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Nucleic Acids Research Advance Access published online on June 12, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm421
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chemistry

A chemical synthesis of LNA-2,6-diaminopurine riboside, and the influence of 2'-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes

Anna Pasternak1, Elzbieta Kierzek1, Karol Pasternak1, Douglas H. Turner2 and Ryszard Kierzek1,*

1Institute of Bioorganic Chemistry, Polish Academy of Sciences, 60-714 Poznan, Noskowskiego 12/14, Poland and 2Department of Chemistry and Department of Pediatrics, University of Rochester, RC Box 270216, Rochester, NY 14627-0216, USA

*To whom correspondence should be addressed. Tel: 48 62 852 85 03; Fax: 48 62 852 05 32; Email: rkierzek{at}ibch.poznan.pl

Received October 19, 2006. Revised March 28, 2007. Accepted May 8, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Modified nucleotides are useful tools to study the structures, biological functions and chemical and thermodynamic stabilities of nucleic acids. Derivatives of 2,6-diaminopurine riboside (D) are one type of modified nucleotide. The presence of an additional amino group at position 2 relative to adenine results in formation of a third hydrogen bond when interacting with uridine. New method for chemical synthesis of protected 3'-O-phosphoramidite of LNA-2,6-diaminopurine riboside is described. The derivatives of 2'-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides were used to prepare complete 2'-O-methyl RNA and LNA-2'-O-methyl RNA chimeric oligonucleotides to pair with RNA oligonucleotides. Thermodynamic stabilities of these duplexes demonstrated that replacement of a single internal 2'-O-methyladenosine with 2'-O-methyl-2,6-diaminopurine riboside (DM) or LNA-2,6-diaminopurine riboside (DL) increases the thermodynamic stability ({Delta}{Delta}G°37) on average by 0.9 and 2.3 kcal/mol, respectively. Moreover, the results fit a nearest neighbor model for predicting duplex stability at 37°C. D-A and D-G but not D-C mismatches formed by DM or DL generally destabilize 2'-O-methyl RNA/RNA and LNA-2'-O-methyl RNA/RNA duplexes relative to the same type of mismatches formed by 2'-O-methyladenosine and LNA-adenosine, respectively. The enhanced thermodynamic stability of fully complementary duplexes and decreased thermodynamic stability of some mismatched duplexes are useful for many RNA studies, including those involving microarrays.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Modified nucleotides are useful tools to study the structures, biological functions and chemical and thermodynamic stabilities of nucleic acids (1–7). Recently, microarray methods were introduced to study the structure of nucleic acids (8–11). In native RNA, a majority of nucleotides form canonical pairs and single-stranded regions are typically short, roughly 5–7 nucleotides long. Detection of these single-stranded regions by RNA binding to probes on microarrays requires that the hybrid formed be thermodynamically sufficiently stable to capture the RNA. The thermodynamic stability of nucleic acid duplexes is strongly dependent on sequence, however. For example, duplexes of RNA heptamers composed of only A-U or G-C base pairs can differ in stability ({Delta}{Delta}G°37) by up to 15 kcal/mol, which is over 10 orders of magnitude in Kd (12). This complicates interpretation of microarray data. Incorporation of modified nucleotides in microarray probes can increase the thermodynamic stability of hybrid duplexes and make the thermodynamic stability relatively independent of sequence. Consequently the single-stranded character of potential binding sites in target RNA becomes the dominant factor determining binding, thus simplifying interpretation to deduce target RNA secondary structure.

There are many ways to adjust the stabilities of nucleic acid duplexes (1,13–22). Initial microarray experiments to deduce RNA secondary structure (11) used 2'-O-methyl RNA probes because 2'-O-methyl RNA/RNA duplexes are more thermodynamically stable than DNA/RNA duplexes (14,17,23) and 2'-O-methyl RNA probes are also chemically stable. The thermodynamic stability of 2'-O-methyl RNA/RNA duplexes can be enhanced by incorporation of LNA nucleotides (16), much as LNA stabilizes DNA/DNA (15,18,19) and DNA/RNA (15,18) hybrids. Here, we show that 2,6-diaminopurine substitution for A in 2'-O-methyl RNA or LNA nucleotides can further increase thermodynamic stabilities of hybrids with RNA and thereby reduce the sequence dependence of hybrid formation.

