Published online 12 June 2007Nucleic Acids Research, 2007, Vol. 35, No. 124055–4063
A chemical synthesis of LNA-2,6-diaminopurine
riboside, and the influence of 20-O-methyl-2,
6-diaminopurine and LNA-2,6-diaminopurine
ribosides on the thermodynamic properties
of 20-O-methyl RNA/RNA heteroduplexes
Anna Pasternak1, Elzbieta Kierzek1, Karol Pasternak1, Douglas H. Turner2and
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
Received October 19, 2006; Revised March 28, 2007; Accepted May 8, 2007
Modified nucleotides are useful tools to study the
structures, biological functions and chemical and
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 30-O-phosphorami-
dite of LNA-2,6-diaminopurine riboside is described.
The derivatives of 20-O-methyl-2,6-diaminopurine
and LNA-2,6-diaminopurine ribosides were used to
prepare complete 20-O-methyl RNA and LNA-20-O-
methyl RNA chimeric oligonucleotides to pair with
RNA oligonucleotides. Thermodynamic stabilities of
these duplexes demonstrated that replacement of a
single internal 20-O-methyladenosine with 20-O-
methyl-2,6-diaminopurine riboside (DM) or LNA-2,6-
diaminopurine riboside (DL) increases the thermo-
dynamic stability (""G837) on average by 0.9 and 2.3
kcal/mol, respectively. Moreover, the results fit a
nearest neighbor model for predicting duplex stabil-
ity at 378C. D-A and D-G but not D-C mismatches
formed by DMor DLgenerally destabilize 20-O-
methyl RNA/RNA and LNA-20-O-methyl RNA/RNA
duplexes relative to the same type of mismatches
adenosine, respectively. The enhanced thermody-
namic stability of fully complementary duplexes and
mismatched duplexes are useful for many RNA
studies, including those involving microarrays.
thermodynamic stabilityof some
Modified nucleotides are useful tools to study the
structures, biological functions and chemical and thermo-
dynamic 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 nucleo-
tides 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
(??G837) 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
*To whom correspondence should be addressed. Tel: 48 62 852 85 03; Fax: 48 62 852 05 32; Email: firstname.lastname@example.org
? 2007 The Author(s)
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deduce RNA secondary structure (11) used 20-O-methyl
RNA probes because 20-O-methyl RNA/RNA duplexes
are more thermodynamically stable than DNA/RNA
are also chemically stable. The thermodynamic stability
of 20-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 substitu-
tion for A in 20-O-methyl RNA or LNA nucleotides can
further increase thermodynamic stabilities of hybrids with
RNA and thereby reduce the sequence dependence of
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 stabi-
lity of RNA and DNA duplexes (20–22). The data
presented here demonstrate that substitution of D for A
increases thermodynamic stability (??G837) of fully
complementary 20-O-methyl RNA/RNA duplexes by
0.4–1.2 and 1.0–2.7 kcal/mol at 378C, respectively, for
each 20-O-methyl-2,6-diaminopurine riboside (DM) or
LNA-2,6-diaminopurine riboside (DL) present in the
duplex. The results for fully complementary 20-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
and D-G pairs are very destabilizing relative to D-U,
thus providing specificity.
MATERIALS AND 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 F254TLC 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 20-O-methyl-
The synthesis of protected 20-O-methyl-2,6-diaminopurine
riboside derivative was performed according to general
procedures of the synthesis of 20-O-methylnucleosides with
some modifications (24). 2,6-Diaminopurine riboside was
treated with 1,3-dichlorotetraisopropyldisiloxane (25) and
nopurine riboside was methylated with iodomethane in
the presence of sodium hydride (24). The 20-O-methylated
derivative was treated with isobutyryl chloride followed by
triethylammonium fluoride (25,26). Treatment of the last
derivative with dimethoxytrityl chloride followed by
overall yield ca. 35%.
