27

Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 29, Issue Number 1, (2011) ©Adenine Press (2011)

*E-mail: marzen@niboch.nsc.ru

R. SerikovV. PetyukYu. VorobijevV. KovalO. FedorovaV. VlassovM. Zenkova*

Institute of Chemical Biology and

Fundamental Medicine SB RAS,

8, Lavrentiev ave., 630090,

Novosibirsk, Russian Federation

Mechanism of Antisense Oligonucleotide Interaction with Natural RNAs

http://www.jbsdonline.com

Abstract

Oligonucleotides find several numbers of applications: as diagnostic probes, RT and PCR primers and antisense agents due to their ability of forming specific interactions with complementary nucleotide sequences within nucleic acids. These interactions are strongly affected by accessibility of the target sequence in the RNA structure. In the present work the mechanism of invasion of RNA structure by oligonucleotide was investigated using a model system: yeast tRNAPhe and oligonucleotides complementary to the 3′-part of this molecule. Kinetics of interaction of oligonucleotides with in vitro transcript of yeast tRNAPhe was studied using stopped-flow technique with fluorescence quenching detection, 5′-DABCYL labeled oligonucleotide was hybridized with 3′-fluorescein labeled tRNAPhe. The results evi-dence for a four-step invasion process of the oligonucleotide-RNA complex formation. The process is initiated by formation of transition complexes with nucleotides in the T-loop and ACCA sequence. This complex formation is followed by RNA unfolding and formation of an extended heteroduplex with the oligonucleotide via strand displacement process. Com-puter modeling of oligonucleotide-tRNAPhe interaction revealed potential factors that could favor transition complexes formation and confirmed the proposed mechanism, showing the oligonucleotide to be a molecular “wedge”. Our data evidence that oligonucleotide invasion into structured RNA is initiated by loop-single strand interactions, similar to the initial step of the antisense RNA-RNA interactions. The obtained results can be used for choosing effi-cient oligonucleotide probes.

Key words: Oligonucleotide; RNA; RNA-oligonucleotide interaction; Strand displacement; Yeast tRNAPhe; Stopped-flow; FRET; Computer simulation.

Introduction

Binding of antisense nucleic acids with their targets is essential for regulation of a number of biological processes. Specific RNA-RNA interactions play important roles in translation machinery (mRNA-rRNA interactions within Shine-Dalgarno sequence, tRNA-rRNA interactions) (1), dimerization of genomic RNAs of HIV-1 and other retroviruses, priming of HIV-1 RNA reverse transcription with tRNALys3 (2), in antisense technology (3), RNA interference (4), ribozymes and DNazymes (5) etc. These complexes are formed due to specific interactions and are stabilized by Watson-Crick base pairs, as well as by non-canonical interactions found in a number of complexes (6). There are also known mechanisms of interaction of RNA molecules based on recognition of three-dimensional structure, for example, rec-ognition of pre-tRNA by RNase P (7). These RNA-RNA interactions are very effi-cient and highly specific.

Interaction of oligonucleotides with RNA is usually much less efficient due to a number of factors (8). RNA molecules possess complex spatial structure, which

Open Access ArticleThe authors, the publisher, and the right holders grant the right to use, reproduce, and disseminate the work in digital form to all users.

28

Serikov et al.

is stabilized by intramolecular interactions: stacking interactions between loops or hairpins, linkages with ions of bivalent metals and proteins (9). Stable spatial struc-ture of RNA is a major factor reducing efficiency of antisense oligonucleotides and their different analogues, DNazymes, ribozymes and all other short nucleic acids targeted to specific sequences within RNA. Under physiological conditions sequences available for interaction with complementary oligonucleotide within natural RNAs are almost absent, and real targets for oligonucleotides or anti-sense RNA represent elements of three-dimensional folding defined by nucleotide sequences (10). Multiple examples are documented that RNA structure near the target sequence strongly affects the efficiency of RNA/antisense oligonucleotide interaction, for reviews see (11-14). RNA interference machinery was shown to be less sensitive to target RNA structure stability (15, 16), at the moment little is known about miRNA/antagoMIRs interactions (17).

The most common elements of RNA secondary structure are hairpins, which are short self–complementary nucleotide sequences consisting of a stem and a loop. Interactions between them are accompanied by formation of pseudo-knots and define formation of biologically active three-dimensionally RNA structure. Stem structures often serve as recognition sites of regulatory proteins in the course of transcription and translation. Interaction between certain RNA hairpins is a key stage at recognition of RNA targets by antisense RNAs (18), and also in the course of retroviral RNA dimerization, leading to formation of biologically active diploid genome of retroviruses (19). The characteristic feature of all these pro-cesses is a rearrangement of hairpin structures during interaction, the mechanism of interaction of various hairpins is defined by the sizes and sequences of their loops and also by sequences of their stem regions (20). Loop sequences demon-strate significant influence on efficiency of interaction with complementary oli-gonucleotides (14). Folding of RNA molecules even under in vitro conditions is difficult to predict (21) and to investigate by physical methods such as NMR (22) or X-ray diffraction (23, 24). Therefore efficient mRNA targeting is largely a random process, which accounts for lots of experiments to find suitable regions in the target or in which addition of either antisense nucleic acid or siRNA will yield no effect (4).

In this study we investigated the process of oligonucleotide invasion into a folded natural RNA using yeast tRNAPhe as a model. This tRNA has rigid and compact spatial structure additionally stabilized by coordination of two magnesium ions (25). It was previously known that only short single-stranded sequences of this mol-ecule (ACCA-end, anticodon loop, a part of the D-loop) are accessible for binding with oligonucleotides under physiological conditions (26). The ability of antisense oligonucleotides to invade 3′-half of the tRNA structure was reported (27, 28). Several synthetic antisense oligonucleotides were targeted to yeast tRNAPhe, tRNAPhe- oligonucleotide interactions were monitored using standard methods (gel-shift analy-sis, chemical and enzymatic probing). Then, the mechanism of interaction of the most efficient antisense oligonucleotide with the tRNA was studied using stopped-flow method with fluorescence quenching detection. Alternatively, formation of the tRNA-oligonucleotide complex was studied by computer simulation technique. We think it was the exceptionally stable tRNAPhe spatial structure that allowed us to “catch” the transition complexes, which were formed in the course of oligonucleotide–tRNA complex formation, in the case of more flexible spatial structures similar transition complexes may form too fast to be detected.

Materials and Methods

General Methods

Ribonucleotide triphosphates, deoxyribonucleotide triphosphates and dideoxyri-bonucleotide triphosphates were from Sigma. [5′-32P]-pCp and [γ-32P]ATP with

29

Antisense Oligonucleotide Interaction with Natural

RNAs

specific activity of ~4000 Ci/mmol were from Biosan (Russia). T4 RNA ligase and ribonuclease T1 were from Boehringer Mannheim (Germany), ribonuclease ONE was from Promega (USA). T4 polynucleotide kinase was from Fermentas (Lithuania). T7 RNA polymerase was isolated by V. Ankilova (this Institute). Yeast tRNAPhe was a generous gift of Dr. G. Keith from IBMC, Strasbourg, France. Oligodeoxyribonucleotides A – M were synthesized by standard phosphoramidite chemistry and purified by ion-exchange and reverse-phase HPLC.

Synthesis of Yeast tRNAPhe In Vitro Transcript

Transformation of E. coli competent cells, isolation and purification of p67YF0 plasmid DNA were carried out using classical procedure. Synthesis of yeast tRNAPhe in vitro transcript was carried out using linearized p67YF0 plasmid con-taining yeast tRNAPhe sequence under control of T7 RNA polymerase promoter as DNA template (29).

[3′-32P] Labeling of tRNAPhe

Yeast tRNAPhe was labeled at 3′ terminus with [5′-32P]-pCp and T4 RNA ligase (30). 15 µl reaction mixture contained 50 mM HEPES-KOH, pH 7.5, 10 mM MgCl2, 100 µg/ml BSA, 10% DMSO, 0.1 mM ATP; 100 pmol of tRNAPhe, 200 µCi [5′-32P]-pCp and 10 units T4 RNA ligase; reaction was carried out at 4°C overnight. tRNA was purified by 12% polyacrylamide/8 M urea gel electrophore-sis, eluted twice with 300 µl 0.3 M sodium acetate pH 5.5 containing 10% phenol during 12 hours at 4ºC, then extracted by phenol-chloroform and precipitated by ethanol.

