Supporting Information
Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2011
C5-Functionalized DNA, LNA, and a-l-LNA: Positional Control of PolaritySensitive Fluorophores Leads to Improved SNP-Typing**
Michael E. Østergaard,[a] Pawan Kumar,[a, b] Bharat Baral,[a] Dale C. Guenther,[a]
Brooke A. Anderson,[a] F. Marty Ytreberg,[c] Lee Deobald,[d] Andrzej J. Paszczynski,[d]
Pawan K. Sharma,[b] and Patrick J. Hrdlicka*[a]
chem_201002109_sm_miscellaneous_information.pdf
Contents
Representative RP-HPLC gradient protocol
S2
MALDI-MS of synthesized ONs
S2
Representative thermal denaturation curves against DNA complements
S3
Representative absorption spectra
S5
Representative excitation spectra
S6
Absorption and excitation maxima
S7
Steady state fluorescence emission spectra against DNA strands
S8
Fluorescence emission maxima
S11
Additional data towards complementary and mismatched RNA
S12
Computational study
S18
References
S20
NMR spectra
S21
S1
Table S1. Representative RP-HPLC gradient protocol.[a]
T (min)
0
2
50
64
69
71
80
Buffer A (V%)
100
100
30
0
0
100
100
Buffer B (V%)
0
0
70
100
100
0
0
[a] Buffer A is 0.05 M triethyl ammonium acetate pH 7, while buffer B is 75% MeCN in H2O v/v. A flow
rate of 1.2 mL/min was used.
Table S2. MALDI-MS of synthesized ONs.[a]
ON
ON5
ON6
ON7
ON8
ON9
ON10
ON11
ON12
ON13
ON14
ON15
ON16
[a]
For structures
Sequences
5’-CG CAA AXA AAC GC
5’-CG CAA CXC AAC GC
5’-CG CAA GXG AAC GC
5’-CG CAA TXT AAC GC
5’-CG CAA AYA AAC GC
5’-CG CAA CYC AAC GC
5’-CG CAA GYG AAC GC
5’-CG CAA TYT AAC GC
5’-CG CAA AZA AAC GC
5’-CG CAA CZC AAC GC
5’-CG CAA GZG AAC GC
5’-CG CAA TZT AAC GC
Calcd m/z [M]
4201.8
4229.8
4233.8
4183.8
4229.8
4182.7
4261.8
4211.8
4229.8
4182.7
4261.8
4211.8
of monomer X, Y, and Z,
S2
+
Found m/z [M]
4202.3
4230.2
4234.3
4184.3
4230.3
4182.0
4262.2
4212.1
4230.3
4182.3
4262.3
4212.3
see Fig. 1 in the
+
main manuscript.
Figure S1. Thermal denaturation curves for duplexes between ON5-ON8 (monomer X) and
complementary DNA.
Figure S2. Thermal denaturation curves for duplexes between ON9-ON12 (monomer Y) and
complementary DNA.
S3
Figure S3. Thermal denaturation curves for duplexes between ON13-ON16 (monomer Z) and
complementary DNA.
S4
S5
Figure S4. Absorption spectra of single stranded ON7, ON11 or ON15 (GBG-context) in absence (SSP) or
presence of matched or mismatched DNA targets (mismatched nucleotide opposite of modification is
mentioned in parenthesis).
S6
Figure S5. Normalized fluorescence excitation spectra of single stranded ON7, ON11 or ON15 (GBGcontext) in absence (SSP) or presence of matched or mismatched DNA targets (mismatched nucleotide
opposite
of
modification
is
mentioned
in
parenthesis).
S7
Table S3. Absorption maxima λmax of single stranded ON5-ON16 (SSPs) and corresponding duplexes with
matched or singly mismatched DNA targets.a
λmax (nm)
ON
5
6
7
8
9
10
11
12
13
14
15
16
Sequences
5’-CG CAA AXA AAC GC
5’-CG CAA CXC AAC GC
5’-CG CAA GXG AAC GC
5’-CG CAA TXT AAC GC
5’-CG CAA AYA AAC GC
5’-CG CAA CYC AAC GC
5’-CG CAA GYG AAC GC
5’-CG CAA TYT AAC GC
5’-CG CAA AZA AAC GC
5’-CG CAA CZC AAC GC
5’-CG CAA GZG AAC GC
5’-CG CAA TZT AAC GC
SSP
349
349
350
350
348
350
350
350
349
349
347
349
B=A
345
346
344
343
344
345
343
343
342
344
343
344
C
349
349
349
350
350
350
351
350
349
349
349
349
G
349
350
349
350
350
350
350
350
349
349
345
349
T
349
349
349
350
350
350
351
350
349
350
349
349
Absorption spectra were recorded in Tm buffer (see Table 1) using 1.0 µM concentrations of each
strand. Absorption was recorded from 220 to 390 nm on a Cary 100 Bio UV/VIS spectrophotometer;
each sample was scanned four times and the resulting averaged spectrum was smoothed using a filter
size of nine. Target sequences: 3’-GC GTT TBT TTG CG-5’ (for ON5/ON9/ON13), 3’-GC GTT GBG TTG CG5’ (for ON6/ON10/ON14), 3’-GC GTT CBC TTG CG-5’ (for ON7/ON11/ON15) and 3’-GC GTT ABA TTG CG5’ (for ON8/ON12/ON16) where B is A, C, G and T.