Synthesis andpurification of oligonucleotides
Biosystems DNA/RNA synthesizer, using b-cyanoethyl
phosphoramidite chemistry (27). Commercially available
A, C, G and U phosphoramidites with 20-O-tertbutyldi-
synthesis of RNA and 20-O-methyl RNA, respectively
(Glen Research, Azco, Proligo). The 30-O-phosphorami-
dites of LNA nucleotides were synthesized according
to published procedures (15,28,29) with some minor
purification of oligoribonucleotides were described pre-
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
were calculated from absorbance above 808C 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 260nm with a heating rate of 18C/min from
0 to 908C 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 ?H8 derived from TM?1versus 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.
Thermodynamic parameters for predicting stabilities of
20-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
RNA/RNA duplexes when the LNAs are separated
by at least one 20-O-methyl nucleotide. Results from
TM?1vs ln (CT/4) plots were used as the data for the
Nucleic Acids Research, 2007, Vol. 35, No. 12
Chemical synthesis ofprotected LNA-2,6-diaminopurine
The derivative of LNA-2,6-diaminopurine was synthe-
sized with an approach similar to that described for
(Figure 1). The derivative of pentafuranose (1) (33) was
condensed with trimethylsilylated 2,6-diaminopurine in
1,2-dichloroethane in the presence of trimethylsilyl tri-
fluoromethanesulfonate as catalyst (34). Treatment of
derivative (2) with lithium hydroxide resulted in the 50-O-
methanesulfonyl derivative (3), which was converted with
lithium benzoate into the 50-O-benzoyl derivative (4).
The application of lithium benzoate instead of sodium
the benzoate salt in N,N-dimethylformamide. Treatment
of 50-O-benzoyl derivative (4) with aqueous ammonia
resulted in formation of (5). Removal of the 30-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 50-O-
dimethoxytrityl derivative (9). The overall yield of
synthesis up to this step was 18%. In reaction of LNA-
riboside (9) with 2-cyanoethyl-N,N,N0,N0-tetraisopropyl-
side-30-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
2,6-diaminopurine riboside-30-O-phosphoramidite (10) is
soluble in acetonitrile. This is in contrast to 50-O-
purine riboside-30-O-phosphoramidite. The details con-
cerning chemical synthesis of derivatives (2–10) are
described in Supplementary Data.
The thermodynamic stability of 20-O-methyl RNA/RNA
duplexescontaining 20-O-methyl-2,6-diaminopurine riboside
replaced singly or completely by 20-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 thermody-
namic data). When only a 50or 30terminal A is substituted
by D with the same type of sugar, the average enhance-
ment in stability at 378C 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
??G837values for the AMto DLreplacements in Table 1
to the sum of corresponding AMto DMand AMto AL
replacements, which in Table 1 are listed immediately
below in square brackets, indicate that the effects of
replacing A with D and 20-O-methyl with LNA are
DLCMUMDLCMCMDLsuggest that the effects of multiple
substitutions are also additive. For these sequences, the
AMCMUMAMCMCMAMdiffers from the sum of enhance-
ments 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
(Table 2). The nearest neighbor parameters with D
are preliminary due to the small number of occurrences
(3) R = Ms, R′ = Bn, R″ = H
(4) R = Bz, R′ = Bn, R″ = H
(5) R = H, R′ = Bn, R″ = H
(6) R = R′ = R″ = H
(8) R = R′ = H, R″ = Ac
(9) R = DMTr, R′ = H, R″ = Ac
(7) R = R′ = R″ = Ac
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,
Nucleic Acids Research, 2007, Vol. 35, No. 124057
50AMCMUMAMGMCMAM/30r(UGAUCGU) were re-mea-
sured 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).