The 2,6-diaminopurine riboside (D) is an analog of adenosine containing an additional amino group at position 2 of the purine ring. The 2-amino group allows formation of a third hydrogen bond with uridine in the complementary strand. Previous studies have shown that 2,6-diaminopurine can increase the thermodynamic stability of RNA and DNA duplexes (20–22). The data presented here demonstrate that substitution of D for A increases thermodynamic stability ({Delta}{Delta}G°37) of fully complementary 2'-O-methyl RNA/RNA duplexes by 0.4–1.2 and 1.0–2.7 kcal/mol at 37°C, respectively, for each 2'-O-methyl-2,6-diaminopurine riboside (DM) or LNA-2,6-diaminopurine riboside (DL) present in the duplex. The results for fully complementary 2'-O-methyl RNA/RNA duplexes fit a nearest neighbor model for predicting stability and the effects of D and LNA substitutions are additive. Measurements of duplexes with mismatches indicate that internal D-A, D-C and D-G pairs are very destabilizing relative to D-U, thus providing specificity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
General methods
Mass spectra of nucleosides and oligonucleotides were obtained on an LC MS Hewlett Packard series 1100 MSD with API-ES detector or an MALDI-TOF MS, model Autoflex (Bruker). Thin-layer chromatography (TLC) purification of the oligonucleotides was carried out on Merck 60 F254 TLC plates with the mixture 1-propanol/aqueous ammonia/water = 55:35:10 (v/v/v). TLC analysis of reaction progress was performed on the same type of silica gel plates with various mixtures of dichloromethane and methanol (98:2 v/v, 95:5 v/v, 9:1 v/v and 8:2 v/v).

Chemical synthesis of phosphoramidite of 2'-O-methyl-2,6-diaminopurine riboside
The synthesis of protected 2'-O-methyl-2,6-diaminopurine riboside derivative was performed according to general procedures of the synthesis of 2'-O-methylnucleosides with some modifications (24). 2,6-Diaminopurine riboside was treated with 1,3-dichlorotetraisopropyldisiloxane (25) and then 5',3'-O-(tetraisopropyldisiloxane-1,3-diyl)-2,6-diaminopurine riboside was methylated with iodomethane in the presence of sodium hydride (24). The 2'-O-methylated derivative was treated with isobutyryl chloride followed by triethylammonium fluoride (25,26). Treatment of the last derivative with dimethoxytrityl chloride followed by 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite gave 5'-O-dimethoxytrityl-2'-O-methyl-N2,N6-diisobutyryl-2,6-diaminopurine riboside-3'-O-phosphoramidite in overall yield ca. 35%.

Synthesis and purification of oligonucleotides
Oligonucleotides were synthesized on an Applied Biosystems DNA/RNA synthesizer, using ß-cyanoethyl phosphoramidite chemistry (27). Commercially available A, C, G and U phosphoramidites with 2'-O-tertbutyldimethylsilyl or 2'-O-methyl groups were used for synthesis of RNA and 2'-O-methyl RNA, respectively (Glen Research, Azco, Proligo). The 3'-O-phosphoramidites of LNA nucleotides were synthesized according to published procedures (15,28,29) with some minor modifications. The details of deprotection and purification of oligoribonucleotides were described previously (12).

UV melting
Oligonucleotides were melted in buffer containing 100 mM NaCl, 20 mM sodium cacodylate, 0.5 mM Na2EDTA, pH 7.0. The relatively low sodium chloride concentration kept melting temperatures in the reasonable range even when there were multiple substitutions and also allowed comparison with previous experiments (14,16). Oligonucleotide single-strand concentrations were calculated from absorbance above 80°C and single-strand extinction coefficients were approximated by a nearest-neighbor model with D approximated as A (30,31). Absorbance vs temperature melting curves were measured at 260 nm with a heating rate of 1°C/min from 0 to 90°C on a Beckman DU 640 spectrophotometer with a thermoprogrammer. Melting curves were analyzed and thermodynamic parameters were calculated from a two-state model with the program MeltWin 3.5 (32). For most sequences, the {Delta}H° derived from TM–1 versus ln (CT/4) plots is within 15% of that derived from averaging the fits to individual melting curves, as expected if the two-state model is reasonable.

