Synthesis of photolabile transcription initiators and preparation of photocleavable functional RNA by transcription

Article abstract of DOI:10.1016/j.bmcl.2012.05.028

Two new photolabile adenosine-containing transcription initiators with terminal thiol and amino functionalities are chemically synthesized. Transcription in the presence of the transcription initiators under the T7 phi2.5 promoter produces 5′ thiol- and amino-functionalized RNA conjugated by a photocleavable (PC) linker. Further RNA functionalization with biotin may be achieved through acyl transfer reactions from either biotinyl AMP to the RNA thiol group or biotin NHS to the RNA amino group. Photocleavage of the PC linker displays relatively fast kinetics with a half-life of 4-5 min. The availability of these transcription initiators makes new photolabile RNA accessible for affinity purification of RNA, in vitro selection of functional RNAs, and functional RNA caging.

Full text of DOI:10.1016/j.bmcl.2012.05.028

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4254–4258
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of photolabile transcription initiators and preparation
of photocleavable functional RNA by transcription
⇑
Faqing Huang , Yongliang Shi
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406-5043, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new photolabile adenosine-containing transcription initiators with terminal thiol and amino func-
tionalities are chemically synthesized. Transcription in the presence of the transcription initiators under
the T7 phi2.5 promoter produces 50 thiol- and amino-functionalized RNA conjugated by a photocleavable
(PC) linker. Further RNA functionalization with biotin may be achieved through acyl transfer reactions
from either biotinyl AMP to the RNA thiol group or biotin NHS to the RNA amino group. Photocleavage
of the PC linker displays relatively fast kinetics with a half-life of 4–5 min. The availability of these tran-
scription initiators makes new photolabile RNA accessible for affinity purification of RNA, in vitro selection
of functional RNAs, and functional RNA caging.
Received 10 April 2012
Revised 3 May 2012
Accepted 8 May 2012
Available online 17 May 2012
Keywords:
Transcription initiator
RNA labeling
RNA biotinylation
RNA photocleavage
In vitro selection
RNA caging
Ó 2012 Elsevier Ltd. All rights reserved.
RNAs with attached functional groups such as free amino and
thiol groups may be used to conjugate diverse chemical and bio-
chemical functionalities. Such functionalized RNAs have been
commonly used in chemistry and biomedical research.1–9 50
Thiol-labeled RNAs (50 HS-RNA) have been used for RNA purifica-
tion by affinity chromatography,5,10 for RNA immobilization on gold
surfaces,11,12 and for construction of gold nanoparticle-siRNA
(AuNP-siRNA) nanoplexes.13,10 In addition, RNA conjugation with
other macromolecules have been achieved with 50 thiol-functional-
ized RNAs.14–19
polymerization, amide bond cleavage and formation, glycosidic
bond formation, carboxyl activation, coenzyme incorporation and
synthesis, thioester synthesis, carbon–carbon bond formation,
Michael adduct formation, aldol reaction, alkylation, and ‘high
energy’ phosphoanhydride bond formation. In principle, conven-
tional in vitro selection methods can be designed to isolate desired
self-modifying ribozymes that catalyze chemical transformations
between a free substrate (often tagged for isolation, such as bio-
tin-tagging) and an RNA-conjugated chemical functional group.
