An RNA modification with remarkable resistance to RNase A

Article abstract of DOI:10.1039/c3cc44621b

A 3′-deoxy-3′-C-methylenephosphonate modified diribonucleotide is highly resistant to degradation by spleen phosphodiesterase and not cleaved at all by snake venom phosphodiesterase. The most remarkable finding is that, despite the fact that both the vicinal 2-hydroxy nucleophile and the 5′-oxyanion leaving group are intact, the 3′-methylenephosponate RNA modification is also highly resistant towards the action of RNase A.

Full text of DOI:10.1039/c3cc44621b

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An RNA modification with remarkable resistance to
RNase A†
Cite this: Chem. Commun., 2013,
4
9, 9036
a
a
b
a
Alice Ghidini, Charlotte Ander, Anna Winqvist and Roger Str o¨ mberg*
Received 19th June 2013,
Accepted 8th August 2013
DOI: 10.1039/c3cc44621b
www.rsc.org/chemcomm
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0
A 3 -deoxy-3 -C-methylenephosphonate modified diribonucleotide
is highly resistant to degradation by spleen phosphodiesterase and
not cleaved at all by snake venom phosphodiesterase. The most
remarkable finding is that, despite the fact that both the vicinal
0
2
-hydroxy nucleophile and the 5 -oxyanion leaving group are intact,
0
the 3 -methylenephosponate RNA modification is also highly resistant
towards the action of RNase A.
Access to synthetic nucleic acid fragments has been pivotal in
the development of life science research and modified oligo-
nucleotides show considerable promise for disease therapeutics.
A general theme in the different therapeutic approaches, such as
1
2
siRNA (short interfering RNA), antisense technologies, including
3
0
pre-mRNA splice-switching and even in the development of Fig. 1 RNA with a 3 methylenephosphonate linkage.
4
–6
artificial ribonucleases,
are stabilised towards enzymatic degradation and have an
increased affinity for the target. However, due to the presumed H-phosphonate approach and its application in oligonucleotide
lability in biological fluids, the oligoribonucleotide analogues synthesis. After the latter appeared, it seemed likely that an
developed typically do not carry a 2 -hydroxyl. The few examples analogous approach via alkylhydrogenphosphinates should be
is the use of oligonucleotides that
14
1
5
0
include non-natural linkages not recognized by enzymes, possible for incorporation of methylenephosphonate linkages
7
8
such as internucleosidic amides and acetals. Among the most into oligonucleotides. We have since developed synthetic
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0
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common modifications are instead 2 -O-alkyl groups and methods for key steps towards ribonucleoside 3 -deoxy-3 -C-
1
0
0
0
phosphorothioates, mostly without free 2 -hydroxyl groups. methylenephosphinates, i.e., a 3 -carbon extension at the
In addition, modified di- and oligonucleotides have been used nucleoside level, synthesis of methylenephosphinate building
as potential enzyme inhibitors and in investigations of enzy- blocks and oxidation of methylenephosphinate linkages as
matic mechanisms for a number of decades, and the use has well as evaluation of condensing agents for formation of the
increased with the number of analogues available. An inter- methylenephosphinate internucleoside linkage. The potential
esting modification that was introduced in diribonucleotides in biological use of methylenephosphonate RNA is dependent on
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18
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19
1
2
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1
970 was internucleoside 3 -deoxy-3 -C-methylenephosphonate its stability towards nucleases and phosphodiesterases. Here we
linkage (Fig. 1). Later on, this was also incorporated in a present a study on the stability of the methylenephosphonate
1
3
trinucleotide but no data on stability to enzymatic cleavage linkage in a diribonucleotide and towards enzyme catalyzed
have been presented. degradation by two different phosphodiesterases/nucleases and
The early synthesis work is difficult to utilize for making RNase A.
