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© 1996 Oxford University Press 655-661

Footnote

Hybridization properties of oligodeoxynucleotide pairs bridged by polyarginine peptides

Hybridization properties of oligodeoxynucleotide pairs bridged by polyarginine peptides Ziping Wei 1,2 , Ching-Hsuan Tung 1 , Tianmin Zhu 1,2 , Walter A. Dickerhof 2 , Kenneth J. Breslauer 2 , Denise E. Georgopoulos 3 , Michael J. Leibowitz 3 and Stanley Stein 1,2,3, *

1 Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway , NJ 08854, USA , 2 Department of Chemistry, Rutgers University, Piscataway , NJ 08855, USA , 3 Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway , NJ 08854, USA

Received October 30, 1995; Revised and Accepted January 4, 1996

ABSTRACT

The hybridization properties of a series of probes, based on two 9mer oligodeoxynucleotides (designated as I and II) having an appended oligoarginine chain (R n ) to produce peptide-oligonucleotide conjugates or peptide-bridged oligonucleotide pairs (e.g. R n -I or II-R n -I), were investigated. For the double-linked probes, we found that the peptide bridge induces the two 9mers to bind complementary single-stranded DNA or RNA targets with substantially enhanced thermal stability. The resulting hybrid with complementary DNA was found to assume a 1:1 complex in the B conformation as judged by UV mixing curves and CD spectroscopy. Complexes of single or double-linked probes with complementary RNA exhibited sensitivity to RNase H digestion. The influence of the identity and chirality of the repeating unit in the bridge, the length of the bridge, the gap size and the salt concentration on the hybridization properties of this new class of oligonucleotide probes was also studied. Our data reveal that these compounds exhibit properties that should prove useful in the development of antisense strategies.

INTRODUCTION

There have been numerous reports concerning the modification of oligonucleotides for improving the inherent property of binding to a complementary strand. For example, oligonucleotide-acridine conjugates were shown to increase the binding affinity of an oligonucleotide to its complementary single-stranded or double-stranded target ( 1 , 2 ). Cationic polylysine was also conjugated to oligonucleotides to improve binding affinity, as well as cellular uptake and nuclease resistance ( 3 ). A conjugate consisting of a pair of tethered oligonucleotide probes, complementary to two noncontiguous sites on an RNA, has been demonstrated to bind cooperatively and monomerically to the RNA target ( 4 ). One tether compound contained negatively charged phosphodiester units that might have a repulsive effect on the target. A neutral poly(ethylene glycol) tether was used later to minimize electrostatic effects ( 5 ). In these cases, no interaction between the bridge and nucleic acid target was reported. Oligonucleotides linked by a terephthalamide group were found to exhibit marked enhancement in stability of duplex or triplex formation ( 6 ). DNA triplex formation was also enhanced by bridged oligonucleotides with linker groups containing oxyphosphinicooxy-l,3-propanediol ( 7 ) or hexaethylene glycol ( 8 ).

We reported the synthesis of polyarginine-oligonucleotide conjugates and polyarginine-bridged oligonucleotide pairs ( 9 ). Two 9mers complementary to neighboring but noncontiguous regions of target nucleic acids were covalently linked by a series of polyarginine peptides. Under physiological conditions, the polyarginine peptide has positive charges on its side chains, and the nucleic acid has negative charges on the phosphate groups in the backbone. The peptide bridge can interact with nucleic acid targets through electrostatic effects and/or hydrogen bonds, potentially increasing the binding affinity. Unlike single-linked peptide oligonucleotide conjugates, the conformations of the polycationic peptides are more restricted in the peptide-bridged oligonucleotide pairs, possibly increasing the opportunity for interaction with the desired region on the target. To understand better the behavior of this class of compounds, spectroscopic measurements were used to study hybridization to single-stranded DNA, and an in vitro RNase H footprinting assay was used to evaluate hybridization to single-stranded RNA.

