrogen bonds with N7 and O6 atoms of guanine (33–36). Therefore these properties can explain the G–X specificity of RNA cleavage by the conjugate pep-7, but not the poor G–X specificity displayed by pep-4 and pep-16. Probably, the oligonucleotide of conjugate pep-7 affects the structure of the peptide in such a way that an optimal interaction of the guanidinium group of arginine with the guanine nucleobase can take place. Presently we can only speculate on which factors determine the specificity of RNA cleavage at G–X versus Pyr–A sequences.

Our results indicate that within the conjugate both peptide and oligonucleotide function unusually. We have to postulate that the catalytic activity and affinity of our conjugates are provided by the peptide moiety and that the covalently attached oligodeoxyribonucleotides affect the peptide structure and modulate the cleavage specificity. Thus manipulation with the sequence and/or length of the oligonucleotide will probably lead to artificial RNases of high activity and different sequence specificities.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at NAR Online.


   ACKNOWLEDGEMENTS
 
This work was supported by Wellcome Trust grant no. 063630, RAS program ‘Physico-Chemical Biology’ and ‘Basic Research to Medicine’, RFBR grants no. 02–04–48664 and no. 03–04–06238, CRDF NO-008-X1, BRHE no. Y1-B-08–08, a grant from the Ministry of Education of the Russian Federation, President program SS–1384.2003.4, grant SB RAS for Interdisciplinary Investigations no. 50 and a grant in support of young scientists.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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