Parameter fitting
Thermodynamic parameters for predicting stabilities of 2'-O-methyl RNA/RNA with the Individual Nearest Neighbor Hydrogen Bonding (INN-HB) model (12) were obtained by multiple linear regression with the program Analyse-it v.1.71 (Analyse-It Software, Ltd., Leeds, England, www.analyse-it.com) which expands Microsoft Excel. Analyse-It was also used to obtain enhanced stability parameters for LNA-2'-O-methyl RNA/RNA duplexes when the LNAs are separated by at least one 2'-O-methyl nucleotide. Results from TM–1 vs ln (CT/4) plots were used as the data for the calculations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Chemical synthesis of protected LNA-2,6-diaminopurine riboside derivative
The derivative of LNA-2,6-diaminopurine was synthesized with an approach similar to that described for synthesis of natural LNA nucleosides (15,28,29) (Figure 1). The derivative of pentafuranose (1) (33) was condensed with trimethylsilylated 2,6-diaminopurine in 1,2-dichloroethane in the presence of trimethylsilyl trifluoromethanesulfonate as catalyst (34). Treatment of derivative (2) with lithium hydroxide resulted in the 5'-O-methanesulfonyl derivative (3), which was converted with lithium benzoate into the 5'-O-benzoyl derivative (4). The application of lithium benzoate instead of sodium benzoate very significantly improved solubility of the benzoate salt in N,N-dimethylformamide. Treatment of 5'-O-benzoyl derivative (4) with aqueous ammonia resulted in formation of (5). Removal of the 3'-O-benzyl with ammonium formate in the presence of Pd/C (35) resulted in formation of LNA-2,6-diaminopurine riboside (6). Derivative (6) was treated with acetyl chloride to produce (7), which was converted into LNA-N2,N6-diacetyl-2,6-diaminopurine riboside (8), using classical Khorana's procedure (36), and later into the 5'-O-dimethoxytrityl derivative (9). The overall yield of synthesis up to this step was 18%. In reaction of LNA-5'-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside (9) with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite was converted into LNA-5'-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside-3'-O-phosphoramidite (10) in 93% yield. It was possible to use acetyl instead of isobutyryl to protect the 2,6-amino groups of LNA-2,6-diaminopurine riboside because LNA-5'-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside-3'-O-phosphoramidite (10) is soluble in acetonitrile. This is in contrast to 5'-O-dimethoxytrityl-2'-O-methyl-N2,N6-diacetyl-2,6-diaminopurine riboside-3'-O-phosphoramidite. The details concerning chemical synthesis of derivatives (2–10) are described in Supplementary Data.


Figure 1
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  		Figure 1. Synthesis of LNA-2,6-diaminopurine phosphoramidite. Reagents and conditions: (i) 2,6-diaminopurine, HMDS, TMSOTf, dichloroethane; (ii) LiOH·H2O, THF, H2O; (iii) BzOLi, DMF; (iv) conc. NH4OH, Py; (v) Pd/C, HCOONH4, MeOH; (vi) AcCl, Py; (vii) KOH, Py, H2O, EtOH; (viii) DMTrCl, Py; (ix) 4,5-DCI, NC(CH2)2OP[N(iPr)2]2, CH3CN.

 
The thermodynamic stability of 2'-O-methyl RNA/RNA duplexes containing 2'-O-methyl-2,6-diaminopurine riboside or LNA-2,6-diaminopurine riboside
The adenosines in duplexes of the form 5'AMCMWMAMXMCMAM/r(3'UGZUYGU) were replaced singly or completely by 2'-O-methyl D (DM) or LNA D (DL), and the thermodynamics for duplex formation were measured (Table 1). Here WZ and XY are Watson–Crick base pairs. The results can be compared with previous measurements (14,16) for the unsubstituted duplexes and for the AMs substituted by LNA A (Table 1, see also Supplementary Data for complete thermodynamic data). When only a 5' or 3' terminal A is substituted by D with the same type of sugar, the average enhancement in stability at 37°C is 0.37 kcal/mol. If the middle A is substituted by D with the same type of sugar, then the average enhancement is 0.94 kcal/mol. Comparisons of {Delta}{Delta}G°37 values for the AM to DL replacements in Table 1 to the sum of corresponding AM to DM and AM to AL replacements, which in Table 1 are listed immediately below in square brackets, indicate that the effects of replacing A with D and 2'-O-methyl with LNA are essentially additive.


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  		Table 1. Thermodynamic parameters of duplex formation to RNA 7-mers complementary to the sequence showna

 
The results with DMCMUMAMCLCMAM, DLCMUMAMCLCMAM, DMCMUMDMCMCMDM, and DLCMUMDLCMCMDL suggest that the effects of multiple substitutions are also additive. For these sequences, the enhancement in heteroduplex stability relative to AMCMUMAMCMCMAM differs from the sum of enhancements due to the individual replacements by only 0.48, 0.29, 0.27 and 0.07 kcal/mol, respectively.