0
0
oligonucleotides and occurred before the development of the
The model dinucleotide, guanosine 5 -(uridine 3-deoxy-3 -C-
methylenephosphonate) (UCH pG, 1, Scheme 1) was synthesized
and then fully deprotected and purified by HPLC (see ESI†). The
UCH pG dinucleotide was first subjected to snake venom
phosphodiesterase (SVPD, PDE I) from Crotalus adamanteus
2
a
Department of Biosciences and Nutrition, Karolinska Institutet, Novum,
2
H a¨ lsov a¨ gen 7, 14183, Huddinge, Sweden. E-mail: Roger.Stromberg@ki.se
b
Medivir AB, Lunastigen 7, 141 44, Huddinge, Sweden
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc44621b and analysed by HPLC at different times. There was no cleavage
9
036 Chem. Commun., 2013, 49, 9036--9038
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for incorporation into therapeutic oligonucleotides. What then is
the stability towards an RNA-degrading endonuclease?
When UCH pG and the native diribonucleotide UpG were
2
subjected to RNase A, the difference in rate of enzyme catalyzed
cleavage was even more pronounced than with the spleen
phosphodiesterase. HPLC analysis revealed that the methylene-
2
phosphonate analogue UCH pG is hardly cleaved at all over two
days and under conditions where the natural dimer UpG is
almost completely degraded after a few minutes (Fig. 2b). In fact,
even after seven days incubation with RNase A there was only a
few percent cleavage of UCH pG (see ESI†).
2
The methylenephosphonate modification clearly seems to be
interesting for further exploration. But what also struck us is the
2
Scheme 1 Pathways for the cleavage of UCH pG and UpG dinucleotides with
RNase A or base (monitoring is only for the first step since the ratio of quite remarkable resistance to the RNase A catalyzed cleavage,
dinucleotide to the sum of all products was evaluated).
especially since the dinucleotide contains all groups suggested to
2
0,21
0
interact in mechanisms for catalysis,
including the 2 -hydroxy
0
nucleophile, the 5 -oxyanion leaving group and a negatively
charged phosphoryl functionality. The only difference between
the substrates is the methylene group and the large effect on the
rate of cleavage when they are catalyzed by the RNase A, which is
not what can be expected by intrinsic chemical reactivity upon
cyclization (Scheme 1). It is known that the rate of alkaline
hydrolysis for cyclic phosphonate esters is only about a factor
observed with SVPD after two days of incubation (see ESI†),
which is expected since the enzyme cleaves internucleosidic
0
phosphate esters with the 3 -oxyanion as the leaving group, but
in the modified dinucleotide this is replaced by a methylene
group.
Both UCH pG and the native diribonucleotide UpG were
2
then subjected to spleen phosphodiesterase (PDE II), which is a
22
of two lower than for the corresponding cyclic phosphate esters.
0
5
-exonuclease. HPLC analysis of the mixtures revealed that the
pG is considerably more
23
The ring strain effect makes cyclic esters somewhat different so,
methylenephosphonate analogue UCH
2
to make sure that there was no major intrinsic chemical difference
stable than the native UpG (Fig. 2a), with about 75% remaining
of the modified dimer under conditions where UpG is almost
completely degraded.
between UCH pG and UpG, we performed a study on the rate of
2
hydrolysis under alkaline conditions (where a more or less
concerted mechanism is expected).
Both dinucleotides were subjected to hydrolysis in 0.01 M
and 0.05 M NaOH solutions (I = 0.2 M with NaCl) at 50 1C
and monitored by RP-HPLC analysis at different times. The
natural RNA dimer UpG was cleaved somewhat faster than the
0
The complete resistance towards a 3 exonuclease and high
0
0
0
resistance towards a 5 -exonuclease suggest that the 3 -deoxy-3 -
C-methylenephosphonate modification can be highly interesting
2
modified UCH pG but the rate difference in both solutions is
only a factor of about 1.7 (Fig. 3a). This clearly does not explain
the large difference in rate found in the enzyme catalyzed
cleavages, especially by RNase A. The methylenephosphonate
RNA is interesting for incorporation in oligonucleotides but
it is also intriguing why it is so stable towards RNase A catalyzed
cleavage. Could the binding to the enzyme be hampered in
some way?
Fig. 3 (a) Graph showing the natural logarithm of the fraction of remaining
2
dinucleotide (UpG in red and UCH pG in blue) when subjected to hydrolysis in
Fig. 2 Graphs showing % remaining dinucleotide (UCH
red) at different times, when subjected to spleen exonuclease, PDE II (a) or RNase A
b) at 37 1C. Quantification of dinucleotide and product was done by integration of
2
pG in blue and UpG in
0.01 M (lower two lines) and 0.05 M NaOH solutions (upper two lines) at 50 1C.
(b) Graph showing % remaining UpG at different times, when subjected to RNase A,
(
2
in the presence of different amounts of UCH pG at 37 1C. (Red line: only UpG, i.e.,
the RP-HPLC analysis of the mixtures.