[5′-32P] Labeling of tRNAPhe

[5′-32P] labeling of yeast tRNAPhe was carried out employing the modified pro-cedure (31). Prior to labeling tRNA was dephosphorylated by bacterial alkaline phosphatase (BAP). 50 µl reaction mixture containing 40 µg tRNA, 20 µg of oli-gonucleotide M (Table I), 50 mM Tris-HCl pH 9.0, 25% formamide was consecu-tively incubated for 2 min at 60ºC, then for 2 min at 0ºC. Then 3 units BAP were added and the mixture was incubated for 60 min at 37ºC. Dephosphorylated tRNA was isolated by 10% polyacrylamide/8 M urea gel electrophoresis. tRNA was visu-alized in the gel by UV shadow. Dephosphorylated tRNA was eluted from the gel as described above and precipitated with ethanol.

15 µl labeling reaction mixture, containing 50 mM Tris-HCl pH 7.6, 10 mM MgCl2, 1 mM DTT, 0.1 mM spermidin, 4 µg of oligonucleotide A (Table I) containing 5′-phosphate, 4 µg of dephosphorylated tRNA, was incubated for 2 min at 60ºC, then for 2 min at 0ºC, then 5 units of T4 polynucleotide kinase were added and the mixture was incubated for 1 hour at 37ºC. [5′-32P]-labeled tRNA was isolated, eluted from the gel and precipitated as described above.

Hybridization of tRNAPhe with Oligonucleotides

Hybridization of oligonucleotides to tRNAPhe was monitored by gel mobility shift assay (28).

a) RNA Pre-Treatment Prior to hybridization, a mixture of labeled [3′-32P]-tRNA and unlabeled tRNA (final concentration in the mixture, 5 3 10–7 M) was heated at 90°C in 3 µl of water for 1 min, then cooled down and incubated at 20°C or 37°C for 10 min. The 53 hybridization buffer “HB” (13: 50 mM HEPES-KOH, pH 7.5, 200 mM KCl, 0.1 mM EDTA, +/–5 mM MgCl2) was added and the solution was incubated for 20 min at 20°C or 37°C.

30

Serikov et al.

b) Equilibrium Binding ON solution ranging in concentration from 1 3 10–6 M to 1 3 10–3 M was added to the tRNA and the yielded mixture (V 5 10 µl) was incubated at 20 or at 37°C for 4 hours. After incubation, 5 µl of loading buf-fer (20% Ficoll, 0.025% bromophenol blue, 0.025% xylene cyanol) was added to each probe and 8 µl of the resulted mixture was applied onto native 10% PAAG equilibrated at 4°C with 100 mM Tris-borate pH 8.3 as running buffer, and resolved at 10 V/cm for 6 h. To get quantitative data, the gels were dried, radioactive bands were cut out of the gel, and their radioactivity was determined by Cherenkov’s counting.

c) Binding Kinetics RNA solution was prepared as described above. The reaction mixture (V 5 100 µl) contained tRNA at concentration 0.5 µM, oligonucleotide at concentration 70 µM and hybridization buffer HB (see above). At given time a 5 µl aliquot of the reaction mixture was taken, mixed with 2 µl of loading buffer and immediately loaded onto running native 10% PAAG. Electrophoresis was car-ried out as described above.

d) Association and Association Rate Constants Equilibrium association constants (Ka) were calculated by minimizing mean square deviation between experimental and calculated curves, obtained with the equation [1]:

a

K ON

K ONa

a

=⋅

+ ⋅[ ]

[ ],

1 [1]

where Ka is equilibrium association constant, a – binding extent, [ON] – oligo-nucleotide concentration.

The direct and reverse association rate constants were calculated by minimizing mean square deviation between experimental curves and calculated ones, using the equation [2]:

a

k ON

k ON ke t k ON k=

⋅⋅ +

⋅ −( )+

+ −

− ⋅ ⋅ ++ −[ ]

[ ]( [ ] )1 [2]

where a is binding extent, k+ and k– – direct and reverse associating rate constant, respectively, [ON] – oligonucleotide concentration. All mean square calculations were performed using Origin 7.5 software.

Chemical and Enzymatic Probing of tRNAPhe Structure During Binding with Oligonucleotide A

Dimethylsulphate (DMS), RNase T1 and ONE were used for probing tRNA struc-ture upon hybridization with oligonucleotide A (here and after ON for oligonucle-otide). tRNA was hybridized with ON A as described above, in the case of RNase ONE either [3′-32P] or [5′-32P]-labeled tRNA was used. At the needed time 1 a.u. of RNase T1, or 0.1 a.u. of RNase ONE, or 0.5 µl of DMS was added to a 10 µl aliquot of the reaction mixture, the mixtures were incubated for 2.5 min in the case of RNases or for 5 min in the case of DMS. After probing with ribonucleases, RNA was precipitated with 2% LiClO4 in acetone followed by analysis of RNA cleavage products in 18% PAAM/8 M urea gel. In the case of DMS the reaction was quenched by addition of 0.5 µl of tRNA carrier (10 µg/ml) prior to ethanol precipitation (1 µl of 3 M sodium acetate pH 5.5 and 100 µl EtOH). After pre-cipitation RNA samples were treated with NaBH3CN and aniline (32). After the treatment samples were precipitated with ethanol and analyzed by electrophoresis in 18% PAAM/8 M urea gel.

31

Antisense Oligonucleotide Interaction with Natural

RNAs

Stopped-flow Experiments

a) Synthesis of 3′ Fluorescein Labeled tRNAPhe In Vitro Transcript tRNA was labeled with fluorescein using sodium periodate oxidation of the RNA 3′-end with subsequent treatment with ethylenediamine, NaBH3CN and FITC (isomer I) (33). Fluorescein labeled tRNA was purified in 12% PAAM/8 M urea gel. The RNA band was visualized by its UV fluorescence, cut out of the gel, eluted twice with 300 µl of 0.3 M sodium acetate pH 5.5, containing 10% phenol, during 12 hours at 4ºC, and precipitated with ethanol.

b) Synthesis of 5′ DABCYL Labeled ON A Conjugate of ON A with DABCYL (N-(3-aminopropyl)-4-(4-dimethylaminophenylazo)-benzamide) was prepared using standard procedure exploiting activation of the oligonucleotide 5′ phosphate by the Py2S2 – Ph3P in the presence of nucleophilic catalyst dimethylaminopyridine (DMAP) (34). DABCYL labeled ON A was purified by electrophoresis in 12% PAAM/8 M urea gel. Oligonucleotide-DABCYL conjugate has orange color; it was eluted from the gel and precipitated as described above.

c) Stopped-flow Kinetic Measurements For the experiment two solutions (V 5 1 ml) were prepared: 0.3 µM 3′-fluorescein tRNAPhe in the buffer HB and 5′-DABCYL-A solution in the buffer HB ranging in concentration from 1.5 310–6 to 10–4 M. Two syringes of the instrument for “stopped-flow” SX.18MV were filled up with these solutions. Syringes and reactor were thermostatically controlled at 20ºC. The fluorescence transitions during hybridization was monitored at λex 5 491 nm and λem ≥ 530 nm. To allow registration of both rapid and slow stages of hybrid-ization, measurements were performed in two time intervals: 0-0.5 and 0-1000 s, each containing 2000 measuring points of fluorescence. For each time interval of any given concentration of oligonucleotide 9 separate “shots” were performed, the first of which was rejected, since it was usually characterized by large experimen-tal error. Thus, each experimental curve was the averaged data on eight indepen-dent experiments. The control fluorescence curve was obtained with the “shot” of 0.3 µM 3′-fluorescein tRNAPhe with the buffer HB in the absence of oligonucle-otide, this control curve was subtracted from the obtained kinetics. The results were processed further using programs “Microsoft Excel 2003” and “Origin 7.5”.

d) Stopped-flow Kinetic Data Processing The hybridization kinetics data for each concentration of ON A contained 2000 measurement points. In order to simplify preliminary calculations the decrease of the number of points in the data array was performed. First, the value of each point was recalculated according to the equation [3] to remove noise:

y

y y y y y y y y y y yn

n n n n n n n n n n n′ −51 1 1 1 1 1 1 1 1 12 2 2 2 1 1 1 1 1( )5 4 3 2 1 1 2 3 4 5

111 [3]

Using this equation the data were averaged-out taking into account five points before the given one, and five ones after it. In the case n 5 and n 1995 it was considered that yn25 5 yn24 5 yn23 5 yn22 5 yn21 5 yn and yn 5 yn11 5 yn12 5 yn13 5 yn14 5 yn15. Then a new data array was formed consisting of every tenth point: y1, y11. y1991, y2000. The subsequent calculations were performed with the use of this shortened array, which contained 200 points for each time interval of 0-0.5 and 0-1000 s and for each concentration of ON A.