a
Table S4. Fluorescence excitation maxima λex,max of ON5-ON16 in absence (SSP) or presence of matched
or singly mismatched DNA targets (λem = 404 nm).a
λex, max (nm)
ON
5
6
7
8
9
10
11
12
13
14
15
16
Sequences
5’-CG CAA AXA AAC GC
5’-CG CAA CXC AAC GC
5’-CG CAA GXG AAC GC
5’-CG CAA TXT AAC GC
5’-CG CAA AYA AAC GC
5’-CG CAA CYC AAC GC
5’-CG CAA GYG AAC GC
5’-CG CAA TYT AAC GC
5’-CG CAA AZA AAC GC
5’-CG CAA CZC AAC GC
5’-CG CAA GZG AAC GC
5’-CG CAA TZT AAC GC
SSP
348
347
346
349
348
348
346
350
348
347
345
348
B=A
344
344
343
343
344
344
343
343
343
344
342
343
C
348
344
345
349
350
344
343
351
347
345
345
347
G
346
343
343
349
347
342
342
349
346
345
342
347
T
348
343
346
349
350
344
344
350
347
346
345
348
a
Conditions as described in footnote of Table 1. Target sequences: 3’-GC GTT TBT TTG CG-5’ (for
ON5/ON9/ON13), 3’-GC GTT GBG TTG CG-5’ (for ON6/ON10/ON14), 3’-GC GTT CBC TTG CG-5’ (for
ON7/ON11/ON15) and 3’-GC GTT ABA TTG CG-5’ (for ON8/ON12/ON16) where B is A, C, G and T.
Interestingly, only mismatched duplexes involving ONs in the ABA- and TBT-contexts display significant
bathochromic shifts (Table S4).
S8
Figure S6. Steady state fluorescence emission spectra of ON5, ON9 or ON13 (ABA-context) in absence
(SSP) or presence of matched or mismatched DNA targets (mismatched nucleotide opposite of
modification is mentioned in parenthesis).
S9
Figure S7. Steady state fluorescence emission spectra of single stranded ON6, ON10 or ON14 (CBCcontext) in absence (SSP) or presence of matched or mismatched DNA targets (mismatched nucleotide
opposite of modification is mentioned in parenthesis).
S10
Figure S8. Steady state fluorescence emission spectra of single stranded ON8, ON12 or ON16 (TBTcontext) in absence (SSP) or presence of matched or mismatched DNA targets (mismatched nucleotide
opposite of modification is mentioned in parenthesis).
S11
Table S5. Fluorescence emission maxima λem,max of ON5-ON16 in absence (SSP) or presence of matched
or singly mismatched DNA targets (λem = 404 nm).a
λex, max (nm)
ON
5
6
7
8
9
10
11
12
13
14
15
16
Sequences
5’-CG CAA AXA AAC GC
5’-CG CAA CXC AAC GC
5’-CG CAA GXG AAC GC
5’-CG CAA TXT AAC GC
5’-CG CAA AYA AAC GC
5’-CG CAA CYC AAC GC
5’-CG CAA GYG AAC GC
5’-CG CAA TYT AAC GC
5’-CG CAA AZA AAC GC
5’-CG CAA CZC AAC GC
5’-CG CAA GZG AAC GC
5’-CG CAA TZT AAC GC
SSP
407
406
405
407
408
407
405
408
408
406
406
407
a
B=A
403
402
402
402
403
402
401
402
403
403
401
402
C
405
405
403
406
407
400
404
407
405
403
404
404
G
405
404
403
408
407
404
403
406
408
405
403
406
T
406
404
404
406
407
400
403
407
407
405
404
407
Conditions as described in footnote of Table 1. Target sequences: 3’-GC GTT TBT TTG CG-5’ (for
ON5/ON9/ON13), 3’-GC GTT GBG TTG CG-5’ (for ON6/ON10/ON14), 3’-GC GTT CBC TTG CG-5’ (for
ON7/ON11/ON15) and 3’-GC GTT ABA TTG CG-5’ (for ON8/ON12/ON16) where B is A, C, G and T.
S12
Figure S9. Thermal denaturation curves for duplexes between ON2, ON6, ON10 or ON14 and
complementary RNA (CBC-context).