The results in Table 1 can be combined with previous results (14,16) to obtain nearest neighbor parameters for 2'-O-methyl RNA/RNA duplexes (Table 2). The nearest neighbor parameters with D are preliminary due to the small number of occurrences for them. The thermodynamic parameters for 5'AMCMUMAMGMCMAM/3'r(UGAUCGU) were re-measured and the values in Table 1 and Supplementary Data were used for deriving the nearest neighbor parameters. The parameters for nearest neighbors without D are similar to those reported previously (14).


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  		Table 2. Thermodynamic parameters for INN-HB nearest neighbor model applied to 2'-O-methyl RNA/RNA heteroduplexes in 0.1 M NaCl, pH 7a

 
The thermodynamic stability of 2'-O-methyl RNA/RNA duplexes containing mismatches formed by DM and DL nucleotides
Some single mismatches in RNA/RNA duplexes are particularly stable thermodynamically due to hydrogen bonding (37). Interpretation of microarray and other data must consider potential hybridization involving mismatches. Because it is important to determine the specificity of base pairing to modified nucleotides, mismatches with DM, DL, AM and AL were studied. Most mismatches were placed at an internal position within duplexes because that is statistically the most likely occurrence. Some terminal mismatches were also measured, however. The results from optical melting experiments are listed in Table 3 (see also Supplementary Data for complete thermodynamic data) and the differences between free energies of duplex formation with A-U or D-U and mismatch pairing for single internal mismatches at 37°C are summarized in Table 4.


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  		Table 3. Effects of mismatches on thermodynamic parameters of helix formationa

 

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  		Table 4. Summary of destabilization ({Delta}{Delta}G°) at 37°C due to internal mismatchesa

 
Many of the duplexes had melting temperatures <20°C, which makes measurements difficult. This is one reason that some of the transitions appear non-two-state as indicated by more than a 15% difference between {Delta}H° values derived from fitting the shapes of the melting curves or from TM–1 vs ln (CT/4) plots. Slightly, non-two-state melts were also found for four duplexes with melting temperature >31°C. For these sequences, there was at most a 19% difference between the derived {Delta}H° values. The {Delta}G°37 values for these sequences are still reliable because errors in {Delta}H° and {Delta}S° compensate making {Delta}G°37 values near the TM reliable (38). Values from non-two-state melts are listed in parentheses in Tables 3 and 4.

Mismatches at 5'- or 3'-terminal positions are typically less destabilizing than internal mismatches (16). To evaluate this effect, DM-G and DL-G mismatches were placed at 5'- or 3'-terminal positions of 2'-O-methyl RNA/RNA duplexes (Table 3C). For 5'XCMUMAMCMCMAM/3'rGGAUGGU duplexes, where X is DM or DL, the destabilization ({Delta}{Delta}G°37) is 0.78 and 0.98 kcal/mol for DM-G and DL-G, respectively. This is similar to the destabilizations of 0.37 and 0.58 kcal/mol when X is AM or AL, respectively. For 5'AMCMUMAMCMCMX/3'rUGAUGGG duplexes, where X is DM or DL, the destabilization is 0.67 and 0.56 kcal/mol for DM-G and DL-G, respectively. This is similar to the destabilization of 0.32 and 0.48 kcal/mol when X is AM or AL, respectively. While the differences between destabilizing by terminal A-G and D-G mismatches are within experimental error, the D-G mismatches are all more destabilizing than A-G suggesting that this is a real, albeit small, effect. As expected, the destabilization effect ({Delta}{Delta}G°37) is reduced compared with the same mismatches at an internal position as listed in Table 4.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY DATA
 REFERENCES
 
There are many reasons to modify the thermodynamic stabilities of nucleic acid duplexes. The application of microarrays of short oligonucleotides to probe RNA secondary structure (11) is one case where it is particularly useful to have sequences that base pair strongly and isoenergetically to RNA targets. Strong pairing permits the use of short oligonucleotides so that self-folding of probe is largely avoided. Moreover, short oligonucleotides provide enhanced specificity of binding (10). Isoenergetic binding further simplifies interpretation of data because binding will be primarily dependent on target structure rather than probe sequence. The synthesis of oligonucleotides with 2,6-diaminopurine described here provides a way to improve recognition of U in RNA targets by enhancing both binding and specificity. Moreover, the thermodynamic results provide approximations that allow design of isoenergetic probes. The design is relatively straightforward because the effects of non-adjacent modifications are usually additive. Short modified oligonucleotides could also be applied as antisense oligonucleotides (ASO) (3,39,40). They could also be useful to modulate binding and biological activity related to single nucleotide polymorphism (SNP) (41,42) and microRNAs (43–47).