The effective rate constants were calculated by fitting experimental data to the equa-tion [4], used to describe the dependence of experimental values of fluorescence F on time t for two parts of experimental curve 0.. 0.5 s and 0.5..1000 s.

F t a be ctkt( )5 1 12 [4]

32

Serikov et al.

where k is the effective rate constant of fluorescence change; t – time; a, b and c – independent coefficients.

The four-step mechanism of interaction of oligonucleotide A with the tRNA was proposed on the basis of the analysis of effective rate constants dependence on oli-gonucleotide concentration (see Results and Discussion). We stated that each curve of fluorescence change during the hybridization had 2 characteristic times: ~0.1 s and 100-400 seconds. The difference between them makes it possible to process the regions of experimental curve 0..0.5 s and 0.5..1000 s independently from each other according to two-step mechanism.

Kinetic scheme for the 0..0.5 s region is:

P B1 1k

k

k

kP P1

2

3

42 3

→← →←

where P1, P2, P3 are different forms of the tRNA, free and bound to ON A, B – oligonucleotide.

For calculating the rate constants k1 – k4 the system of differential equations [5] was solved, corresponding to the given kinetic scheme:

∂∂∂∂

( )∂∂

P

tk BP k P

P

tk BP k k P k P

P

tk P k P

11 1 2 2

21 1 2 3 2 4 3

33 2 4 3

52 1

5 2 1 1

5 2

[5]

In this system P1 – P3 are concentrations of different forms of tRNA, B – oligonucleotide concentration, k1 – k4 – rate constants for the kinetic scheme used.

Taking into account that the initial concentration of oligonucleotide in all experi-ments greatly exceeded the initial concentration of tRNA (B P0

10 ), we suppose

oligonucleotide concentration to remain constant (B 5 B0). Also, in the course of hybridization the sum of all forms of tRNA is equal to its initial concentration: P P P P1 2 3 1

01 1 5 . So, the system of differential equations [5] can be brought to single differential equation [6] of the second order.

∂∂

( ) ∂∂

( )( )2

32 1 2 3 4

31 3 4 2 4 3 1 3 1

0 0P

tk B k k k

P

tk B k k k k P k k BP1 1 1 1 1 1 1 2 5 [6]

In accordance with the theory of linear differential equations, the solution of the equation [6] takes the form [7]:

P C C e C et t3 0 1 2

1 25 1 1l l [7]

where

l1 21 2 3 4 1 2 3 4

2

1 3 4 2 42 4, 521 1 1

1 1 1

2 1 2k B k k k k B k k k

k B k k k k( ) ( )

33

Antisense Oligonucleotide Interaction with Natural

RNAs

Coefficients C0, C1, C2 of the equation [7] are determined from initial conditions.

At t 5 0, P3 5 0 and ∂∂P

t3 05 , ∂

∂

23

2 1 3 10P

tk k BP5 , so the system of equations [8] is

formed to find the coefficients C0, C1, C2:

C C C

C C

C C k k BP

0 1 2

1 1 2 2

12

1 22

2 1 3 10

0

0

1 1 5

1 5

1 5

l l

l l

[8]

Solving the system [8] and using substitution of the obtained coefficients C0, C1, C2 into the equation [7], we obtain the dependences of the concentrations of tRNA forms P1, P2, P3 on time:

P tk P B

k k e k k et t1

1 10

1 2 1 22 1 3 4 1 2 3 4

1 2( )( )

( ) ( )5 2 1 1 1 1 1 1l l l l

l l l ll l

−kk k

k B2 4

1 01 2( )l l2

[9]

P tk P B

k e k e kt t2

1 10

1 2 1 22 1 4 1 2 4 4 1 2

1 2( )5 1 2 1 1 2l l l l

l l l l l ll l

−( ) ( ) ( ) (( )

[10]

P tk k P B

e et t3

1 3 10

1 2 1 22 1 1 2

1 2( ) =−( ) − + − l l l l

l l l ll l [11]

The dependence of fluorescence F on substance concentrations in the solution is shown by the equation [12]:

F F P F P F P e B5 1 1 2( )1 1 2 2 3 30e [12]

where F1, F2, F3 are the fluorescence amplitudes of tRNA forms P1, P2, P3, respec-tively; ε – the oligonucleotide absorption coefficient within the employed wavelength range; B0 – initial concentration of the oligonucleotide.

The values of rate constants k1 – k4 were calculated by fitting experimental curves to the equations [9-12] using Origin 7.5 software.

For the second region 0.5..1000s the kinetic scheme can be written as:

P B3 1k

k

k

kP P5

6

7

84 5

→← →←

where P3′ P4′ P5 are different forms of the tRNA, B – oligonucleotide. The values of rate constants k5 – k8 were calculated by fitting experimental curves to the equations 9-12 with the only difference of using P3′ P4′ P5 instead of P1′ P2′ P3 respectively.

Computer Simulation Experiments

Modeling of atomic structure of a metastable primary complex of the tRNAPhe with ON A was performed via optimization technique based on molecular dynamics and simulated annealing method. It was assumed that a formation of primary complexes follows by negligible conformational changes of the tRNAPhe structure. The opti-mal structure of molecular complex corresponds to a deepest or global minima on the potential energy surface (35). The problem of the global conformational energy minima search for a macromolecular system does not have a general solution (35).

34

Serikov et al.

We used a method of oligonucleotide chain growth, which is believed to mimic the natural process of oligonucleotide docking on the tRNA via a zipper mechanism. The zipper mechanism is common for biopolymer chain interactions and have been proven for a triplex formations (36). It can be assumed that the zipper mechanism has two main stages, 1) the initiation complex formation, 2) complex elongation. A formation of the tRNA-oligonucleotide initiation complex starts from a formation of several complementary base pairs oligonucleotide-tRNAPhe with single-stranded tRNA nucleotides. The complex elongation occurs through a zipper mechanism, i.e. each next nucleotide is searching for its optimal position on the tRNA taking into account a restriction due to a link with the previous nucleotide. In general the modeling of the complexes tRNA-oligonucleotides are done with the protocol: 1) modeling of a initiation complex, i.e. complementary complex between acceptor end ACCA tRNAPhe and oligonucleotide U1G2G3U4, 2) preliminary docking of the nucleobase of the oligonucleotide on the fixed tRNAPhe structure, 3) reconstruc-tion of d-ribose-phosphate backbone linking the nucleic base with the last docked oligonucleotide, 4) optimization of the structure via molecular dynamic simulated annealing method coupled with gradually disappearing harmonic constraints on the move of the oligonucleotide atoms, 5) optimization of the complex via molecular dynamic simulated annealing coupled with gradually disappearing harmonic con-straints on the move of the atoms of tRNA and oligonucleotide, 6) go back to step 2 for the next cycle of oligonucleotide grow, 7) final optimization of the complex via molecular dynamic method at constant T 5 300 K, and calculation of aver-age potential energy over trajectory of 250 ps. An optimization protocol consists of 100 steps of energy optimization by the method of conjugated gradients and subsequent simulated annealing protocol includes four temperature intervals with gradual change of thermostat temperature linearly with simulation time, 1) from 10 K to 150 K during 100-200 ps, 2) from 150 K to 350 K during 300-500 ps, 3) from 350 K to 300 K during 300-500 ps, 4) at 300 K during 500 ps. Control of temperature is done by the Berendsen thermostat method (37), the MD time step is equal to 0.001 ps. Typically optimization has been run 3 times to get convergence of the total energy of molecular complex. The modeling was done via program BISON (37) with Amber99 force field and Gaussian shell solvation model (38) and distant dependent electrostatic dielectric screening (37). The Gaussian solvation shell model (38) was extended for nucleic acids assuming solvation parameters of a phosphor atom to be equal to that of the sulphur atom. Molecular Dynamics simulation at various levels of sophistication can be applied to investigate structure and motion within nucleic acids, proteins and membranes and for details see a few of the representative papers published this Journal (39-60).