However, there are multiple potential reaction sites on the RNA
molecule, including the hydroxyl groups on the ribose, the nitro-
gen sites on the nucleobase, and the 50 phosphate. Consequently,
unintended side reactions within RNA internal sites can often over-
shadow the intended chemistry at a predefined site.31–33
In many RNA applications, it is highly desirable that conjugated
chemical/biochemical functionalities be cleaved from RNA so that
captured RNA is released from resins/surfaces20 or that caged inac-
tive RNA becomes active.21–26 A cleavable chemical linker may be
used to connect a chemical/biochemical group and RNA. To facili-
tate linker cleavage, the linker may be chosen such that it is sensi-
tive to either reducing agents, acidic solutions, or photons. Among
the different cleavage methods, photocleavage has several distinct
advantages including clean cleavage at precise sites, avoidance of
using cleavage chemicals, and easy realization of spatial and tem-
poral cleavage control.21–25,27,26
The intrinsic limitation of conventional in vitro selection meth-
ods may be overcome by introducing a photocleavable linker (PC)
between a pendant reactant group and RNA, which may be
achieved by either photolabile guanosine derivative-initiated tran-
scription34–36 under the class III promoter (
U6.5) or ligation with a
oligoribonucleotide containing a reactant group and a PC.37,38
Expanding our previously developed high efficiency RNA labeling
methods6,8,9,39,10 based on adenosine derivative-initiated tran-
Generation of functional ribozymes by in vitro selection28–30
has been very successful. In vitro-isolated ribozymes can catalyze
impressive diverse chemistries, including phosphodiester bond
cleavage, phosphorylation, aminoacylation, RNA ligation, RNA
scription under the T7
U2.5 promoter, we describe the chemical
synthesis of two new photolabile adenosine derivatives (6 and
10) that are efficient transcriptional initiators for the preparation
of photocleavable RNAs. Compound 6 is a symmetrical adenosine
derivative that contains two adenosines, two PC groups, and a
disulfide bond. Photocleavage of 6 using a portable UV lamp
⇑
Corresponding author.
E-mail address: Faqing.Huang@usm.edu (F. Huang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.028

F. Huang, Y. Shi / Bioorg. Med. Chem. Lett. 22 (2012) 4254–4258
4255
(4 W) displays a relatively fast kinetics. Under standard transcrip-
tion conditions, the disulfide bond is reduced to a free thiol group.
As a result, the RNA transcript (7) initiated by 6 has a free thiol
group at the 50 end through a PC linker. Built on the half unit of
6, 10 contains an adenosine and a free terminal amino group linked
by a PC group. In vitro transcription in the presence of 10 results in
amino-functionalized RNA transcript (11) which can be cleaved off
by photon irradiation.
a NaOH solution (1.2 mL, 0.3 M, stirring for 1 h). Water (3 mL) was
then added to the solution to precipitate 4, which was collected by
centrifugation and washed by water (3 ꢀ 6 mL). The light yellow
solid was dried under vacuum until reaching a constant weight
(0.138 g, 74% yield). Both NMR and MS (Supplementary data) were
consistent with the structure of 4. The Boc protecting groups of 4
were removed by TFA treatment (1 mL of dichloromethane, 1 mL
of TFA, and 0.1 mL of triisopropylsilane, 45 min at room tempera-
ture). The resulting 5 was precipitated by ether (30 mL) and col-
lected by centrifugation, followed by washing with ether
(3 ꢀ 20 mL). It was then dried under vacuum until constant weight
(98 mg, 69% yield). The identity of 5 was confirmed by both NMR
and MS (in Supplementary data).
Following RNA preparation, both 50 thiol and amino functional-
ized RNAs can be further modified by other chemical groups through
acyl transfer reactions. Using biotin as an example, we have demon-
strated that two forms of biotin-tagged RNA—50 BiotinCO-S-PC-RNA
(8) and 50 BiotinCO-NH-PC-RNA (12), may be prepared through
either imidazole-catalyzed thioesterization reaction7 or biotinyla-
tion with an activated biotin ester. Finally, photocleavage kinetics
of biotin-conjugated RNAs 8 and 12 are presented to show relatively
fast PC linker cleavage upon photon irradiation.
The final step to 6 was achieved by conjugation of 5 with aden-
osine 50-phosphorimidazolide (ImdAMP).40,10 Compound 5 (80 mg,
0.12 mmol) was dissolved in 280
lL DMF. ImdAMP (800 lL, 1 M in
DMF) and triethylamine (120 L) were added and the mixture was
l
Synthesis of symmetrical thiol-containing transcription initiator—
HS-PC-AMP dimer (6): As shown in Scheme 1, the synthesis of 6
involved six chemical steps. To the starting 3-amino-3-(2-nitro-
phenyl)-propionic acid 1 (ANPPA, 110 mg, 0.52 mmol) dissolved in
1 mL of DMF, were added Boc-Gly-NHS (136 mg, 0.50 mmol) and
triethylamine (0.075 mL, 0.54 mmol). The reaction was stirred over-
night at room temperature to yield 2. N-hydroxysuccinimide
stirred for 4 days at room temperature to afford the photocleavable
symmetrical thiol-containing transcription initiator 6, which was
then purified by HPLC to yield the pure product (52 mg, 40
33% yield) (NMR, MS, in Fig. 1S in Supplementary data).