Synthesis of LNA-2,6-diaminopurine riboside was reported by Rosenbohm et al. (20) and Koshkin et al. (21). Both used 2-amino-6-chloropurine as precursor of 2,6-diaminopurine. Rosenbohm used a saturated solution of ammonia in methanol to convert derivative of 2-amino-6-chloropurine riboside into 2,6-diaminopurine riboside and this transformation was accompanied by formation of 6-O-methyl derivative. Efficient synthesis (65% yield) of 2,6-diaminopurine riboside required not only specific temperature but particularly control of the pressure during this reaction. Koshkin proposed to convert the derivative of 2-amino-6-chloropurine riboside into 2-amino-6-azidopurine riboside and then into 2,6-diaminopurine riboside derivative simultaneously with deprotection of 3'-O-benzyl. An advantage of Rosenbohm and Koshkin approaches is universal character of 2-amino-6-chloropurine riboside derivative which beside 2,6-diaminopurine riboside can be transformed into LNA-guanosine and LNA-2-aminopurine riboside. A disadvantage is the much higher price of 2-amino-6-chloropurine than 2,6-diaminopurine. Moreover, both authors propose to use benzoyl as amino protecting group and in consequence using 40% aqueous solution of methylamine at 60–65°C for 2–4 h for deprotection of oligonucleotides containing 2,6-diaminopurine riboside. The method described herein is based on standard and much cheaper substrate as well as many well established procedures and is therefore a simple and efficient method for synthesizing LNA-2,6-diaminopurine riboside. Moreover, the chemical synthesis and deprotection of many oligonucleotides carrying LNA-2,6-diaminopurine riboside demonstrate that acetyl is very suitable for protection of amino groups in 2,6-diaminopurine residue.

Facile synthesis and incorporation of 2'-O-methyl-2,6-diaminopurine riboside and LNA-2,6-diaminopurine riboside into oligonucleotides allowed measurements of the thermodynamics for formation of 2'-O-methyl RNA/RNA and LNA-2'-O-methyl RNA/RNA duplexes containing DM and DL. The results show that incorporation of 2,6-diaminopurine into oligonucleotides allows modulation of duplex stability over a wide range.

Replacement of adenosine by DM and DL always enhances the thermodynamic stability of fully complementary 2'-O-methyl RNA/RNA and LNA-2'-O-methyl RNA/RNA duplexes. The largest stabilization is observed at internal positions where enhancements range from 0.7 to 1.2 kcal/mol with an average of 0.9 kcal/mol and 1.7–2.7 kcal/mol with an average of 2.3 kcal/mol, respectively, for DM and DL substituting for AM. The DM stabilization is in the range expected for addition of a hydrogen bond in RNA (48). The DL stabilization is the sum of the effects of an extra hydrogen bond and of the LNA. The enhancement ({Delta}{Delta}G°37) for DM and DL relative to AM and AL is less at 5'- and 3'-terminal positions where it averages 0.4 kcal/mol. This difference in stabilization at terminal and internal positions is likely due to the competition between stacking and hydrogen bonding at terminal base pairs (48) and to the particular sequences studied. Other sequences may show larger effects for 2,6-diaminopurine substitutions at terminal positions.

The stabilities of fully complementary 2'-O-methyl RNA/RNA and LNA-2'-O-methyl RNA/RNA duplexes at 37°C can be predicted reasonably well with simple models. The nearest neighbor parameters in Table 2 allow prediction of stabilities for 2'-O-methyl RNA/RNA duplexes using the INN-HB model (12) and the additional enhancement, {Delta}{Delta}G°37 (chimera/RNA), due to an LNA sugar can be predicted from:

Formula 1

(1)

Here n5'tL is the number of 5' terminal LNAs, niAL/UL, niDL and niGL/CL are the number of internal LNAs in A-U, D-U and G-C pairs, respectively, n3'tU and n3'tAL/CL/GL/DL are the number of 3' terminal LNAs that are U or not U, respectively. The equation is similar to that suggested previously (14,16), but has been updated to include the new results in Table 1. The predicted values are listed in square brackets in Table 1.

Mismatches formed by DM and DL destabilize duplexes (Tables 3 and 4). At the central position of duplexes with seven pairs that melt in a two-state manner, the destabilization ({Delta}{Delta}G°37) ranges between 2.3 and 5.2 kcal/mol at 37°C when D was only present as a mismatch. This corresponds to Kd's less favorable by 42 to 4600-fold at 37°C.