Results and Discussion

Secondary structure of yeast tRNAPhe and the oligonucleotides (ONs) used in this study are shown on the Figure 1. Oligonucleotides A – M were from 11 to 18 nucleotides long, their binding sites include 3′-ACCA sequence, the acceptor stem and part of the T-hairpin. Sequences of oligonucleotides (here and after ON) were derived by stepwise shortening (B, C and D) or lengthening (K, L and M) of ON A from 3′-end, shortening (E, F, G and H) of this ON from 5′-end. In all experiments ON A was used as a reference oligonucleotide.

Binding of ONs to tRNAPhe. Gel-shift Assay

To determine equilibrium binding constants (Ka) and association rate constants (keff), [3′-32P] tRNAPhe was incubated in the appropriate buffer in the presence of oligonucleotide in concentration ranging from 1.7 µM to 0.8 mM for different times and under different conditions (see Table I). Reaction was quenched by addi-tion of the loading buffer, analyzed by gel-shift assay and quantified as described in experimental section. Figure 2 displays representative autoradiographs of this analysis. Equilibrium binding constants and rate constants obtained using gel-shift

35

Antisense Oligonucleotide Interaction with Natural

RNAs

ability to invade tRNA structure depends both on oligonucleotide length and com-plementary site within tRNA. For example ON E differs from ON A only in one base in the part complementary to ACCA-end of yeast tRNAPhe, but this shorten-ing results in 14-fold decrease of Ka value. On the contrary, ON A shortening from the 3′-end (ON B) results in some increase of Ka. Comparison of ON binding with tRNA under similar conditions show that only oligonucleotides, whose comple-mentary site comprises ACCA sequence at the 3′-end and at least two bases of T-stem efficiently bind to the tRNA.

For these oligonucleotides binding extents are shown to decrease sharply, when they are shortened at the 5′-part complementary tRNA ACCA-end that seems to be impor-tant for initial binding and initiation of the strand displacement process. From the obtained results it is seen that oligonucleotides complementary to region comprising ACCA-sequence bound to the tRNA with high efficiencies. Stepwise lengthening of ON A within its 3′-end (ONs K, L and M) does not significantly increase their

GCGGAUU

m22GCCAGA

20

10

30

60

CU

Cm AU YGm A A

Gm7G

UA

G A G C

GC U C m2G

GG

DAD

G A

40

70

50m5C U G U G

Y

CG A C A C G

Um1A

CT

ACCACGCUUAA

AGGU

m5CY

5' U C C A C A G A A U U C G C A C C A 3' tRNA7060

H G F E A

M L K A B C D

3' 5'

Figure 1: Secondary structure of the yeast tRNAPhe and combined complementary site (shown in grey) of the oligonucleotides A–M (shown as solid line). Sequences of oligonucleotides B, C, D and K, L and M were derived by a stepwise shortening or lengthening of the oligonucleotide A from the 3′-end, respectively. Sequences of oligonu-cleotides E, F, G and H were derived by a stepwise shortening of oligonucleotide A from the 5′-end. The sites of tRNAPhe cleavage in the course of ON A binding by ribonucleases are designated by squares for RNase A, triangles for RNase T1, circles for RNase ONE in the case of [3′-32P]-tRNA, and asterisks for RNase ONE in the case of [5′-32P]-tRNA. The open and filled symbols show a rapid decrease and a gradual increase of the sensitivity of respec-tive phosphodiester bond to RNases. Guanines exhibiting increased sensitivity to DMS modification are marked by grey circles.

assay for oligonucleotides A–M are listed in Table I. Binding extents of ONs H and G was extremely low within used concentration interval, so that their Ka could not be determined. On the other hand, ONs M, L and K efficiently hybridized to the tRNA even at minimal concentration used (1.75 µM), thus only Ka lower estimate was calculated. From the data shown in Table I it is evident that oligonucleotides

36

Serikov et al.

binding, which indicates lesser important role of this region for hybridization. This observation correlates well with previously reported data. On the contrary, shorten-ing of ON A from 3′-end (ONs B, C and D) leads to sharp decrease of their binding ability (excluding ON B) thus demonstrating that the stem region has to comprise at least 9-10 nucleotides to provide sufficient base-pairing. It is worth to mention that

Table ISequences, binding extents, association and kinetic constants of the oligonucleotides, obtained using gel-shift analysis.

ON Sequence 5′ → 3′ Length Binding conditions1) Binding extent2) Ka, M21 k1, M21c21

M TGGTGCGAATTCTGTGGA 18 20°C 0.97 4.0 3 105 3.8L TGGTGCGAATTCTGTGG 17 20°C 0.96 3.9 3 105 2.3K TGGTGCGAATTCTGTG 16 20°C 0.96 3.4 3 105 1.8

A3) TGGTGCGAATTCTGT 15 20°C20°C, 5 mM

MgCl2

37°C37°C, 5 mM

MgCl2

20°C (tr)20°C, 5 mM MgCl2 (tr)

0.830.020.940.05

7.1 3 104

3.3 3 102

2.1 3 105

6.6 3 102

1.9 2.2 3 1022

2.8 3 102

2.5 3 101

5.3 3 101

1.0

B TGGTGCGAATTCTG 14 20°C 0.87 9.5 3 104 1.5C TGGTGCGAATTCT 13 20°C 0.53 1.7 3 104 1.9D TGGTGCGAATTC 12 20°C 0.02 2.4 3 102 24)

E GGTGCGAATTCTGT 14 20°C 0.25 4.9 3 103 24)

F GTGCGAATTCTGT 13 20°C 0.02 2.9 3 102 24)

G TGCGAATTCTGT 12 20°C ~05)

H GCGAATTCTGT 11 20°C ~05)

1) For the ONs except A data were obtained at 20ºC in the absence of Mg21.2) Binding extents were obtained at concentration of oligonucleotide 70 µM and tRNA concentration 0.5 µM.3) Since ON A is a reference oligonucleotide, its binding with tRNAPhe is studied in details.4) Kinetic constants were not determined due to poor binding of the oligonucleotides with the tRNA.5) ONs G and H did not bind to tRNAPhe under experimental conditions.

oligonucleotide complementary to tRNAPhe region 61-72 does not bind with this tRNA (primary data not shown). tRNA:ON B complex is characterized by abnor-mally low electrophoretic mobility (Figure 2A), which is explained by formation of tandem duplex with two ON B molecules bound tail to head (the second ON B molecule is bound at partially complementary sequence in T-loop) (27).

Figure 2: tRNAPhe:ON complex formation studied by gel-shift assay. Autoradiographs of 10% native polyacrylamide gel showing free and oligonucleotide bound [3′-32P] labeled tRNAPhe. (A) Equilibrium binding of the oligonucleotides with tRNAPhe. 0.5 µM tRNAPhe was incubated in the presence of respective ON (70 µM) for 4 h at 20°C in 50 mM HEPES-KOH pH 7.2, containing 200 mM KCl and 0.1 mM EDTA. (B) Kinetics of ON A binding with the tRNAPhe. The buffer and temperature are the same as in (A).

37

Antisense Oligonucleotide Interaction with Natural

RNAs

Kinetics of ON binding was studied at the ON concentration 70 µM (Table I, Figure 2B). It is seen, that ON hybridization with tRNA at 20ºC proceeds slowly, with characteris-tic time values in the range 0.5-2 h, apparently due to the rigid tRNAPhe structure (25). For mature yeast tRNAPhe and its transcript, lacking modified bases, effective rate constants differ by 28 times. It is well known, that yeast tRNAPhe contains four sites of specific magnesium coordination. Addition of magnesium to the hybridization buffer stabilizes spatial structures of both transcript and mature tRNA and results in strong decrease of the effective rate constant (53 and 86 times, respectively). When the tem-perature is rised from 20º to 37ºC, significant increase of the rate constants is observed both in the absence and in the presence of Mg2+: 150 and 1000 times, respectively. Thus, the factors stabilizing tRNA structure decrease hybridization rate, indicating that unfolding of tRNA structure upon hybridization is a rate-limiting step.