lmol,
Synthesis of photocleavable amino-containing transcription initi-
ator—NH2-PC-AMP (10): Based on 6, a photocleavable amino deriv-
ative 10 was prepared in 2 simple steps, as shown in Scheme 2. In
(69.0 mg, 0.60 mmol) and
N,N0-dicyclohexylcarbodiimide
the first step, 6 (20 mg, 15
the solution, were added mercaptoethanol (60
amine (30 L). After overnight reaction, compound 9 was precipi-
l
mol) was dissolved in 160
lL DMF. To
(206 mg,1.0 mmol) were then added to the above solution. The mix-
ture was stirred for 4 h at room temperature. The insoluble urea
derivative from the reaction was removed by centrifugation and
the resulting clear solution 3 was used directly for the next step
reaction.
l
L) and triethyl-
l
tated by ether and recovered by centrifugation. After multiple
washing with ether, followed by centrifugation and finally drying,
9 was recovered in high yield (20 mg). The second step involved
conjugation of an aminoethyl group with 9 through a thioether
Cystamine (32 lL, 7 M, 0.22 mmol) was added to the solution 3
and stirred overnight at room temperature to yield disulfide-linked
bond. Compound 9 (20 mg) was first dissolved in water (100
lL),
symmetrical 4. Any unreacted NHS ester was hydrolyzed by adding
following by adding 2-bromoethylamine (24 L, 5 M) and NaOH
l
Scheme 1. Chemical synthesis of symmetrical HS-PC-AMP conjugate (6), enzymatic preparation of HS-PC-RNA (7) by one-step in vitro transcription, and posttranscriptional
RNA biotinylation by imidazole-catalyzed thioesterification. (i) Boc-Gly-NHS, (ii) NHS, DCC, (iii) cystamine, (iv) TFA, (v) adenosine 50-phosphorimidazolide (ImdAMP).

4256
F. Huang, Y. Shi / Bioorg. Med. Chem. Lett. 22 (2012) 4254–4258
Scheme 2. Chemical synthesis of NH2-PC-AMP conjugate (10), preparation of NH2-PC-RNA (11) by in vitro transcription, and biotin-labeling of RNA (12) by chemical
conjugation. (i) mercaptoethanol, (ii) 2-bromoethylamine.
(70
l
L, 2 M). After 1 h reaction at room temperature, the solution
from transcription yielded 68% biotinylated RNA (Fig. 1B, lane 4).
Following HS-PC-RNA purification from RNA/HS-PC-RNA mixture
pH was adjusted to 7.0 by using acetic acid. Compound 10 was iso-
lated by HPLC to high purity (18 mg, 84% yield) (NMR, MS, in
Fig. 2S in Supplementary data).
Photocleavage kinetics of 6: A sample of 6 was dissolved in D2O
and placed in a quartz NMR tube. After UV irradiation at 360 nm
for different duration using a portable UV lamp (4 W), NMR spectra
were taken. By quantitating four well-resolved NMR peaks (Fig. 3S
in Supplementary data) that decreased over photo irradiation time,
a photocleavage kinetic curve was obtained (Figure 4S in Supple-
mentary Data). A half-life of 5–6 min under the photo irradiation
conditions was obtained from the curve. The result in general
agrees with those for other o-nitrobenzyl photocleavage.41
Preparation of 50 HS-PC-RNA (7): Using our standard transcrip-
tion protocol,5,42,43,39 50-thiol-PC labeled RNA (HS-PC-RNA, 7) was
prepared by in vitro transcription under the T7
U2.5 promoter
and in the presence of HS-PC-AMP dimer 6 (Scheme 1). For rela-
tively small RNA (<100 nt), the unlabeled RNA transcripts (pppRNA
as a result of ATP initiation) and thiol-PC-labeled RNA transcripts
can be separated directly by high resolution polyacrylamide gel
electrophoresis (PAGE). Figure 1A shows RNA thiol-PC-labeling un-
der different labeling conditions by a high resolution gel (single
nucleotide resolution). The gel indicates that RNA thiol-PC-labeling
yields can vary, depending on the concentration ratio of HS-PC-
AMP dimer 6 and ATP. At 2:1 and 4:1 ratios, 50-HS-PC-RNA yields
can reach 70–80%.