With the possible exception of the 5'CAA/3'GGU context, mismatches of DM and DL with G and A destabilize 2'-O-methyl RNA/RNA and LNA-2'-O-methyl RNA/RNA duplexes more than similar mismatches formed by AM and AL, respectively (Table 4). The trend of destabilization is reversed for D-C mismatches. The D-C mismatches might be stabilized by a hydrogen bond between the 2-amino group of D and the O2 of C.

Interestingly, the effect of a central D-G mismatch is enhanced when both terminal base pairs are D-U in the context 5'DCMUMDCMCMD/3'rUGAGGGU (Table 3). Here, the destabilization is 4.03 and 4.59 kcal/mol when each D is 2'-O-methyl or LNA, respectively, compared with 1.57 and 2.99 kcal/mol when the terminal nucleotides of the probe are 2'-O-methyl A. For mismatches at terminal positions, the destabilization ranges from 0.6 to 1.0 kcal/mol at 37°C. Mismatches with an LNA nucleotide are usually more destabilizing than those with a 2'-O-methyl nucleotide (Table 4).

The enhanced, variable and predictable duplex stability available from 2,6-diaminopurine substitutions with either 2'-O-methyl or LNA sugars makes them valuable for designing isoenergetic duplexes. The large destabilizations from internal mismatches means that oligonucleotides with 2,6-diaminopurine will be highly specific for their complementary sequence. Thus they should facilitate many applications of oligonucleotides, including microarray methods for probing RNA structure (11) and design of nanostructures (49–51).


   SUPPLEMENTARY DATA
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
REFERENCES
 
Supplementary Data are available at NAR Online.


   ACKNOWLEDGEMENTS
 
This work was supported by Polish State Committee for Scientific Research (KBN) Grant No 2 PO4A 03729 to R.K. and NIH grant 1R03 TW1068 to R.K. and D.H.T. A.P. is a recipient of a fellowship from the President of the Polish Academy of Sciences. The authors thank Walter Moss for writing the program to calculate thermodynamic stability of modified RNA duplexes. Funding to pay the Open Access publication charges for this article was provided by KBN.

Conflict of interest statement. None declared.


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

  1. Freier SM, Altman K-H. The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. (1997) 25:4429–4443.[Abstract/Free Full Text]

  2. Kurreck J. Antisense technologies. Improvement through novel chemical modifications. Eur. J. Biochem. (2003) 270:1628–1644.[ISI][Medline]

  3. Testa SM, Disney MD, Turner DH, Kierzek R. Thermodynamics of RNA-RNA duplexes with 2- or 4-thiouridines: Implications for antisense design and targeting a group I intron. Biochemistry (1999) 38:16655–16662.[CrossRef][Medline]

  4. Tolstrup N, Nielsen PS, Kolberg JG, Frankel AM, Vissing H, Kauppinen S. OligoDesign: optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling. Nucleic Acids Res. (2003) 31:3758–3762.[Abstract/Free Full Text]

  5. Darfeuille F, Hansen JB, Orum H, Primo CD, Toulme JJ. LNA/DNA chimeric oligomers mimics RNA aptamers targeted to the TAR RNA element of HIV-1. Nucleic Acids Res. (2004) 32:3101–3107.[Abstract/Free Full Text]

  6. Braasch DA, Corey DR. Locked nucleic acid (LNA): fine tuning the recognition of DNA and RNA. Chem. Biol. (2001) 8:1–7.[CrossRef][ISI][Medline]

  7. Kvaerno L, Wengel J. Antisense molecules and furanose conformations - is it really that simple? Chem. Commun. (2001) 1419–1424.

  8. Ooms M, Verhoef K, Southern EM, Huthoff H, Berkhout B. Probing alternative foldings of the HIV-1 leader RNA by antisense oligonucleotide scanning arrays. Nucleic Acids Res. (2004) 32:819–827.[Abstract/Free Full Text]

  9. Sohail M, Akhtar S, Southern EM. The folding of large RNAs studied by hybridization to arrays of complementary oligonucleotides. RNA (1999) 5:646–655.[Abstract]

  10. Gamper HB, Arar K, Gewirtz A, Hou YM. Unrestricted accessibility of short oligonucleotides to RNA. RNA (2005) 11:1441–1447.[Abstract/Free Full Text]