Probing of RNA Structure in the Course of ON Binding

We followed changes of tRNAPhe structure upon hybridization with ON A by prob-ing with dimethylsulphate (DMS) and with RNases ONE and T1 (Figure 3). Analysis of pattern of RNA modifications or cleavages was performed using electrophoresis in 18% PAA/8 M urea gels. Native tRNAPhe, tRNA treated with corresponding RNases or DMS in the absence of ON A and tRNA incubated with ON A were used as controls. Probing data are summarized in Figure 1. The data show ON A binding with yeast tRNAPhe to be accompanied by changes of the structure within all tRNA regions: acceptor stem, D-loop, anticodon hairpin, variable loop, T-hairpin and ACCA-end. The dynamics of changes of the tRNA structural elements sensitivity to chemical and enzymatic probes can be divided in two processes: fast and slow.

After ON A is added to the reaction mixture, immediate inhibition of cleavages at the ACCA-end, acceptor stem and anticodon loop is observed. The inhibition of the reactions at the acceptor stem (G4, A5, G71, C72) and ACCA-end (A73, C74, C75) can be explained by tRNA:ON transition complex formation, where unfold-ing of the tRNA structure did not yet occur. As it is seen from the Figure 3, ON A only partially protects nucleobases of tRNAPhe from cleavage by ribonucleases. This partial protection of A73, C74, C75 from RNase ONE cleavage is caused by incomplete ON A binding with the tRNA: binding extent does not exceed 0.60 at ON A concentration 70 µM used for probing, thus 40% of the tRNA is not hybrid-ized with the oligonucleotide. Moreover, the end base pairs of the heteroduplex are likely “breathing” (are periodically opened for a small time intervals) and therefore are accessible to cleavage with RNase ONE. Besides, partial protection of RNA nucleotides from cleavage with RNases is the result of exchange between free and ON A bound tRNA species. Decrease in sensitivity of nucleotides A5, G71 and C72 is likely caused by shielding of this tRNA region by oligonucleotide A involved in transition complex formation with ACCA-sequence and interaction with the adja-cent region via non-canonical hydrogen bonding (non-canonical triplex formation cannot be excluded). Surprisingly, immediate inhibition of the cleavage at the anti-codon loop (A31, U33, A35) is observed. We suppose, that transition complex of ON A with this tRNA region is formed, but this complex half-life time is short, so it is not registered by RNase H footprinting (data not shown) (27). At the tRNA region 31-36 the hexameric heteroduplex with ON A r(ACUGAA)/d(TGTCTT) containing one mismatch U/T can be formed. This heteroduplex can be additionally stabilized by specific structure of anticodon loop, that favors heteroduplex formation (61).

Slow changes of sensitivity of tRNA nucleotides to the probes are observed in acceptor stem, D-loop, anticodon stem and T-stem. Increase of cleavage at the T-hairpin is the result of complementary duplex formation and displacement of the second strand of the T-hairpin. Increase of cleavage at several phosphodiester bonds located in the acceptor stem (G1, A5, U6, U7) is also the result of homolo-gous strand displacement upon oligonucleotide binding. Appearance of several cuts in D-loop could be the result of disruption of its tertiary structure upon tRNAPhe

Figure 3: Chemical and enzymatic probing of tRNAPhe in the course of hybridization with oligonucleotide A (autoradiographs of 18% denaturing PAAG). The time of tRNA incubation with ON A (prior to cleavage) is shown in the top. Lane L - tRNAPhe alkaline ladder, T1 – RNase T1 ladder. Major cleavage sites are shown sideways. (A) Modification with dimethylsulphate ([3′-32P] labeled tRNA). Lanes C1, C2, C3 – free tRNA, tRNA treated with DMS, NaBH4 and aniline in the absence of ON A and incubation control, respectively. (B) RNase T1 probing ([3′-32P] labeled tRNA). Lanes C1, C2 and C3 – free tRNA, RNase T1 cleavage of tRNAPhe under native conditions in the absence of ON A and incubation control, respectively. (C) RNase ONE probing ([5′-32P] labeled tRNA). Lanes C1, C2 and C3 – free tRNA, RNase ONE cleavage of tRNAPhe under native conditions and incubation control, respectively. (D) RNase ONE probing ([3′-32P] labeled tRNA). Lanes C1, C2 and C3 – free tRNA, RNase ONE cleavage of tRNAPhe under native conditions in the absence of ON A and incubation control, respectively.

39

Antisense Oligonucleotide Interaction with Natural

RNAs

hybridization with ON A, while new cleavage sites at the positions C40, U41, G42, G43 of the anticodon stem indicates both destabilization of the stem and rearrange-ment of three-dimensional structure of the tRNA.

Upon chemical probing of tRNA:ON A complex with DMS we observed increased reactivity of G57 due to T-loop unfolding. In the T-loop of tRNAPhe N7 atom of G57 forms a hydrogen bond with 2′-OH group of pseudouridine Ψ55 (62). Increas-ing of sensitivity of G51 and G53 to modification with DMS can be explained by rearrangement of double stranded RNA helix, which is involved in tertiary interac-tions, to an opened state.

Kinetics of ON Binding to tRNAPhe

In order to elucidate mechanism of tRNAPhe interaction with oligonucleotides, kinetics of tRNAPhe:ON A complex formation under different oligonucleotide con-centrations was studied using gel-shift assay to follow slow stages and stopped-flow method to monitor fast kinetic stages. A hyperbolic dependence of k+eff value on oligonucleotide A concentration can not be described either by equation of sec-ond order reaction (in this case k+eff would have linear dependence on ON concen-tration) or by equation of first order reaction (in this case k+eff would not depend on ON concentration).

Thus, based on probing data and kinetics measurements, we suppose that ON bind-ing to tRNAPhe occurs via one or several transition complexes characterized by low stability and short half-life times (so-called metastable complexes). In the case, when one metastable complex [tRNA:ON A is formed, the hybridization of ON A with tRNAPhe is described by scheme 1:

tRNA ON tRNA ON tRNA ON 1

1

2

A A AK k

k

x : ] : →← →← [

Scheme 1

where k+eff is a function of ON A concentration, equation [13]:

k kK ON A

K ON AeffX

X1 5 3

3

3+ +

[ ]

[ ]1 [13]

k+ and k– are rate constants of direct and reverse intramolecular transition of partial complex to full-length duplex, respectively, Kx – equilibrium constant for transition complex formation.

The suggested mechanism includes fast transition complex formation at the first stage and then, at the slow stage, reversible intramolecular transition of metastable complex into full-length complementary complex. According to the probing data and gel-shift analysis of ON binding we suppose that the metastable complex is formed by Watson-Crick base pairing between 5′-end of the oligonucleotide and single-stranded tRNA regions (ACCA-end and part of T-loop).

For the detailed study of ON A binding with tRNA and especially formation of short-living transition complexes, stopped-flow method in combination with Fluo-rescence Resonance Energy Transfer (FRET) was used (see Experimental part for details). The fluorophores labeled ON A and tRNAPhe used for this experiment are shown in the Figure 4A. 3′-end of the yeast tRNAPhe transcript (tRNA transcript was used in this experiment instead of mature tRNA since it can be easily prepared in big amounts) was labeled with fluorescein (fluorescence energy donor) using sodium periodate oxidation of 3′-terminal ribose residue (33). Antisense oligo-nucleotide A contained DABCYL label (fluorescence energy acceptor), attached

40

Serikov et al.

at 5′-end according to standard protocol using activation of 5′-phosphate by tri-phenylphosphine (Ph3P) – dipyridyldisulphide (Py2S2) RedOx pair (34). Interac-tion between labeled tRNAPhe and ON A should lead to quenching of fluorescence allowing direct real time monitoring of the hybridization. Stopped-flow (Figure 4B) was performed on SX.18MV equipment (Applied Photophysics Limited, Great Britain). Portions of 50 µl of tRNA and ON solutions were “shot” from the syringes 1 and 2 (Figure 4B) and immediately mixed in the reaction chamber, where the fluorescence measurements were performed.