Figure 1. (A) High resolution polyacrylamide gel electrophoresis (PAGE, 8% with
7 M urea) analysis of 50 thiol-PC-labeling of RNA in the presence of different
concentrations of HS-PC-AMP conjugate (6). In addition to NTPs and 6, a trace
Synthesis of 50 BiotinCO-S-PC-RNA (8): Biotinylation of thiol-PC-
labeled RNA (HS-PC-RNA, 7) may be achieved quantitatively in
aqueous solutions by reaction with biotinyl adenylate (Biotin-
AMP) to form a biotinyl thioester in the presence of imidazole.7,10
As can be seen from Figure 1B (a low resolution gel), 50 biotin-PC-la-
beled RNA (BiotinCO-S-PC-RNA, 8) can bind streptavidin to form sta-
ble BiotinCO-S-PC-RNA-streptavidin complexes, which can be easily
separated from unlabeledRNA by PAGE.5,7,10 Under thebiotinylation
conditions, unlabeled RNA produced no RNA-streptavidin com-
plexes (Fig. 1B, lane 2), whereas RNA/HS-PC-RNA mixture directly
amount of [a-
32P] ATP was included in the transcription solution to label RNA
internally by 32P for the purpose of RNA analysis by PAGE and phosphorimaging.
Bands of HS-PC-RNA were completely separated from those of unlabeled RNA
(pppRNA). This high resolution (single nucleotide resolution) gel was achieved by
long gel-running time (1 h at 15 W for a 20 cm ꢀ 15 cm ꢀ 0.4 mm gel). (B) Low
resolution PAGE (8% with 7 M urea) analysis of streptavidin-Biotin–RNA complex
formation under different conditions. Biotinylated RNA-streptavidin complex (top
band) was easily fractionated from unlabeled RNA (bottom band). This low
resolution gel resulted from short gel-running time (10 min at 15 W for
a
20 cm ꢀ 15 cm ꢀ 0.4 mm gel). The RNA in the experiments contained 35 nucleo-
tides (nt) as described previously.43

F. Huang, Y. Shi / Bioorg. Med. Chem. Lett. 22 (2012) 4254–4258
4257
by thiolpropyl Sepharose 6B affinity column,7,10, high yield RNA bio-
tinylation can be obtained (Fig. 1B, lane 6).
Preparation of 50 NH2-PC-RNA (11): Following the same tran-
scription protocol,5,42,43,39 50-amino-PC labeled RNA (NH2-PC-
RNA, 11) was synthesized by in vitro transcription in the presence
of NH2-PC-AMP (10) (Scheme 2). High resolution PAGE analysis
indicated efficient RNA labeling by 50-amino-PC (Fig. 2A, lanes 2, 3).
Synthesis of 50 BiotinCO-NH-PC-RNA (12): An alternative way to
achieve RNA biotin-labeling is through Biotin-NHS reaction with a
free amine on the RNA (Scheme 2, step iv). As can be seen in Figure
2B, Biotin-NHS reaction (5 mM in 50:50 DMSO:water, 30 min at
room temperature) with RNA/NH2-PC-RNA mixture directly from
transcription resulted in high yield RNA biotinylation (Fig. 2B, lane
4), while unlabeled RNA produced very little biotinylated RNA un-
der the same conditions (Fig. 2B, lane 2).
Photocleavage kinetics of 8 and 12: After RNA biotinylation
reactions (Scheme 1, step vii and Scheme 2, step iv), 32P-labeled
BiotinCO-S-PC-RNA (8) and BiotinCO-NH-PC-RNA (12) were puri-
fied by denaturing PAGE to remove unreacted Biotin-AMP and
Biotin-NHS. Streptavidin was then mixed with BiotinCO-S-PC-
RNA and BiotinCO-NH-PC-RNA separately. After UV irradiation of
the streptavidin-RNA complexes for different duration, the sam-
ples were analyzed by PAGE. As shown in Figure 3, the PC linker
in both BiotinCO-S-PC-RNA and BiotinCO-NH-PC-RNA was rapidly
cleaved with similar kinetics to release free RNA, with a half-life of
4–5 min, similar to that of transcriptional initiator 6 photoclea-
vage. From the kinetic curves, it appeared that a small fraction of
Biotin-PC-RNA (ꢁ20%) had slower kinetics. This biphasic photoc-
leavage kinetics (Fig. 3) of Biotin-PC-RNA (8 or 12) differs to some
extent from transcriptional initiator 6 photocleavage kinetics
(Figure 4S in Supplementary data). It is unclear what factor(s) from
RNA contributed to such a difference.