  11. Kierzek E, Kierzek R, Turner DH, Catrina IE. Facilitating RNA structure prediction with microarrays. Biochemistry (2006) 45:581–593.[CrossRef][Medline]

  12. Xia TB, SantaLucia J, Burkard ME, Kierzek R, Schroeder SJ, Jiao XQ, Cox C, Turner DH. Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry (1998) 37:14719–14735.[CrossRef][Medline]

  13. Obika S, Nanbu D, Hari Y, Morio K, In Y, Ishida T, Imanishi T. Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C-3'-endo sugar puckering. Tetrahedron Lett. (1997) 38:8735–8738.[CrossRef][ISI]

  14. Kierzek E, Mathews DH, Ciesielska A, Turner DH, Kierzek R. Nearest neighbor parameters for Watson-Crick complementary heteroduplexes formed between 2'-O-methyl RNA and RNA oligonucleotides. Nucleic Acids Res. (2006) 34:3609–3614.[Abstract/Free Full Text]

  15. Koshkin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, Olsen CE, Wengel J. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron (1998) 54:3607–3630.[CrossRef][ISI]

  16. Kierzek E, Ciesielska A, Pasternak K, Mathews DH, Turner DH, Kierzek R. The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes. Nucleic Acids Res. (2005) 33:5082–5093.[Abstract/Free Full Text]

  17. Inoue H, Hayase Y, Imura A, Iwai S, Miura K, Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. (1987) 15:6131–6148.[Abstract/Free Full Text]

  18. Vester B, Wengel J. LNA (Locked nucleic acid): High-affinity targeting of complementary RNA and DNA. Biochemistry (2004) 43:13233–13241.[Medline]

  19. McTigue PM, Peterson RJ, Kahn JD. Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation. Biochemistry (2004) 43:5388–5405.[CrossRef][Medline]

  20. Rosenbohm C, Pedersen DS, Frieden M, Jensen FR, Arent S, Larsen S, Koch T. LNA guanine and 2,6-diaminopurine. Synthesis, characterization and hybridization properties of LNA 2,6-diaminopurine containing oligonucleotides. Bioorg. Med. Chem. (2004) 12:2385–2396.[CrossRef][Medline]

  21. Koshkin AA. Syntheses and base-pairing properties of locked nucleic acid nucleotides containing hypoxanthine, 2,6-diaminopurine, and 2-aminopurine nucleobases. J. Org. Chem. (2004) 69:3711–3718.[CrossRef][ISI][Medline]

  22. Gaffney BL, Marky LA, Jones RA. The influence of the purine 2-amino group on DNA conformation and stability - II. Synthesis and physical characterization of d[CGT(2-NH2)ACG], d[CGU(2-NH2)ACG], and d[CGT(2-NH2)AT(2-NH2)ACG]. Tetrahedron (1984) 40:3–13.[CrossRef][ISI]

  23. Sugimoto N, Nakano S, Katoh M, Matsumura A, Nakamuta H, Ohmichi T, Yoneyama M, Sasaki M. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry (1995) 34:11211–11216.[CrossRef][Medline]

  24. Shohda K, Okamoto I, Wada T, Seio K, Sekine M. Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine. Bioorg. Med. Chem. Lett. (2000) 10:1795–1798.[CrossRef][Medline]

  25. Markiewicz WT. Tetraisopropyldisiloxane-1,3-diyl, a group for simultaneous protection of 3'- and 5'-hydroxy functions of nucleosides. J. Chem. Res. (S) (1979) 24–25.

  26. Markiewicz WT, Biala E, Kierzek R. Application of the tetraisopropyldisiloxane-1,3-diyl group in the chemical synthesis of oligoribonucleotides. Bull. Pol. Acad. Sci. (1984) 32:433–451.

  27. McBride LJ, Caruthers MH. An investigation of several deoxyribonucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides. Tetrahedron Lett. (1983) 24:245–248.[CrossRef][ISI]

  28. Koshkin AA, Fensholdt J, Pfundheller HM, Lomholt C. A simplified and efficient route to 2'-O, 4'-C-methylene- linked bicyclic ribonucleosides (locked nucleic acid). J. Org. Chem. (2001) 66:8504–8512.[CrossRef][ISI][Medline]

  29. Pedersen DS, Rosenbohm C, Koch T. Preparation of LNA phosphoramidites. Synthesis (2002) 802–808.

  30. Borer PN. CRC Handbook of Biochemistry and Molecular Biology: Nucleic Acids—Fasman GD, ed. (1975) 1, 3rd. Cleveland, OH: CRC Press. 589–595.