1 : 0; black line: equimolar amounts, i.e., 1 : 1; blue line: 1 : 5; green line: 1 : 10.)
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The methylene modification should stereoelectronically an RNase endonuclease activity makes methylenephosphonate
0
enforce a preference for the north (3 -endo) conformations of RNA interesting for incorporation into therapeutic oligo-
2
4,25
26
the sugar
as it is found in a difluromethylenephosphonate,
nucleotides. In particular, it would be interesting to evaluate
even though this has a more electron withdrawing group methylenephosphonate RNA in the antisense strand of thera-
0
attached to the 3 -position. If the dinucleotide binding to RNase peutic siRNAs.
0
A would occur in another conformation, e.g., 2 -endo (south), one
We gratefully acknowledge financial support from The Swedish
could expect that the overall rate would be higher with the native Research Council and EU Marie Curie network funding
UpG that is less locked to a north conformation. If this were the (EC-FP7-ITN-2008-238679).
reason for a much lower rate of cleavage with UCH pG, one
2
would not expect this compound to be able to compete efficiently
with UpG in binding to the active site. If, however, the dimer
Notes and References
1
Y. Dorsett and T. Tuschl, Nat. Rev. Drug Discovery, 2004, 3, 318;
M. Manoharan, Curr. Opin. Chem. Biol., 2004, 8, 570.
binds in the north conformation one would expect UCH pG to
2
be a competitive inhibitor. UCH pG is cleaved but at such a rate
2
2 S. T. Crooke, Annu. Rev. Med., 2004, 55, 61; S. T. Crooke, Curr. Mol.
Med., 2004, 4, 465.
that it would essentially act as an inhibitor for the UpG substrate
3
J. Bauman, N. Jearawiriyapaisarn and R. Kole, Oligonucleotides,
009, 19, 1.
J. R. Morrow and O. Iranzo, Curr. Opin. Chem. Biol., 2004, 8, 192.
(
at ten times higher concentration of RNase A the cleavage of
UCH pG is still slow but more prominent, see ESI†).
Competition experiments, where different amounts of
UCH pG were added to incubations of UpG with RNase A, were
2
2
4
5 H. Åstr o¨ m, N. H. Williams and R. Str o¨ mberg, Org. Biomol. Chem.,
2
003, 1461; H. Åstr o¨ m and R. Str ¨o mberg, Org. Biomol. Chem., 2004,
2
2, 1901.
performed. It is evident that already at equimolar amounts of
the two dinucleotides the rate of cleavage of UpG is substan-
6
7
M. Murtola and R. Str ¨o mberg, Org. Biomol. Chem., 2008, 6, 3837;
M. Murtola, M. Wenska and R. Str o¨ mberg, J. Am. Chem. Soc., 2010,
132, 8984.
tially retarded (Fig. 3b, black line). As the amount of UCH pG
2
E. Rozners, D. Katkevica, E. Bizdena and R. Str ¨o mberg, J. Am. Chem.
Soc., 2003, 125, 12125.
present is increased to 5 and 10 equivalents compared to UpG,
the rate of cleavage of the natural dimer decreases even further
8 E. Rozners and R. Str ¨o mberg, J. Org. Chem., 1997, 62, 1846.
S. M. Freier and K. H. Altmann, Nucleic Acids Res., 1997, 25, 4429.
9
(Fig. 3a, the two upper blue and green lines). This indicates that
1
0 R. S. Geary, R. Z. Yu, A. Siwkowski and A. Levin, Antisense Drug
Technology, 2nd edn, 2008, 305; P. Guga and M. Koziolkiewicz,
Chem. Biol. Artif. Nucleic Acids, 2012, 259; H. Almer, J. Stawinski
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R. Str ¨o mberg, J. Am. Chem. Soc., 1996, 118, 7921.
UCH pG efficiently competes with UpG for the binding in the
2
active site and the magnitude of the suppression even suggests
0
that UCH pG binds tighter than the native dimer. If the 3 -endo
2
conformer is adopted upon binding, it would explain why
1
1
1
1 M. Warashina, Y. Takagi, W. J. Stec and K. Taira, Curr. Opin.
Biotechnol., 2000, 11, 354.