N

N

N

N

NOP

O

O

H

S H

OO

O

O

O

O

N

N

N

H2-

RNA

-

-

N

ON

N N

NH

HP

OO Oligonucleotide

RNA

ON

λex= 491 nm

≥ 530 nmλem

1

2

3(B)

(A)

Figure 4: Principal scheme of the “stopped-flow” experiment. (A) The FRET employs tRNA labeled with fluorescein at 3′-end and ON A labeled with DABCYL at 5′-end. Formation of the tRNAPhe:ON complex results in quenching of fluorescein fluorescence. (B) The experiment was carried out using the SX.18MV apparatus made by “Applied Photophysics Limited”, Great Britain. 1 – syringe with tRNA solution, 2 – syringe with oligonucleotide solution, 3 – reactor chamber. The hybridization was monitored by total level of fluorescence of labeled ON A within the range λemission ≥ 530 nm (530 nm high-pass optical filter was used), excitation wavelength λex = 491 nm (found experimentally).

Typical experimental curves of fluorescence quenching are shown in Figure 5. Each curve appears to have two characteristic times: ~0.1 s and 100-400 s (Figure 5A and 5B). A typical experimental curve can be divided into three parts. The first part lies between 0 and 30 ms, when no fluorescence change occurs. The second one – between 30 ms and 0.5 s, when a very small but fast exponential decrease of the fluorescence intensity is observed. The third part has characteristic time 100-400 seconds depending on oligonucleotide concentration; during this time the major exponential decrease of the fluorescence intensity occurs. Data sets for the experi-mental curves were fit to the equation [4] (see Experimental part). Dependencies of effective rate constants on ON A concentration are shown in Figure 5C and 5D. Calculated value k1′ corresponds to measurements within 0..0.5 s, k2′ is effective rate constant for processes occurring within 0.5..1000 s time range. Data obtained in each of the two time ranges can be computed independently due to at least three orders of magnitude difference in the values of effective rate constants. As it is seen (Figure 5C), k1′ value does not depend on oligonucleotide concentration. It indicates that the observed fluorescence changes are caused by intramolecular rear-rangement of tRNAPhe structure. This rearrangement is expected to be the result of

41

Antisense Oligonucleotide Interaction with Natural

RNAs

some short-living complex formation occurring within 0..30 ms and the quencher (5′-end of ON A bearing DABCYL) is located far from the complementary site (since no fluorescence changes are observed during this time). Then, the stage char-acterized by very low fluorescence change (less than 5% of total) take place, which means the quencher (and the oligonucleotide molecule) is still located far from the complementary site.

The k2′ value has a complex dependence on ON A concentration that is close to hyperbole (Figure 5D). This indicates the following: i) during the time interval 0.5..1000 s at least two individual interaction steps occur, providing k2′ value the complex dependence on ON A concentration; ii) the second oligonucleotide A mol-ecule is involved in one of these steps; iii) interaction with the oligonucleotide occur at the complementary site, since the fluorescence change is very noticeable; iv) during this interaction some transition complex forms; v) this transition com-plex slowly processes to fully complementary duplex.

Taking all observations into account the overall hybridization mechanism consisting of four stages can be supposed (Figure 6A). In order to prove the suggested mecha-nism for oligonucleotide hybridization with tRNA we processed experimental data by fitting the data to the equations 6-12 (see Experimental section), Figure 6B. The correlation coefficient (R2) between obtained data and theoretical curves is 0.998, which apparently indicates correct selection of the kinetic scheme (Figure 6C). The value of k1 5 (1.4 ± 0.4) 3107 M–1s–1 is characteristic to a typical diffusion kinetic

3

3,5

4

4,5

5

5,5(A) (B)

(C) (D)

0 0,1 0,2 0,3 0,4 0,5

Time, s

Fluo

resc

ence

, a. u

.

1.5 µΜ2.5 µΜ4.3 µΜ7.2 µΜ12.2 µΜ20.7 µΜ35 µΜ59.2 µΜ100 µΜ

1,5

2

2,5

3

3,5

4

4,5

5

5,5

0 200 400 600 800 1000

Time, s

Fluo

resc

ence

, a. u

.

1.5 µΜ2.5 µΜ4.3 µΜ7.2 µΜ12.2 µΜ20.7 µΜ35 µΜ59.2 µΜ100 µΜ

0

10

20

30

40

50

60

0 20 40 60 80 100

[A], µM

k1',

s-1

0

0,005

0,01

0,015

0,02

0 20 40 60 80 100

[A], µM

k2',

s-1

Figure 5: Changes in fluorescence of the 3′-end fluorescein labeled tRNA upon hybridization with DABCYL labeled oligonucleotide A. Two time scales are shown, up to 0.5 (A) and up to 1000 s (B), reactions were performed in 50 mM HEPES-KOH, pH 7.0, 0.2 M KCl, 0.1 mM EDTA, λex = 491 nm, λem > 530 nm. The concentration of the tRNA was 0.3 µM, oligonucleotide A concentration was varied from 1.5 to 100 µM (shown on the right of the graphs). Both data sets were fit to the equation F A Be

ktCt5 1

21 [4], where F is relative fluorescence, k – effective rate constant, t – time; A, B and C – independent coefficients. The dependen-

cies of effective rate constants on the ON A concentration for k1′ and k2′ are shown on the graphs (C) and (D), respectively.

42

Serikov et al.

constant. At relatively low ON A concentrations (<5 µM) the overall hybridization rate is limited by k5 5 (1.1 ± 0.1) 3103 M–1s–1 and k7 5 (6.4 ± 3.3) 310–3 s–1. At higher ON A concentrations the overall hybridization rate is limited only by tRNA structure rearrangement during complex formation and is characterized by k7 5 (6.4 ± 3.3) 310–3 s–1. This slow RNA structure rearrangement makes the hybridization to be relatively slow at any ON concentration.

In the course of tRNAPhe:ON A duplex formation two molecules of ON A are involved in the process, while the final product contains only one ON A molecule bound with tRNA. The first stage is formation of a short-living intermediate (during the first 30 ms) between several bases of the oligonucleotide and tRNA bases located far from the fluorophores. We searched for possible candidates within tRNAPhe and found short region in the T-loop TGGTGCG/ACCUAGC (oligonucleotide part is underlined, mis-matches are shown in italic). This interaction occurs far from tRNA ACCA-end and conjugated fluorescein and is not characterized by noticeable fluorescence change.

Constant Value

k1 (1.4±0.4) ×107 M-1s-1

k2 42±26 s-1

k3 12±9 s-1

k4 12±7 s-1

k5 (1.1±0.1)×103 M-1s-1

k6 (6.1±1.5)×10-3 s-1

k7 (6.4±3.3)×10-3 s-1

k8 not determined1,5

2

2,5

3

3,5

4

4,5

5

5,5

0 200 400 600 800 1000

Time, s

Fluo

resc

ence

, a. u

.(A)

(B) (C)

Figure 6: Kinetic scheme for oligonucleotide A binding with yeast tRNAPhe (A), calculated rate constants (B) and comparison of the theoretical curves of fluo-rescence quenching with the experimental data (C). The first step is formation of a collision complex between bases of the oligonucleotide and T-loop of the tRNA. The formation of the collision complex partially melts the tRNA stems (the second step) causing the tRNA structure breathing. Then the “correct” collision complex is formed between the second oligonucleotide molecule and single stranded ACCA sequence. The last step is extended heteroduplex formation via strand displace-ment. Rate constants k1 – k8 were calculated as described in Materials and Methods. Correlation coefficient (R2) between obtained data and theoretical curves is 0.998, which apparently indicates correct selection of the model.