The thermodynamic stability of20-O-methyl RNA/RNA
duplexes containing mismatches formed by DMandDL
Some single mismatches in RNA/RNA duplexes are
particularly stable thermodynamically due to hydrogen
Table 1. Thermodynamic parameters of duplex formation to RNA 7-mers complementary to the sequence showna
LNA-20OMe RNA (50–30)
A. Duplex formation with DM-U or DL-U at terminal positions.
B. Duplex formation with DM-U or DL-U at internal positions.
C. Duplex formation with three DM-U or DL-U base pairs.
aSolutions are 100 mM NaCl, 20 mM sodium cacodylate and 0.5 mM Na2EDTA, pH 7. Values are from TM?1versus log (CT/4) plots. Values in
parentheses are from non-two-state melts.
bValues in square brackets are predicted on the basis of the INN-HB model (Table 2) and equation 1.
cCalculated for 10?4M total oligonucleotide strand concentration.
dThe differences in ?G837compared with completely 20-O-methyl RNA strand without any D (14,16).
eThe differences in ?G837due to substitution DLfor ALin the same 20-O-methyl RNA strand (16).
fThe differences in ?G837due to substitution of LNA for 20-O-methyl RNA,
gThe sum of differences in ?G837for AMto DMand AMto ALsubstitutions. The thermodynamic data of some reference 20-O-methyl RNA/RNA
duplexes were published earlier (14,16).
Nucleic Acids Research, 2007, Vol. 35, No. 12
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, AMand ALwere studied.
Most mismatches were placed at an internal position
within duplexes because that is statistically the most likely
occurrence. Some terminal mismatches were also mea-
sured, however. The results from optical melting experi-
ments 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 378C are summarized in Table 4.
Many of the duplexes had melting temperatures5208C,
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 ?H8
values derived from fitting the shapes of the melting curves
or from TM?1vs ln (CT/4) plots. Slightly, non-two-state
melts were also found for four duplexes with melting
temperature 4318C. For these sequences, there was at
most a 19% difference between the derived ?H8 values.
The ?G837values for these sequences are still reliable
making ?G837values near the TMreliable (38). Values
from non-two-state melts are listed in parentheses in
Tables 3 and 4.
Mismatches at 50- or 30-terminal positions are typically
less destabilizing than internal mismatches (16). To
evaluate this effect, DM-G and DL-G mismatches were
placed at 50- or 30-terminal positions of 20-O-methyl RNA/
RNA duplexes (Table 3C). For 50XCMUMAMCMCMAM/
30rGGAUGGU duplexes, where X is DMor DL, the
destabilization (??G837) 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
30rUGAUGGG duplexes, where X is DMor 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 AMor 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 (??G837) is reduced
compared with the same mismatches at an internal
position as listed in Table 4.
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
Table 2. Thermodynamic parameters for INN-HB nearest neighbor model applied to 20-O-methyl RNA/RNA heteroduplexes in 0.1M NaCl, pH 7a
?S8b(eu) Number of occurrences
Per Terminal AU
aDigits beyond experimental error are provided to allow better predictions of melting temperature. See (12) for INN-HB model.
bCalculated from ?S8=(?H8 ? ?G8)/310.15.
Nucleic Acids Research, 2007, Vol. 35, No. 124059
binding will be primarily dependent on target structure
rather than probe sequence. The synthesis of oligonucleo-
tides 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 oligo-
nucleotides could also be applied as antisense oligonucleo-
tides (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
Table 3. Effects of mismatches on thermodynamic parameters of helix formationa
LNA-20OMe RNA (50–30) RNA (50–30)
A. Effects of DM-A, DL-A, AM-A, and AL-A mismatches at internal positions.
B. Effects of DM-C, DL-C, AM-C, and AL-C mismatches at internal positions.
C. Effects of DM-G, DL-G, AM-G, and AL-G mismatches at internal and terminal positions.
D. Effects of DM-G, DL-G, AM-G, and AL-G mismatches in the presence of DM-U and DL-U base pairs.
aSolutions are 100 mM NaCl, 20 mM sodium cacodylate and 0.5 mM Na2EDTA, pH 7. Values are form TM?1vs log (CT/4) plots. Values in
parentheses are from non-two state melts.
bCalculated for 10?4M oligonucleotide strand concentration.
cDifference compared with duplex formation when bold A or D is paired with U.