MATERIALS AND METHODS

Preparation of oligonucleotide conjugates and bridged pairs

Oligonucleotides were synthesized by phosphoramidite methodology using a Model 380B DNA Synthesizer (Applied Biosystems, Foster City, CA). All oligonucleotides were purified by a Trityl-on and Trityl-off method on a Hamilton PRP- 1 column using an acetonitrile gradient. The oligonucleotide derivatives are listed in Table 1 . Oligonucleotide conjugates and pairs bridged by polyarginine peptides were made as described ( 9 ).

Table 1 . The structures and abbreviations of single-linked oligonucleotide conjugates and bridged oligonucleotide pairs Abbreviation

X =

Cys-(L-Arg) 3 -Cys

R 3 - I

Cys-(L-Arg) 7 -Cys

R 7 - I

Cys-(L-His) 3 -Cys

H 3 - I

Y =

Cys-(L-Arg) 3 -Cys

II -R 3 - I

Cys-(L-Arg) 5 -Cys

II -R 5 - I

Cys-(L-Arg) 7 -Cys

II -R 7 - I

Cys-L-Arg-D-Arg-L-Arg-Cys

II D/L -R 3 - I

Cys-(L-Arg-D-Arg) 2 -L-Arg-Cys

II D/L -R 5 - I

Cys-(L-His) 3 -Cys

II -H 3 - I

(OCH 2 CH 2 CH 2 OPO 2 ) 3 (no amino linker)

II -L 3 - I

X --5'-TAA TGT GAT-3' 9mer- I 5'-GAC TAG GTG-3'-- Y --5'-TAA TGT GAT-3' 9-mer- I I 9-mer- I

Polyhistidine conjugates, H 3 - I , II -H 3 - I , as well as I -R 3 - II were made similarly. II -L 3 - I , which has three oxyphosphinicooxy-1, 3-propanediol linkers (defined as L , Table 1 ) between the two 9mers (without amino linker), was machine synthesized using spacer phosphoramidites (Glen Research, Sterling, VA). The extinction coefficients of all single-stranded oligonucleotides and oligonucleotide conjugates or pairs at 260 nm, 25oC and neutral pH were calculated from dimer and monomer values by using the nearest-neighbor method ( 10 , 11 ).

Mixing curves

Stock solutions of 3.3 [mu]M 9mer- I plus 9mer- II (i.e. non-bridged), II -R 3 - I , and the DNA target T 24 (Table 2 ) were prepared in 10 mM sodium phosphate (pH 7.0), 0.1 M NaCl, 0.1 mM EDTA. The mixing curves of duplex formation were measured essentially as described ( 12 ). The 9mer- I plus 9mer- II , or II -R 3 - I stock solution was added to a T 24 solution to initiate duplex formation. After addition, the cuvettes were equilibrated at 15oC for 20 min, and then absorption readings at 260 nm were recorded. In another set of data, a T 24 solution was added to the 9mer- I plus 9mer- II , or II -R 3 - I stock solutions.

Melting temperature studies

The melting studies were done on a computer-interfaced Perkin Elmer spectrophotometer Coleman 575 (Norwalk, CT) at 260 nm. The samples were 3.3 [mu]M of each oligomer in 1 ml buffer composed of 10 mM sodium phosphate (pH 7.0), 0.1 M NaCl and 0.1 mM EDTA, unless otherwise specified. After the samples were annealed by cooling from 90 to 5oC at 0.5oC/min, the T m curves were measured using a temperature gradient from 5 to 90oC at 0.5oC/min. The cuvettes were kept under dry nitrogen gas to prevent water condensation at low temperature. The sequences of the DNA targets used in the studies are given in Table 2 . All T m data are estimated to be accurate +-0.5oC.

Circular dichroism (CD) spectroscopy

CD spectra were recorded on a Cary 60 spectropolarimeter equipped with a Cary 6001 CD accessory and thermostatically controlled cell holder (AVIV Associates, Lakewood, NJ). The samples were 2.2 [mu]M of each oligomer in the same buffer as used for the melting studies. Each sample was kept for 15 min at each specified temperature (5, 25, 45 or 65oC), and then scanned from 350 to 200 nm, with a 5 s averaging time at each wavelength. The spectra were subtracted from the spectrum of the buffer alone at the same temperature.