Figure S10. Thermal denaturation curves for duplexes between ON4, ON8, ON12 or ON16 and
complementary RNA (TBT-context).
S13
Table S6. Thermal denaturation temperatures of representative duplexes between probes and
complementary (B=A) or mismatched RNA targets.[a]
ON
2
6
10
14
4
8
12
16
Sequences
5’-CG CAA CTC AAC GC
5’-CG CAA CXC AAC GC
5’-CG CAA CYC AAC GC
5’-CG CAA CZC AAC GC
5’-CG CAA TTT AAC GC
5’-CG CAA TXT AAC GC
5’-CG CAA TYT AAC GC
5’-CG CAA TZT AAC GC
Tm (∆Tm)
[oC]
B=A
51.5
46.5 (-5.0)
49.5 (-2.5)
48.5 (-3.0)
40.0
35.5 (-4.5)
34.5 (-5.5)
37.0 (-3.0)
mismatch ∆Tm
[oC]
C
G
U
-15.5 -3.0 -13.5
-10.5 -5.0 -8.5
-14.0 -5.5 -11.5
-18.0 -4.5 -14.5
-19.0 -5.0 -18.0
-8.0
-8.5 -5.5
-12.0 -8.5 -10.5
-16.0 -8.0 -13.5
[a] For conditions see footnote of Table 1.
Incorporation of a single X-Z monomer into ONs with the representative CBC- and TBT-sequence
contexts, results in similar decreases in thermal affinity toward RNA complements as observed toward
DNA complements (compare data Table S6 and Table 1). Hybridization to mismatched RNA targets
results in decreased thermostability of the resulting duplexes. As with DNA, the following trend in target
specificity is observed in the TBT-context: thymidine > α-L-LNA monomer Z > LNA monomer Y > DNA
monomer X (Table S6) with a minor exception - α-L-LNA monomer Z displays better mismatch
discrimination in the CBC-context than thymidine.
Fluorescence properties of duplexes with complementary or mismatched RNA targets were evaluated in
an equivalent manner as for the corresponding DNA targets (see main manuscript). As seen for
DNA:DNA homoduplexes, the DNA:RNA heteroduplexes modified with monomers X-Z exhibit highly
unstructured fluorescence emission maxima centered at ~387 nm and ~402 nm indicating multiple
electronic interactions between the pyrene and surrounding nucleobases (Figs. S6-S8). Smaller increases
in fluorescence intensity were observed upon hybridization of ONs to RNA targets (2.2- to 4.3-fold, Fig.
S13) than upon hybridization with DNA targets. Just as for DNA:DNA duplexes, the duplexes between
probes and RNA targets display higher fluorescence emission quantum yields when ONs are modified
with LNA monomer Y or α-L-LNA monomer Z (ΦF = 0.50-0.69, Table S7). Similar trends as in studies with
DNA targets were observed in the emission spectra except for smaller bathochromic shifts of excitation
maxima in the TBT-contexts (Table S8).
S14
Figure S11. Steady state fluorescence emission spectra of single stranded ON6, ON10 or ON14 (CBCcontext) in absence (SSP) or presence of matched or mismatched RNA targets (mismatched nucleotide
opposite of modification is mentioned in parenthesis).
S15
Figure S12. Steady state fluorescence emission spectra of ON8, ON12 or ON16 (TBT-context) in absence
(SSP) or presence of matched or mismatched RNA targets (mismatched nucleotide opposite of
modification is mentioned in parenthesis).
S16
Table S7. Fluorescence quantum yields (ΦF) of probes with complementary or mismatched RNA targets.a
ΦF
ON
6
8
10
12
14
16
a
Sequences
5’-CG CAA CXC AAC GC
5’-CG CAA TXT AAC GC
5’-CG CAA CYC AAC GC
5’-CG CAA TYT AAC GC
5’-CG CAA CZC AAC GC
5’-CG CAA TZT AAC GC
B=A
0.48
0.44
0.69
0.51
0.68
0.50
C
0.11
0.23
0.27
0.36
0.46
0.18
G
0.26
0.27
0.27
0.30
0.31
0.39
U
0.11
0.19
0.21
0.24
0.36
0.14
Conditions as described in footnote of Table 1.
Table S8. Fluorescence excitation maxima of probes in absence (SSP) or presence of complementary
(B=A) or singly mismatched RNA targets (λem = 404 nm).a
λex,max (nm)
a
ON
6
8
Sequences
5’-CG CAA CXC AAC GC
5’-CG CAA TXT AAC GC
SSP
347
349
B=A
345
345
C
345
350
G
346
349
U
345
350
10
12
5’-CG CAA CYC AAC GC
5’-CG CAA TYT AAC GC
348
350
343
345
346
350
346
348
347
350
14
16
5’-CG CAA CZC AAC GC
5’-CG CAA TZT AAC GC
347
348
344
346
345
348
345
348
346
348
Conditions as described in footnote of Table 1.