2 H. P. Albrecht, G. H. Jones and J. G. Moffatt, J. Am. Chem. Soc., 1970,
UCH pG is an efficient binding competitor since it is more
2
locked in that conformation.
92, 5511.
2
So why then is UCH pG cleaved at such a low rate by RNase A?
3 A. Mazur, B. E. Tropp and R. Engel, Tetrahedron, 1984, 40, 3949.
A couple of options involving a conformational change can be 14 P. J. Garegg, T. Regberg, J. Stawinski and R. Str o¨ mberg, Chem. Scr.,
2
0
1985, 25, 280–282; P. J. Garegg, T. Regberg, J. Stawinski and
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5 P. J. Garegg, C. Henrichson, I. Lindh, T. Regberg, J. Stawinski and
R. Str ¨o mberg, Tetrahedron Lett., 1986, 27, 4051; B. C. Froehler and
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R. Str ¨o mberg, Tetrahedron Lett., 1986, 27, 4055.
considered. The Breslow mechanism involves a phosphorane
intermediate and if initial attack does not take place with the
-oxygen in an apical position, pseudorotation will be necessary
for completion of the reaction. This would be severely retarded
since it would involve an apical methylene substituent that has
a very low apicophilicity. Another option is that the initially
0
5
1
2
7
0
0
bound 3 -endo conformer has to flip to 2 -endo before the attack
on phosphorus can take place, and this is retarded by the
1
6 A. Winqvist and R. Str o¨ mberg, Eur. J. Org. Chem., 2001, 4305.
conformational preference of the methylenephosphonate, perhaps 17 A. Winqvist and R. Str o¨ mberg, Eur. J. Org. Chem., 2002, 1509.
1
1
2
8 A. Winqvist and R. Str o¨ mberg, Eur. J. Org. Chem., 2002, 3140.
9 A. Winqvist and R. Str o¨ mberg, Eur. J. Org. Chem., 2008, 1705.
0 R. Breslow and W. H. Chapman Jr, Proc. Natl. Acad. Sci. U. S. A.,
1996, 10018.
accentuated when bound to the enzyme. It is also plausible that
lysine-41, suggested to stabilize charge build up on the non-
2
8
bridging phosphoryl oxygens, is somehow involved and inter-
0
21 C. M. Cuchillo, M. V. Nogues and R. T. Raines, Biochemistry, 2011,
0, 7835; R. T. Raines, Chem. Rev., 1998, 98, 1045.
2 R. Kluger and S. D. Taylor, J. Am. Chem. Soc., 1990, 112, 6669.
acts with the 3 -oxygen. This could also be connected to one of
5
the conformational changes above.
2
In view of what is known about the RNase A mechanism it is 23 A. Eberhard and F. H. Westheimer, J. Am. Chem. Soc., 1965, 87, 253;
R. Kluger and S. D. Taylor, J. Am. Chem. Soc., 1991, 113, 5714.
4 Use of NMR for conformational analysis (see ref. 26) was attempted
surprising that the methylenephosphonate dinucleotide is
2
highly resistant to RNase A catalyzed cleavage. It is also inter-
esting that there still seem to be details in the mechanism of
catalysis for RNase A that are not yet resolved and hopefully the
methylenephosphonate modification can contribute to further
studies, e.g., by co-crystallization with the enzyme and further
enzymology.
but we were unable to resolve the coupling constant for coupling
0
0
between 1 -H and 2 -H. That this coupling is so small is in itself an
indication of a high preference for the north conformers.
5 C. Altona and M. Sundaralingam, J. Am. Chem. Soc., 1973, 95, 2333.
6 C. Fressigne, S. Piettre, E. Condamine, C. Altona and A. Gautier,
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8 J. M. Messmore, D. N. Fuchs and R. T. Raines, J. Am. Chem. Soc.,
1995, 117, 8057.
2
2
2
That the modification leads to a higher resistance towards
2
0
0
degradation by both 3 -exo and 5 -exonucleases as well as
9
038 Chem. Commun., 2013, 49, 9036--9038
This journal is c The Royal Society of Chemistry 2013