Figure 1 . Mixing curves for formation of complexes. ([circle]) Mixing of 9mer- I plus 9mer- II with T 24. (-) Mixing of II -R 3 - I with T 24.

RNase H assay

A fragment (bases 1-152 from the 5' end) of m transcript of the M 1 dsRNA genomic segment of killer virus of yeast Saccharomyces cerevisiae was generated as before ( 13 ) with the following modifications. Instead of primer 2375 used for generating full length M 1 cDNA by PCR amplification, a 21mer (5'-GCG CTT CAC GAG GTA GTA ATG-3'), complementary to base 131-152 of M 1 cDNA, was now used in PCR to generate a transcription template of shorter length. The in vitro transcript generated with bacteriophage SP6 RNA polymerase was labeled with [[gamma]- 33 P]ATP (NEN Dupont, Boston, MA) instead of [[gamma]- 32 P]ATP using cloned T4 polynucleotide kinase (Pharmacia, Piscataway, NJ) after alkaline phosphatase dephosphorylation. In the RNase H assay, 1 nM [5'- 33 P]mRNA (5000 c.p.m.) was mixed with 1 [mu]M of an oligonucleotide or a bridged oligonucleotide pair, as specified, in 20 mM Tris-HCl (pH 7.5), 0.1 M KCl, 10 mM MgC1 2 , 0.1 mM DTT and 5% (w/v) sucrose. The reaction mixtures were incubated at 65oC for 5 min and then at 37oC for 5 min to facilitate hybridization. Escherichia coli RNase H (0.5 U) was then added (final volume, 10 [mu]l) and incubations were continued for 2 h. After incubation, 10 [mu]l loading buffer (8 M urea, 5 mM EDTA, 0.025% each of xylene cyanol and bromophenol blue) was added. A RNase T 1 ladder of the same transcript was generated to indicate the size of each fragment ( 14 ). Reactions were analyzed by electrophoresis on 1.2 mm-thick 15% polyacrylamide-8 M urea electrophoresis gels. The gels were dried at 80oC under vacuum after soaking in 5% acetic acid, 15% methanol and 5% glycerol for 1 h, and exposed to X-OMAT film (Kodak, Rochester, NY) at -70oC.

RESULTS

Mixing curves

Oligonucleotide sequences used in the preparation of peptide-oligonucleotide conjugates were complementary to two noncontiguous sequences on a single-stranded 24mer DNA target ( T 24). The stoichiometry of duplex formation was studied by the method of continuous fractions ( 12 ). The comparison between untethered 9mer- I plus 9mer- II / T 24 di-duplex and tethered II -R 3 - I / T 24 duplex was made; inflection points at mole fractions of 0.48 and 0.47 respectively, were obtained (Fig. 1 ). The mixing curves proved that two 9mers, linked by a peptide bridge, are able to bind cooperatively to the target by Watson-Crick duplex formation. According to the absorbance at the inflection points, the extinction coefficient of 9mer- I plus 9mer- II / T 24 di-duplex and II -R 3 - I / T 24 duplex can be estimated to be 6.99 M -1 cm -l and 6.73 M -1 cm -l respectively.

Effect of peptides on hybridization of single-linked conjugates

The polyarginine peptides of single-linked conjugates were found to provide a greater increase in hybridization thermal stability in comparison with polyhistidine (Fig. 2 ) or poly([delta]-ornithine) ( 15 ) peptides. Three arginine residues on 9mer- I increased the T m of 9mer- I from 28.0 to 34.0oC, and seven arginine residues from 28.0 to 41.0oC. This gave an average increase of 2.0oC in T m per arginine residue in the duplex of 9mer- I and T 24. Poly([delta]-ornithine) peptides were linked to a 12mer oligonucleotide and reported to have an average increase of 0.5oC in T m per ornithine residue with the complementary strand ( 15 ). Although different DNA sequences were used in this comparison between polyarginine and polyornithine peptides, the melting temperature range (within 28-41oC) was about the same. The polyhistidine peptide of H 3 - I had an effect similar to that of polyornithine.