In summary, we have synthesized two new photolabile adeno-
sine-containing transcription initiators (HS-PC-AMP dimer 6 and
NH2-PC-AMP 10) with terminal thiol and amino functionalities.
High yield labeling of RNA (>80%, depending on the 6:ATP or
10:ATP ratio) by either thiol-PC or amino-PC may be achieved by
Figure 3. Kinetics of photocleavable linkage of Biotin-PC-RNA–streptavidin com-
plexes under UV irradiation. The RNA-streptavidin samples (2 lL) were contained
in 0.5 ml eppendorf tubes and placed on ice. A portable UV lamp (UVL-56, UVP,
4 W) was then placed in contact above the tubes. At the indicated time, the tubes
were removed from UV irridiation. At the completion of UV-irradiation experi-
ments, the samples were analyzed by low resolution PAGE (8% with 7 M urea) for
photocleavage of Biotin-PC-RNA at different UV irradiation time. (A) BiotinCO-S-PC-
RNA photocleavage, (B) BiotinCO-NH-PC-RNA photocleavage.
transcription under the T7
U2.5 promoter and in the presence of
6 or 10. Following RNA preparation, the RNA may be further deriv-
atized post-transcriptionally with a broad range of acyl groups
through the terminal thiol and amino groups by well-established
acyl transfer reactions involving activated carboxylates (carboxyl-
ate-adenylates and carboxylate-NHS). In vitro cleavage of the
photocleavable linker between RNA and the terminal functional
group can be easily achieved by using simple portable long wave-
length UV lamps (360 nm). The availability of these two adeno-
sine-based transcription initiators makes new photolabile RNA
accessible for affinity purification of RNA, in vitro selection of func-
tional RNAs (aptamers and ribozymes), and functional RNA caging.
Acknowledgment
This work was supported by an NASA grant NNX07AI98G.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
028.
Figure 2. (A) High resolution PAGE (8% with 7 M urea) analysis of 50 amino-PC-
labeling of RNA in the presence of different concentrations of NH2-PC-AMP
conjugate (10). (B) Low resolution PAGE (8% with 7 M urea) analysis of streptavi-
din-Biotin–RNA complex formation. Transcription and PAGE conditions were the
same as those in Figure 1.
References and notes
1. Zhang, B.; Cech, T. R. Nature 1997, 390, 96.
2. Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389, 54.
3. Unrau, P. J.; Bartel, D. P. Nature 1998, 395, 260.

4258
F. Huang, Y. Shi / Bioorg. Med. Chem. Lett. 22 (2012) 4254–4258
23. Casey, J. P.; Blidner, R. A.; Monroe, W. T. Mol. Pharm. 2009, 6, 669.
4. Seelig, B.; Jaschke, A. Chem. Biol. 1999, 6, 167.
5. Coleman, T. M.; Huang, F. Chem. Biol. 2002, 9, 1227.
6. Huang, F.; Wang, G.; Coleman, T.; Li, N. RNA 2003, 9, 1562.
7. Coleman, T. M.; Li, N.; Huang, F. Tetrahedron Lett. 2005, 46, 4307.
8. Li, N.; Yu, C.; Huang, F. Nucleic Acids Res. 2005, 33, e37.
9. Guo, S.; Huang, F.; Guo, P. Gene Ther. 2006, 13, 814.
10. Huang, F.; Shi, Y. Bioorg. Med. Chem. Lett. 2010, 20, 6254.
11. Seetharaman, S.; Zivarts, M.; Sudarsan, N.; Breaker, R. R. Nat. Biotechnol. 2001,
19, 336.