43

Antisense Oligonucleotide Interaction with Natural

RNAs

This intermediate complex formation leads to partial destabilization of the aminoac-ceptor stem and, probably, of the T-stem, making these regions more accessible for further interaction with oligonucleotide. This partial melting is also characterized by negligible fluorescence change but requires more time to proceed (up to 0.5 s). The following two stages lead to formation of the fully complementary duplex. The other intermediate is formed by interaction of the second oligonucleotide molecule and single-stranded 3′-ACCAC-5′ sequence, which might be stabilized by triplex base-pairing between oligonucleotide and aminoacceptor stem of the tRNA. Formation of the metastable complex initiate an intramolecular rearrangement, leading to fully complementary duplex tRNA:ON A by a stepwise displacement of the RNA strand from the complex. This rearrangement proceeds slowly and is characterized by sig-nificant fluorescence decrease, which indicates close interaction of tRNA 3′-end with fluorescein and oligonucleotide 5′-end with DABCYL. Characteristic times of this stage correlate well with similar characteristic time values obtained using gel-shift analysis and tRNA structure probing.

Computer Simulation Experiments of Formation of Complexes Between tRNAPhe and Antisense Oligonucleotides

According to the Figure 6, the first fast stage produces a transition short-living complex tRNAPhe – ON A involving the T-loop of the tRNA, which is converted to another intermediate. After interaction with the second oligonucleotide molecule, the slow last stage proceed, that involves a large conformational rearrangements of the tRNAPhe i.e. double strand segments unwinding, native base pairs brake and formation of new complementary pairs. The final stage corresponds to the global energy minima on the surface of conformational energy of complex tRNAPhe – ON A. A formation of the initial complexes of the tRNAPhe – ON A follows minor con-formational changes of the tRNAPhe structure. These complexes have metastable structures and correspond to the favorable complexes on the conformational frozen native structure of the tRNAPhe. Computational modeling of probable structures of a primary complexes tRNA – ON A provide a structural basis for interpretation of a experimental data on the mechanism of interactions of tRNAPhe with antisense oligonucleotide and structural rearrangements and understanding of a principles of construction of optimal antisense oligonucleotides.

Primary Complex with T-loop

The native X-ray structure of the tRNAPhe demonstrates a tertiary interactions between nucleotides of D- and T-loops: G19-C56 Watson-Crick, canonical base-pair (here and after WC), D16-U59 and G18-Ψ55 forms a mismatch pair with several H-bonds, G57 intercalates between G18/G19 making a good stacking interactions. Inspection of the atomic structure of the tRNAPhe have revealed that the six-bases sequence of G56G57U55A58C60U59 in the 3D-space is similar to the single strand structure in 5′-3′ direction having nucleic bases in the stack-ing arrangement with orientations favorable to form coplanar interactions with the complementary single strand sequence of bases. Under a low salt conditions in the absence of Mg+2 the D-, T-loop interaction is weak, the D-, T-loop junc-tion can be opened and the stacked T-loop sequence G56G57T55A58C60U59 can be accessible for interaction with ON A. In such condition the sequence 5′-G2G3U4G5C6G7 of the ON A can make an initiation complex with the T-loop via formation of complementary G-C and mismatched G:U pairs and then pro-ceeds to form primary complex with tRNAPhe. The computational model of the atomic structure of the T-loop complex (TL) is shown in Figure 7. It can be seen that ON A forms a well organized structure with T-loop in its native structure and shifted D-loop. Several bases of ON A replace similar bases of the native D-loop and simultaneously interact with the D-loop in rearranged conforma-tion. The nucleic bases of the ON A involved in the H-bonding interactions with D and T-loop bases making 14, 15 hydrogen bonds, namely U17-G2-U59

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(3 H-bonds), G18-G3-U59 (2 H-bonds), G5-U55 (2 H-bonds), G5 intercalates between A58 and G57 bases of T-loop, C6-G57 and G7-C56 are the WC base pairs. The overall shape of ON A in the T-loop complex is a loop, Figure 7A and 7B. The base A9(N7) of the ON A makes internal H-bonds with U11(N3), G14 makes H-bonds with the nucleotides G19 and A21 of the D-loop. In overall the T-loop complex consists of three interacting loops of nucleotide strands and is formed under favorable conditions.

Major Groove Triple Complexes

Formation of complementary base pairs between T1G2G3T4 sequence from the 5′-end of ON A and tRNAPhe ACCA 3′-end can proceed without conformational changes of the tRNA and therefore serves as natural initiation stage of transition complex formation (Figure 6). The main energetic determinants of a nucleic acid structure formation are coplanar base-base interactions via formation of hydrogen bonds due to electrostatic complementarity and stacking base-base interactions due to favorable Van der Waals interactions between two parallel aromatic ring sys-tems. Optimization of these energy determinants can be done via interaction of the nucleic bases of ON A with major groove of the acceptor stem of the tRNAPhe

and formation of the recombinant triplex with two identical parallel nucleic chains. Docking of the oligonucleotide ON A on the tRNAPhe is done by the next build up protocol: 1) modeling complementary complex between acceptor end ACCA tRNAPhe and oligonucleotide U1G2G3U4, 2) preliminary docking of the next nucleic base of the ON A on the fixed structure tRNAPhe, 3) reconstruction of the d-ribose-phosphate backbone linking the nucleic base with the docked oligonucle-otide, 4) optimization of the structure via the molecular dynamic simulated anneal-ing method coupled with the gradually disappearing harmonic constraints on the move of the oligonucleotide atoms, 5) final optimization of the complex via the molecular dynamic method, 6) go back to step 2 for the next cycle of oligonucle-otide grow. It was found that the oligonucleotides up to the length of 15–mer fit the major groove of the acceptor stem and coaxial T-stem quite well. Nine triplets of nucleobases are formed: four triplets ↓U•A↑⊕ U↓, three triplets ↓G•C↑⊕ G↓ having three hydrogen bonds, two triplets ↓C•G↑⊕ C↓ and two less effective trip-lets ↓G•U↑⊕ A↓ and ↓A•U↑⊕ A↓ (Figure. 8).

Figure 7: (A) T-loop complex formation. Complementary Watson-Crick base pairs C56-G7, G57-C6 — low right corner, ON A is shown in red. (B) Conforma-tional changes of the D-loop. ON A — blue, D-, T-loops in complex — red, native conformation of the D-loop — green. Complementary pairs C56-G7, G57-C6 — low side.

45

Antisense Oligonucleotide Interaction with Natural

RNAs

The final energy of the major groove complexes are shown in the Figure 9. The potential energy of the T-loop complex is about 50 kcal lower than the energy of the unfavorable major groove complex of tRNAPhe – ON A. The computational model of the T-loop complex indicate that oligonucleotide ON A is able to form a more favorable T-loop complex instead of the major groove triple strand complex. It can be seen that the energy of the tRNAPhe•oligonucleotide N metastable complex is getting low in general linearly with the length N of the oligonucleotide up to the N equal to 13 nucleotides. The 3′-end nucleotide of the 14-mer and last two nucle-otides of the ON A do not contribute into the stabilization energy of the primary complex tRNAPhe•oligonucleotide. The destabilization of the complex result in the unfavorable interaction of the nucleotides 14 and 15 with the frozen global structure of the tRNAPhe, with the tRNA nucleotides in the vicinity of the D-, T-loop interac-tion (Figure 10). These interactions perturb the D-,T-loop junction and has lower

Figure 8: (A) A part of the secondary structure of the tRNAPhe and ON A, forming a major groove complex; ON A bases shown in italic indicate the position of ON A forming the T-loop complex. (B) Coplanar triplets of ON A bases with complementary base pairs of acceptor and T-stem of tRNAPhe. ON A bases are shown in bold, • for WC, A, ⊗ for major groove interaction, 3 2 G-U base pair.