Figure 2 . Melting curves of single-linked peptide-9mer- I conjugates compared with 9mer- I with T 24 target. T m values are: 1: 9mer- I , 28.0oC; 2: H 3 - I , 30.0oC; 3: R 3 - I , 34.0oC; 4: R 7 - I , 41.0oC, as numbered from left to right.

Effect of peptides on hybridization of bridged oligonucleotide pairs

The hybridization of the peptide-bridged oligonucleotide pairs and the free oligonucleotides to T 24 was determined by melting curves (Fig. 3 ). The 9mer- I plus 9mer- II had two separate binding sites on T 24, and gave two thermal transitions on melting curve 1. The thermal stability of a DNA duplex depends on its base sequence ( 16 ); 9mer- II had 55% GC content with both 5' and 3' end terminated by GC, whereas 9mer- I only contained 22% GC content. After 9mer- I was conjugated to a peptide (as R 3 - I ), the positively charged peptide increased the T m of 9mer- I with T 24, and, therefore, one broad thermal transition was observed (curve 2). In both cases, cooperative binding of 9mer- I and 9mer- II was not apparent.


Figure 3 . Melting curves of peptide-bridged oligonucleotide pairs compared with 9mer- I or R 3 - I plus 9mer- II with T 24 target. 1: 9mer- I plus 9mer- II ; 2: R 3 - I plus 9mer- II ; 3: II -R 3 - I , 48.5oC; 4: II -R 5 - I , 49.5oC; 5. II -R 7 - I , 51.0oC, as numbered from left to right at the level of 1.52 absorbance units.

The peptide-bridged oligonucleotide pairs, 9mer- II -peptide-9mer- I , had a much higher T m (Fig. 3 , curves 3-5). The transition curves were also considerably steeper. These results indicate that the peptide bridge allows the two 9mers to bind to the complementary strand in a cooperative manner. Melting temperatures increased with the addition of positive charges on the peptide bridges, H-Cys-(Arg) n -Cys-NH 2 ( n = 3, 5, 7). The II -R 5 - I / T 24 duplex had a T m 1oC higher than the II -R 3 - I / T 24 duplex. The II -R 7 - I / T 24 duplex had an even higher T m , which was 2.5oC higher than the II -R 3 - I / T 24 duplex. Thus, the peptide bridge increases the duplex thermal stability through interaction with the complementary target oligonucleotide. At physiological temperature (37oC), neither 9mer would be expected to hybridize to the target, whereas strong hybridization would occur when these two 9mers are bridged by polyarginine.

The importance of orientation of the guanidine groups in the peptide was investigated using both D- and L-arginine residues (Table 1 ). II D/L -R 3 - I and II D/L -R 5 - I contained alternating residues of D- and L-arginines. They produced the same melting temperatures as did II -R 3 - I , II -R 5 - I respectively (Table 3 ). The bridged oligonucleotide pair, I -R 3 - II , in which the positions of the two 9mers were reversed compared with that of II -R 3 - I , showed the same T m as did II -R 3 - I (Table 3 ).

Furthermore, the polyarginine bridge was compared with a polyhistidine and a non-peptide (oxyphosphinicooxy-l,3-propanediol) bridge (Table 3 ). The order of duplex thermal stability was II -R 3 - I > II -H 3 - I > II -L 3 - I . Compared with II -L 3 - I , the arginine peptide bridge showed an average of 1.0oC T m increase per arginine residue in the bridged oligonucleotide pair in duplexes with T 24. This value is lower than that of single-linkage polyarginine-oligonucleotide conjugates (2.0oC per residue), perhaps because the interaction of arginine was restricted to the single-stranded gap region on the targets.