24. Jain, P. K.; Shah, S.; Friedman, S. H. J. Am. Chem. Soc. 2010.
25. Yu, H.; Li, J.; Wu, D.; Qiu, Z.; Zhang, Y. Chem. Soc. Rev. 2010, 39, 464–473.
26. Matsushita-Ishiodori, Y.; Ohtsuki, T. Acc. Chem. Res. ASAP article, February 24,
2012, doi: http://dx.doi.org/10.1021/ar200227n.
27. Luo, Y.; Eldho, N. V.; Sintim, H. O.; Dayie, T. K. Nucleic Acids Res. 2011, 39, 8559.
28. Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818.
29. Tuerk, C.; Gold, L. Science 1990, 249, 505.
30. Robertson, D. L.; Joyce, G. F. Nature 1990, 344, 467.
31. Wilson, C.; Szostak, J. W. Nature 1995, 374, 777.
32. Jenne, A.; Famulok, M. Chem. Biol. 1998, 5, 23.
12. McCarthy, J. E.; Marsden, S.; von der Haar, T. Methods Enzymol. 2007, 430, 247.
13. Giljohann, D. A.; Seferos, D. S.; Prigodich, A. E.; Patel, P. C.; Mirkin, C. A. J. Am.
Chem. Soc. 2009, 131, 2072.
33. Li, N.; Huang, F. Biochemistry 2005, 44, 4582.
14. Heredia, K. L.; Nguyen, T. H.; Chang, C. W.; Bulmus, V.; Davis, T. P.; Maynard, H.
D. Chem. Commun. (Camb) 2008, 3245.
34. Eisenfuhr, A.; Arora, P. S.; Sengle, G.; Takaoka, L. R.; Nowick, J. S.; Famulok, M.
Bioorg. Med. Chem. 2003, 11, 235.
15. Lee, J. S.; Green, J. J.; Love, K. T.; Sunshine, J.; Langer, R.; Anderson, D. G. Nano
Lett. 2009, 9, 2402.
35. Fusz, S.; Eisenfuhr, A.; Srivatsan, S. G.; Heckel, A.; Famulok, M. Chem. Biol. 2005,
12, 941.
16. Vazquez-Dorbatt, V.; Tolstyka, Z. P.; Chang, C. W.; Maynard, H. D.
Biomacromolecules 2009, 10, 2207.
36. Fusz, S.; Srivatsan, S. G.; Ackermann, D.; Famulok, M. J. Org. Chem. 2008, 73,
5069.
17. Boyer, C.; Bulmus, V.; Davis, T. P. Macromol. Rapid Commun. 2009, 30, 493.
18. Xu, J.; Boyer, C.; Bulmus, V.; Davis, T. P. J. Polym Sci., Part A: Polym. Chem. 2009,
47, 4302.
37. Hausch, F.; Jaschke, A. Bioconjugate Chem. 1997, 8, 885.
38. Sengle, G.; Eisenfuhr, A.; Arora, P. S.; Nowick, J. S.; Famulok, M. Chem. Biol.
2001, 8, 459.
19. York, A. W.; Zhang, Y.; Holley, A. C.; Guo, Y.; Huang, F.; McCormick, C. L.
Biomacromolecules 2009, 10, 936.
20. Olejnik, J.; Krzymanska-Olejnik, E.; Rothschild, K. J. Nucleic Acids Res. 1996, 24,
361.
39. Huang, F.; He, J.; Zhang, Y.; Guo, Y. Nat. Protoc. 1848, 2008, 3.
40. Joyce, G. F.; Inoue, T.; Orgel, L. E. J. Mol. Biol. 1984, 176, 279.
41. Kim, M. S.; Diamond, S. L. Bioorg. Med. Chem. Lett. 2006, 16, 4007.
42. Huang, F. Nucleic Acids Res. 2003, 31, e8.
21. Shah, S.; Rangarajan, S.; Friedman, S. H. Angew. Chem., Int. Ed. 2005, 44, 1328.
22. Shah, S.; Jain, P. K.; Kala, A.; Karunakaran, D.; Friedman, S. H. Nucleic Acids Res.
2009, 37, 4508.
43. Coleman, T. M.; Wang, G.; Huang, F. Nucleic Acids Res. 2004, 32, e14.