  31. Richards EG. CRC Handbook of Biochemistry and Molecular Biology: Nucleic Acids—Fasman GD, ed. (1975) 1, 3rd. Cleveland, OH: CRC Press. 596–603.

  32. McDowell JA, Turner DH. Investigation of the structural basis for thermodynamic stabilities of tandem GU mismatches: Solution structure of (rGAGGUCUC)2 by two-dimensional NMR and simulated annealing. Biochemistry (1996) 35:14077–14089.[CrossRef][Medline]

  33. Pfundheller HM, Lombolt C. Current Protocols in Nucleic Acid Chemistry.—Harkins EW, ed. (2002) 1. New York, NY, USA: John Wiley & Sons, Inc. 4.12.11–14.12.16.

  34. Vorbrüggen H, Krolikiewicz K. New catalysts for the synthesis of nucleosides. Angew. Chem. Int. Ed. Engl. (1975) 14:421–422.[CrossRef][ISI]

  35. Bieg T, Szeja W. Removal of O-benzyl protective groups by catalytic transfer hydrogenation. Synthesis (1985) 76:317–318.

  36. Schaller H, Weimann G, Lerch B, Khorana HG. Studies on polynucleotides. XXIV. The stepwise synthesis of specific deoxyribopolynucleotides (4). Protected derivatives of deoxyribonucleosides and new syntheses of deoxyribonucleoside-3' phosphates. J. Am. Chem. Soc. (1963) 85:3821–3827.[CrossRef][ISI]

  37. Kierzek R, Burkard ME, Turner DH. Thermodynamics of single mismatches in RNA duplexes. Biochemistry (1999) 38:14214–14223.[CrossRef][Medline]

  38. Freier SM, Petersheim M, Hickey DR, Turner DH. Thermodynamic studies of RNA stability. J. Biomol. Struct. Dyn. (1984) 1:1229–1242.[ISI][Medline]

  39. Lim TW, Yuan J, Liu Z, Qiu DX, Sall A, Yang DC. Nucleic-acid-based antiviral agents against positive single- stranded RNA viruses. Curr. Opin. Mol. Ther. (2006) 8:104–107.[ISI][Medline]

  40. Warfield KL, Panchal RG, Aman MJ, Bavari S. Antisense treatments for biothreat agents. Curr. Opin. Mol. Ther. (2006) 8:93–103.[ISI][Medline]

  41. Anthony RM, Schuitema ARJ, Chan AB, Boender PJ, Klatser PR, Oskam L. Effect of secondary structure on single nucleotide polymorphism detection with a porous microarray matrix; Implications for probe selection. Biotechniques (2003) 34:1082–1089.[ISI][Medline]

  42. Hong BJ, Sunkara V, Park JW. DNA microarrays on nanoscale-controlled surface. Nucleic Acids Res. (2005) 33:e106.[Abstract/Free Full Text]

  43. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science (2001) 294:853–858.[Abstract/Free Full Text]

  44. Hannon GJ. RNA interference. Nature (2002) 418:244–251.[CrossRef][Medline]

  45. Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science (2001) 294:858–862.[Abstract/Free Full Text]

  46. Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, Wojcik SE, Iorio MV, Visone R, Sever NI, et al. A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N. Engl. J. Med. (2005) 353:1793–1801.[Abstract/Free Full Text]

  47. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science (2001) 294:862–864.[Abstract/Free Full Text]

  48. Turner DH, Sugimoto N, Kierzek R, Dreiker SD. Free energy increments for hydrogen bonds in nucleic acid base pairs. J. Am. Chem. Soc. (1987) 109:3783–3785.[CrossRef][ISI]

  49. Sherman WB, Seeman NC. Design of minimally strained nucleic acid nanotubes. Biophys. J. (2006) 90:4546–4557.[CrossRef][ISI][Medline]

  50. Nasalean L, Baudrey S, Leontis NB, Jaeger L. Controlling RNA self-assembly to form filaments. Nucleic Acids Res. (2006) 34:1381–1392.[Abstract/Free Full Text]

  51. Lu Q, Moore JM, Huang G, Mount AS, Rao AM, Larcom LL, Ke PC. RNA polymer translocation with single-walled carbon nanotubes. Nano Lett. (2004) 4:2473–2477.[CrossRef][ISI]


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E. Kierzek, R. Kierzek, W. N. Moss, S. M. Christensen, T. H. Eickbush, and D. H. Turner
Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function
Nucleic Acids Res., April 1, 2008; 36(6): 1770 - 1782.
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