Table 2 . Oligonucleotide targets used for physicochemical studies
Sequences (5' -> 3')

Name

Gap size

ATC ACA TTA CAC CTA GTC

T 18

0

ATC ACA TTA CTA CAC CTA GTC GTA

T 24

3

ATC ACA TTA CTTA CAC CTA GTC GTA

T 25

4

ATC ACA TTA CTTTTA CAC CTA GTC GTA

T 27

6

The boldfaced regions represent the binding sites of 9mer- I and 9mer- II . The gap size refers to the number of nucleotides between the two 9mer binding sites on the oligonucleotide targets.

Effect of gap size on hybridization

The hybridization properties of peptide-bridged oligonucleotide pairs were also investigated by varying the number of nucleotides between the two 9mer binding sites on the oligonucleotide target (Table 2 ). To minimize the influence of the sequence specificity, the sequences of the two 9mer binding sites were kept the same for all the targets. In the above studies (with T 24) the gap size (i.e. number of unhybridized bases between the complementary sequences on the target strand) was 3. Gap sizes 4 and 6 were generated by adding thymidine nucleotides to T 24 in the gap region, whereas removal of the 5'-CTA-3' sequence generated a target with a gap of 0 (Table 2 ).

Table 3 . Melting temperatures of different oligonucleotide pairs with T 24 target
Oligonucleotide pairs

T m (oC)

II -L 3 - I

45.0

II -H 3 - I

47.0

II -R 3 - I

48.5

II D/L -R 3 - I

48.5

I -R 3 - II

49.0

II -R 5 - I

50.0

II D/L -R 5 - I

50.0

II -R 7 - I

51.0

The sizes of the peptide and the gap are all important for hybridization of an oligonucleotide pair. For a particular peptide bridge, a small gap would make the peptide bulge out, not fitting well in the gap region, but too large a gap would make the target strand not interact well with the peptide. In Figure 4 , melting temperatures determined for gap sizes 0, 3, 4 and 6 on the target are presented. The T m was almost the same with gap sizes 3 and 4, but the T m decreased by 2oC with gap size 0, and decreased by 1-2oC with gap size 6. This means that proper spacing on the target helps the hybridization, and a gap that is too small or too large decreases the duplex thermal stability. In a comparison of three peptide-bridged oligonucleotide pairs tested, there was a tendency that conjugates with longer peptide bridges had less decrease in T m with increase of the gap size. The II -R 7 - I oligonucleotide pair had stronger binding for all complementary strands tested than did the II -R 3 - I and II -R 5 - I conjugates. It may be concluded that interactions of guanidino groups at both the single-stranded gap region and at double-stranded regions increase thermal stability. A perfectly matched C 18mer/ T 18 duplex ( C 18mer has a sequence of 5'-GAC TAG GTG TAA TGT GAT-3') under the same conditions had a T m of 57oC. For the bridged oligonucleotide pair/DNA target duplex, the reason for the reduction in duplex thermal stability is probably lack of the nearest-neighbor interaction for the last nucleotides at the 5' end of 9mer- I and 3' end of 9mer- II ( 16 ).


Figure 4 . Effects of peptide and gap size on T m . Duplex of (-) II -R 3 - I , ([circle]) II -R 5 - I , ([squf]) II -R 7 - I , with T 18 (gap = 0), T 24 (gap = 3), T 25 (gap = 4) or T 27 (gap = 6) target is shown.