Nucleic Acids Research, 2007, Vol. 35, No. 12
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
with deprotection of 30-O-benzyl. An advantage of
Rosenbohm and Koshkin approaches is universal char-
which beside 2,6-diaminopurine riboside can be trans-
formed into LNA-guanosine and LNA-2-aminopurine
riboside. A disadvantage is the much higher price of
Moreover, both authors propose to use benzoyl as
amino protecting group and in consequence using
40% aqueous solution of methylamine at 60–658C
for 2–4h 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 synth-
esis and deprotection of many oligonucleotides carrying
LNA-2,6-diaminopurine riboside demonstrate that acetyl
is very suitable for protection of amino groups in
Facile synthesis and incorporation of 20-O-methyl-2,6-
diaminopurine riboside and LNA-2,6-diaminopurine ribo-
side into oligonucleotides allowed measurements of the
thermodynamics for formation of 20-O-methyl RNA/
containing DMand DL. The results show that incorpora-
tion of 2,6-diaminopurine into oligonucleotides allows
modulation of duplex stability over a wide range.
Replacement of adenosine by DMand DLalways
enhances the thermodynamic stability of fully comple-
mentary 20-O-methyl RNA/RNA and LNA-20-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
The DMstabilization is in the range expected for addition
of a hydrogen bond in RNA (48). The DLstabilization
is the sum of the effects of an extra hydrogen bond and
of the LNA. The enhancement (??G837) for DMand DL
relative to AMand ALis less at 50- and 30-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 atterminal base
show larger effects for 2,6-diaminopurine substitutions at
The stabilities of fully complementary 20-O-methyl
RNA/RNA and LNA-20-O-methyl RNA/RNA duplexes
at 378C can be predicted reasonably well with simple
models. The nearest neighbor parameters in Table 2 allow
prediction of stabilities for 20-O-methyl RNA/RNA
duplexes using the INN-HB model (12) and the additional
enhancement, ??G837(chimera/RNA), due to an LNA
sugar can be predicted from:
substituting for AM.
Here n50tLis the number of 50terminal LNAs, niAL/UL,
niDLand niGL/CLare the number of internal LNAs in A-U,
D-U and G-C pairs, respectively, n30tUand n30tAL/CL/GL/DL
are the number of 30terminal 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 DMand DLdestabilize duplexes
(Tables 3 and 4). At the central position of duplexes with
seven pairs that melt in a two-state manner, the
destabilization (??G837) ranges between 2.3 and 5.2
kcal/mol at 378C when D was only present as a mismatch.
This corresponds to Kd’s less favorable by 42 to 4600-fold
With the possible exception of the 50CAA/30GGU
context, mismatches of DMand DLwith G and A
methyl RNA/RNA duplexes more than similar mis-
matches formed by AMand AL, respectively (Table 4).
The trend of destabilization is reversed for D-C mis-
matches. The D-C mismatches might be stabilized by a
hydrogen bond between the 2-amino group of D and the
O2 of C.
Table 4. Summary of destabilization (??G8) at 378C due to internal
30A G U
30A G U
30U G U
30U G U
30C G U
30C G U
30G G U
30G G U
30A G A
30A G A
30A A A
30A A A
30A G G
30A A G
30A C A
30A C A
30A C G
30A C G
aTop value is for AMor DMand bottom value is for ALor DL; values
in parentheses are from non-two-state melts.
Nucleic Acids Research, 2007, Vol. 35, No. 124061
Interestingly, the effect of a central D-G mismatch is
enhanced when both terminal base pairs are D-U in the
context 50DCMUMDCMCMD/30rUGAGGGU (Table 3).
Here, the destabilization is 4.03 and 4.59 kcal/mol when
each D is 20-O-methyl or LNA, respectively, compared
with 1.57 and 2.99 kcal/mol when the terminal nucleotides
of the probe are 20-O-methyl A. For mismatches at
0.6 to 1.0 kcal/mol at 378C. Mismatches with an LNA
nucleotide are usually more destabilizing than those with a
20-O-methyl nucleotide (Table 4).
The enhanced, variable and predictable duplex stability
available from 2,6-diaminopurine substitutions with either
20-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 micro-
array methods for probing RNA structure (11) and design
of nanostructures (49–51).
Supplementary Data are available at NAR Online.
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.
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