S17
Figure S13. Fluorescence intensity, hybridization-induced increases and discrimination factors Im/Imm of
single stranded probes in the absence (SSPs) or presence of complementary RNA or mismatched RNA
targets. Panels depict CBC (upper) and TBT (lower) sequence contexts. Hybridization-induced increases and
discrimination factors (Im/Imm), defined as the fluorescence intensity of duplexes with complementary DNA divided
by the intensity of SSPs or duplexes with mismatched RNA, respectively, are listed above corresponding
histograms. Intensity recorded at λem = 402 nm at T = 5 °C.
S18
Molecular modelling protocol: We computed the Gibbs free energy as a function of glycosidic angle
using umbrella sampling and weighted histogram analysis for all three monomers.S1 Initial coordinates
for all nucleosides were generated using the JME molecular editor and PRODRGS2. Simulations were
performed using the ffAMBER portS3 of the GROMACS 4.0 software packageS4 with the AMBER-99SB
force field.S5 The nucleoside systems were solvated in TIP3P water and Na+ and Cl- ions were added to
neutralize the total charge. The system was then minimized for 500 steps to remove steric clashes,
followed by a 1.0 ns simulation with the position of the nucleoside heavy atoms restrained to allow for
equilibration of the water. For all three nucleosides we computed the free energy using 72 windows (5°
increments) where the glycosidic angle was harmonically restrained using a biasing potential energy
with force constant of 1000 kJ/mol/rad2. Each window was simulated for 1.0 ns; 500.0 ps were discarded
for equilibration, and 500.0 ps were used for the free energy analysis. All other simulation details are the
same as those in reference S6.
Computational studies: Given a) the distinctly different fluorescent properties of ONs modified with
monomers X-Z, b) the proposed importance of anti to syn rotation of the glycosidic torsion angle for
SNP-discrimination by monomer X, and that c) the extreme sugar puckering of LNA and α-L-LNA
nucleotides might influence the anti to syn rotational barrier (Fig. 1), we calculated the Gibbs free
energy as a function of the glycosidic angle for the corresponding O3′,O5′-unprotected nucleosides of
monomers X-Z (for structures, see Fig. S15). These Gibbs free energies were computed via the weighted
histrogram analysis methodS1 using all-atom molecular dynamics simulations in explicit solvent. Figure
S14 shows that, as expected, the anti conformation is the global minimum of all three nucleosides, with
a local minimum corresponding to the syn conformation (i.e., 25°<χ<45°). The syn conformation is the
second lowest minimum for the 5-[3-(1-pyrenecarboxamido)propynyl]-2 ′ -deoxyuridine and the
difference between the anti and syn conformation is approximately 17 kJ/mol, thus the syn
conformation is expected to be well populated. By contrast, the corresponding C5-functionalized LNA
nucleoside has two additional minima at -70° and +115°, while the C5-functionalized-α-L-LNA nucleoside
has one additional local minimum at -60°, all of which are of lower free energy than the syn
conformation. The syn conformation of the C5-functionalized LNA and α-L-LNA nucleosides will
therefore not be as highly populated as for the 5-[3-(1-pyrenecarboxamido)propynyl]-2′-deoxyuridine
nucleoside. While the results suggest that the bicyclic skeletons of the C5-functionalized LNA and α-LLNA nucleosides influence the glycosidic torsional angle profile, it remains unclear how these trends
translate to duplexes without additional molecular modeling and/or NMR studies.
S19
Figure S14. Gibbs free energy as a function of the glycosidic torsion angle for the corresponding free
nucleosides of monomers X-Z nucleotides. Structures of nucleosides used in the calculation are shown in
Fig. S15.
O
O
O
O
O
O
N
H
N
H
N
H
NH
NH
HO
N
O
HO
O
N
NH
O
O
O
OH
O
N
O
HO
OH O
OH
DNA derivative
LNA derivative
α-L-LNA derivative
Figure S15. Chemical structure of nucleosides used for molecular modeling studies shown in Figure S14.
S20
References
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1011–1021.
S2. A. W. Schuettelkopf, D. M. F. van Aalten, Acta Crystallogr. 2004, D60, 1355-1363.
S3. E. J. Sorin, V. S. Pande, Biophys. J. 2005, 26, 2472-2493.
S4. D. van der Spoel, E. Lindahl, B. Hess, G. Groenhof, A. E. Mark, H. J. C. Berendsen, J. Comput. Chem.
2005, 26, 1701–1718.
S5. V. Hornak, R. Abel, A. Okur, B. Strockbine, A. Roitberg, C. Simmerling, Proteins 2006, 65, 712-725.
S6. F. M. Ytreberg, J. Chem. Phys. 2009, 130, 164906 1-8.
S21