Effect of salt on hybridization

To determine the influence of charge interactions of bridged oligonucleotide pairs on duplex formation, the salt dependence of hybridization was evaluated (Fig. 5 ). The melting curves for II -R 3 - I were measured in 10 mM sodium phosphate and 0.1 mM EDTA buffer, pH 7.0, at three different salt concentrations (0, 0.1 and 1.0 M sodium chloride). A large increase in T m (13oC) was observed when salt concentration was changed from 0 to 0.1 M NaCl. There was only an 8oC increase in T m from 0.1 to 1.0 M NaCl concentration. The gap sizes did not influence this T m change. For the perfect match C 18mer/ T 18, the increase was even greater from 41.5oC (no NaCl) to 57oC (0.1 M NaCl), to 68oC (1 M NaCl). That is, the two stage increases were 15.5 and 11oC respectively. Therefore, the polyarginine-bridged oligonucleotide pairs showed less salt dependence than unmodified oligonucleotides. This implied that there was less counter-ion binding in the duplex formed from polyarginine-bridged oligonucleotide pairs, which probably resulted from the presence of cationic arginine residues in the duplex. It has already been shown that cationic peptides are able to stabilize DNA duplex formation similarly to metal ions ( 17 ).


Figure 5 . Effect of salt concentration on T m . Duplex of II -R 3 - I with T 18 (gap = 0), T 24 (gap = 3), T 25 (gap = 4) or T 27 (gap = 6) target is shown. (-) No NaCl, ([circle]) 0.1 M NaCl, ([squf]) 1.0 M NaCl.

CD spectra

Circular dichroism (CD) spectroscopy has been a useful method for distinguishing the structures of peptides and oligonucleotides. This technique was applied to study the conformational changes derived from polyarginine peptides in duplexes (Fig. 6 ). All of the spectra have similar peak positions (276 or 277 nm) and trough positions (247 or 248 nm) within the temperature range of 5-65oC, which indicated B form duplex with all bases perpendicular to the helix axis ( 18 , 19 ). However, CD amplitude and temperature sensitivity varied between duplexes. The R 7 - I / T 24 duplex showed the least sensitivity to temperature change and lower amplitude at low temperatures than shown by 9mer- I / T 24 duplex (Fig. 6 ). II -R 3 - I showed effects similar to R 7 - I , having smaller CD bands and more resistance to temperature change compared to II -L 3 - I . Since the peptide and DNA both participated in the signal changes, the conformation change contributed by the oligonucleotide or peptide alone cannot be determined. The CD spectra of H-Cys(StBu)-(L-Arg) 7 -Cys-NH 2 alone (data not shown) suggested that the polyarginine peptide had a random coil structure in the absence of DNA, which is similar to the conformation of an arginine-rich peptide from the HIV Tat protein ( 20 ).


Figure 6 . CD spectra with T 24 target at 5, 25, 45 and 65oC respectively. The curves may be identified by the order of ellipticity at 276 or 277 nm, which decreases with temperature.

RNase H assay

In systems where antisense activity is mediated by RNase H, which cleaves the RNA strand in an RNA/DNA duplex, the ability of the antisense oligonucleotide/target RNA duplex to act as a substrate is critical. Some modifications of oligonucleotides, such as [alpha]-anomeric ( 21 , 22 ) or methylphosphonate ( 23 , 24 ), may cause loss of RNase H stimulation. In contrast, phosphorothioate oligonucleotides ( 25 ) or terminally modified oligonucleotides, are still able to promote RNase H activity after hybridization to RNA.

An RNase H footprinting assay, in which the direction of cleavage is 3' -> 5', was used to evaluate the hybridization of poly- arginine-bridged oligonucleotide pairs to single-stranded RNA. The T 24 DNA sequence is from a fragment (bases 65-88) of m transcript of killer virus of yeast. Therefore, the same bridged oligonucleotide pairs were used in both the physicochemical studies and in the in vitro bioassay. A fragment (bases 1-152 from the 5' terminus) of m transcript was generated by in vitro transcription. The sequences of 9mer- I and 9mer- II are complementary to bases 65-73 and 77-85 respectively, and there is a gap region of 3 nt between these two binding sites.