(A) A76 • U1 C75 • G2 C74 • G3 A71 • U4

G1 • C72 ⊕ G5C2 • G71 ⊕ C6G3 • C70 ⊕ G7G4 × U69 ⊕ A8A5 • U68 ⊕ A9U6 • A67 ⊕ U10U7 • A66 ⊕ U11

C12 U13 G14 U15 U1G2G3U4G5⊕ ⊕ ⊕ ⊕ C60Um59

G65 A64 C63 A62C61 A58

• • • • •mC49 U50 G51 U52G53 G57• C6 C 48 U15G14U13 T54 C56 •G7U47 Ψ55 A8

G46 C12 U11U10A9A44G45

(B)

↓G1•C72↑⊕ G5↓, ↓G3•C70↑⊕ G7↓,↓C63•G51↑⊕ G14↓

↓C2•G71↑⊕C6↓,↓C49•G65↑⊕C12↓

↓A5•U68↑⊕А9↓ ↓G4•U69↑⊕A8↓

↓U6 • A67↑⊕U10↓,↓U7 • A66↑⊕U11↓,↓U50 • A64↑⊕U13↓,↓U52 • A69↑⊕U13↓

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Serikov et al.

total conformational energy of the complex compared to the energy of the optimal major groove complex tRNAPhe•ON C (Figure 9). This behavior of the energy of the complexes versus oligonucleotide length allows assuming that the mechanism of destruction of tRNAPhe structure by a long complementary oligonucleotide is similar to a molecular wedge. A conformational perturbations in the vicinity of the D-, T-loop junction due to nucleotides 14 (ON B) and 15 (ON A) initiate a large conformational changes in the structure of the tRNAPhe•ON complex and formation of a final complementary complex.

Oligonucleotide Binding Initiate Conformational Changes of the D-, T-loop Junction

To check an ability to initiate conformational changes of the D-, T-loop junction, a modeling of the tRNAPhe – ON M complex has been done. The oligonucleotide M is complementary to the tRNAPhe – sequence U59-A76. The 3′-end of the oligonucle-otide M is complementary to the T-loop bases C60U59. The question is to check an ability of the oligonucleotide complementary to the loop bases switch on con-formational rearrangements and make complementary WC base pairs with the loop bases. Inspection of the atomic model of the tRNAPhe shows that the bases C60U59 are bulged out, interacts with D-loop base U16 and not involved in the local interac-tions with T-loop nucleotides. The final structure of the tRNAPhe – ON M complex is shown in Figure 11. It can be seen that bases G17A18 of the oligonucleotide M form WC base pairs with C60U59 of the tRNAPhe (Figure 11B). Oligonucleotide M invades the region of the D-, T-loops junction, and forces out the D-loop bases. The native structures of the D- and T-loops itself are changed. The potential energy of the complex tRNAPhe – ON M is quite low, Figure 9, suggesting that the loop structure and their junctions are conformationally mobile elements of RNA struc-ture and have ability to rearrange into a new low energy structures.

Triple Complex 2 ON A/tRNAPhe

ON A in the MGC and T-loop complex covers non-overlapping segments of the tRNAPhe spatial structure. Therefore these complexes can exist simultaneously as

Figure 9: Potential energy of the tRNAPhe complexes with antisense oligonucleotides of N nucle-otides of length docked on tRNA. Red filled circles – major groove complexes; diamonds – T-loop complex; red triangle (down) – complex with ON M; star – average energy per ON A for the triple com-plex ON A (MGC) + ON A (TL); square – complementary complex tRNAPhe – ON A.

Figure 10: Major groove complex tRNAPhe – ON A. Oligonucleotide A 5′-3′ (up to down) is shown in red, tRNAPhe – in blue, from top to bottom – acceptor ACCA end, acceptor- and T-loop stems; right low corner – T-loop and D-loop; left low corner – anti-codon loop.

47

Antisense Oligonucleotide Interaction with Natural

RNAs

one triple complex 2 ON A/tRNAPhe. As it was shown above, the formation of the MGC tRNAPhe – ON A encounters some spatial restrictions due to unfavorable interactions the last nucleotide 15 with D-, T-loops junction. A formation of the TL complex of the tRNAPhe-ON A follows by conformational rearrangement of D-loop. This D-loop rearrangement takes off the unfavorable spatial restrictions for formation of the MGC for the ON A. The optimized computational model of the triple complex 2 ON A/tRNAPhe is shown in Figure 12. It can be seen that MGC ON A and TL ON A forms tail-to-head oligonucleotide strands. The potential energy of this MGC/TL complex with tRNAPhe is lower than the average energy of MGC and T-loop complexes (Figure 9). Thereby the conformational changes of the D-loop initiated by the T-loop complex formation makes the tRNAPhe structure more favorable for building of the MGC, compared with that complex on the frozen tRNAPhe structure. The second factor of lowering the potential energy of the double complex is the tail-to-head interactions between two strands of ON A.

Model of the Complementary Complex tRNAPhe ON A

The atomic model of complementary complex tRNAPhe – ON A is build up by an exchange of nucleotides (G1-U7, C49-U52) of the tRNA by correspond-ing nucleotide of the ON A. The structure has been subjected to the simulated

Figure 11: Major groove complex tRNAPhe – ON M. Oligonucleotide M – red, tRNAPhe bound in complex – blue, native tRNAPhe – green. (A) Full structure of complex; (B) T-, D-loop region is shown; complementary pairs of oligonucleotide-tRNA bases, U59-A18, C60-G17 are shown.

Figure 12: (A) Double complex tRNAPhe – ON A. Oligonucleotide of the major groove complex is shown in green, oligonucleotide of the T-loop complex – in red, tRNAPhe – in blue. (B) Complementary complex tRNAPhe – ON A. Antisense ON A complementary bound to the tRNA is shown in red, tRNA sequence unfolded by ON A – in light blue, tRNAPhe – in blue.

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Serikov et al.

annealing molecular dynamic optimization. The final structure of the complementary complex tRNAPhe – ON A is shown in Figure 12B. The structure remains to be stable in MD simulation of 500 ps long at 300 K. The complementary complex tRNAPhe – ON A realize the main principles of the RNA structure, namely, a maxi-mal realization of a coplanar and stacking base-base interactions. The potential energy of the complementary complex tRNAPhe – ON A is less than the energy of MGC or TL complexes tRNAPhe – ON A, Figure 9.

Conclusion

Interaction between two RNA molecules or between RNA and oligonucleotide is often initiated by the formation of only few base pairs or even few hydrogen bonds. Besides, the kinetic constant of association of two RNA molecules virtually does not depend on their sequence and in general is defined by spatial structures of RNA molecules. A strong example is the presence of 7–base loops in RNA (anticodon tRNA loop and loops in RNAI and RNAII coded by ColE1 plasmids), which cre-ates favorable conditions for preferable complex formation by the way of struc-ture recognition (formation of a loop-loop complex), this loop-loop complex is substantially stabilized by effective stacking interactions between the loops and in each of the stems. After formation of the loop-loop complex, interaction of two RNAs among themselves passes through a series of steps leading to formation of the extended stable duplex (ColE1 or dimerization of a retrovirus genome). In our case complex formation between RNA and antisense oligonucleotides show that this process has similar peculiarities.

In the present study, the combination of biochemical and physicochemical methods with computer simulation gave us a deeper insight into the process of nucleic acids interaction with RNA and resulted in revealing detailed mechanism of antisense oli-gonucleotide invasion into stable spatial structure of RNA. The process is initiated by the formation of different transition complexes with nucleotides in the T-loop and ACCA sequence. Formation of these complexes destabilizes RNA structure and favors following formation of an extended heteroduplex with the oligonucleotide via strand displacement process. Computer modeling of oligonucleotide–tRNAPhe interaction revealed potential factors that could favor transition complexes forma-tion and confirmed the proposed mechanism, showing the oligonucleotide to be a molecular “wedge”. Our data evidence that oligonucleotide invasion into structured RNA is initiated by loop-single strand interactions, similar to the initial step of the antisense RNA–RNA interactions. The obtained results complement existing algo-rithms for choosing efficient oligonucleotide or other probes giving the knowledge to a scientist that it is needed to consider not only the factors favoring thermo-dynamics of duplex formation but also the factors favoring transition complexes formation and — if it is possible — attenuation or weakening of the stable spatial RNA–target structure. The results apparently show oligonucleotide invasion into structured RNA to be initiated by loop-single strand interactions, similar to the initial step of the antisense RNA–RNA interactions.

Acknowledgements

This research was supported by the Russian Academy of Sciences under the pro-grams “Molecular and Cell Biology”, and the program for support of leading scientific schools (Grant no. NSh-7101.2010.4), and Ministry of Science and Edu-cation of the Russian Federation (state contract P438), and Russian Fund of Basic Research (Grant no. 09-04-00136).

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Date Received: November 24, 2010

Communicated by the Editor Elena Bichenkova