Figure 7 . ( A ) Autoradiography of a 15% denaturing polyacrylamide gel. Lane T 1 : RNase T 1 ladder. Lanes 1-5: RNase H digestion of RNA in the presence of 9mer- I plus 9mer- II , C 18-mer, II -R 3 - I , II -R 5 - I and II -R 7 - I respectively. Lanes 6-10: RNA in the presence of 9mer- I plus 9mer- II , C 18-mer, II -R 3 - I , II -R 5 - I and II -R 7 - I (without RNase H) respectively. ( B ) Schematic drawing of RNase H digestion of RNA in the presence of a bridged oligonucleotide pair.

The unmodified oligonucleotides or bridged oligonucleotide pairs were allowed to hybridize to 5'-end-labeled RNA and RNase H was then added. Electrophoresis showed the major product in all cases to be the shortest expected cleavage fragments at position 81 (due to 9mer- II ) and 69 (due to 9mer- I ), corresponding to positions 5 nt from the 5' end of either hybridization region on the RNA target (Fig. 7 ). Detection of mostly the shortest predicted fragments may have been due to the processive exonucleolytic activity of RNase H ( 26 ). The bridged oligonucleotide pairs were compared with the normal (non-bridged) 9mer- I plus 9mer- II , and C 18mer in the assay. In the presence of a mixture of 9mer- I and 9mer- II , most of the cleavage fragments were at position 81 (lane 1), because 9mer- II had a sufficiently higher T m (paired more stably to its target) than did 9mer- I . The C 18mer resulted in cleavage mainly at position 69 (lane 2), due to the increased thermal stability of the duplex relative to that with the two 9mers. Similarly, when the bridged oligonucleotide pairs were hybridized to the end-labeled transcript, most of the cleaved fragments were at position 69. Also, less uncleaved RNA transcript was left after RNase H digestion in the presence of C 18mer or bridged oligo- nucleotide pairs, which further supported the stronger binding affinity of the bridged oligonucleotide pairs to the targets. These results indicated the cooperative binding of 9mer- I and 9mer- II in the bridged oligonucleotide pairs to the RNA target, which resembles that of the C 18mer oligonucleotide. The RNase H assay showed that the oligonucleotide pairs can bind sequence-specifically to the desired region on an RNA target, and can stimulate the RNase H-mediated degradation of RNA. In the control samples not having RNase H added (Fig. 7 ), no degradation was observed.

DISCUSSION

We evaluated the hybridization properties of polyarginine-oligonucleotide conjugates and polyarginine-bridged oligonucleotide pairs to single-stranded DNA and RNA. The bridge concept can function to improve strength of hybridization through electrostatic interactions and/or hydrogen bonding, as proven by spectroscopic measurements. The gap size on the target should be taken into consideration to achieve optimal hybridization. A peptide bridge can bring other properties to oligonucleotides, such as the incorporation of a ribonuclease mimic into the bridge ( 27 ). In this potential application, the oligonucleotide would provide strong affinity and high specificity for the substrate, acting as a binding site, and the peptide mimic acting as an active site would cleave the recognized site of the RNA target and then release the cleaved fragments, which should be bound less tightly than the intact target. Bridged oligonucleotide pairs differ from the single-linked conjugates by limiting the interaction region of the conjugated component.

The single-linked polyarginine and the polyarginine bridge may have special attributes in certain situations. For example, single-stranded RNA sequences might be better targets for these conjugates, since arginine has been demonstrated to have specific recognition for some RNA targets ( 28 , 29 ). The arginine bridge should increase the affinity of tethered antisense oligonucleotides to noncontiguous single-stranded regions of an RNA target ( 4 ). Arginine peptides were reported to associate with double-stranded DNA in its major groove ( 30 ), which has the same orientation as the third strand of Hoogsteen triplex. Bridged oligonucleotide pairs are capable of binding noncontiguous regions of duplex DNA to form a triple helix ( 31 - 33 ), and the polyarginine or another polycation might provide additional binding energy.

ACKNOWLEDGEMENTS

This work was supported by a grant to S. Stein from Gene Shears (Australia) Pty. Ltd, and grant number DAAL03-92-G-0312 to M. J. Leibowitz from the US Army Research Office.

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*To whom correspondence should be addressed at: Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854, USA
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