A non-enzymatic, dna template-directed morpholino primer extension approach

Article abstract of DOI:10.1002/chem.200902237

Extension, please ! A non-enzymatic DNA-template-directed synthesis approach has been developed that allows the morpholino-nucleoside extension of a PNA primer. Equilibration of the ribonucleosides with the template-primer complex, in a noncovalent fashion (see scheme), was shown to be essential for high selectivity. It is envisaged that this chemistry might be used to transcribe nature's genetic material into alternative abiological polymers. (Chemical Equation Representation).

Full text of DOI:10.1002/chem.200902237

DOI: 10.1002/chem.200902237
A Non-Enzymatic, DNA Template-Directed Morpholino Primer Extension
Approach
Neil M. Bell, Raymond Wong, and Jason Micklefield*[a]
Non-enzymatic RNA- and DNA-template-directed syn-
thesis of oligonucleotides has been a focus of research for
many years, particularly in the context of prebiotic chemis-
try. Notably, 5’-phosphorimidazolide-activated nucleotides
can be assembled on nucleic acid templates to produce com-
plementary oligonucleotide products.[1–3] In addition, modi-
fied systems including PNA,[4] have been used as templates
for the non-enzymatic assembly of complementary oligonu-
cleotides, by using similar 5’-activated nucleotides.[5] More
recently, helper strands have been used to increase the se-
lectivity and rates of non-enzymatic DNA primer extension
reactions,[6] allowing detection of single-nucleotide poly-
morphisms with 5’-activated nucleotides labelled with fluo-
rophores.[7] One problem associated with the use of 5’-acti-
vated nucleotides in the template-directed, non-enzymatic
assembly of oligonucleotides is the fact that the formation
of the phosphodiester bond is kinetically controlled and es-
sentially irreversible. Of course nature has overcome this
problem through the evolution of polymerases with addi-
tional hydrolytic proofreading function. However, in the ab-
sence of a proofreading polymerase, mismatches inevitably
accumulate and can lead to complex mixtures of truncated
products. In the case of ribonucleotides the possibility of
forming 2’!5’ phosphodiester bonds can further complicate
product analysis.[8] To overcome problems associated with
the formation of phosphodiester bonds, thermodynamically
controlled imine formation has been explored. For example,
thymidine monomers with 5’-NH2 and 3’-CH2CHO groups
were assembled on a (dA)8 template and the imine-linked
oligomers were trapped by NaCNBH3 to give a polyamine
product.[9] Whilst this approach has not been extended to in-
clude the other three nucleoside monomers (A, C and G),
similar reductive amination chemistry was used to effect
DNA-template-directed assembly of PNA tetramers, with
N-terminal free amino and C-terminal aldehyde groups.[10]
In this paper we introduce an alternative approach to-
wards the non-enzymatic transcription of DNA into comple-
mentary “non-natural” morpholino-amide oligomers (Sche-
me 1a). This system was investigated because a range of
morpholine-based oligonucleotide mimics have been shown
to form stable duplexes with complementary nucleic
acids.[11] Also, unlike the well known phosphorodiamidate
morpholino oligonucleotides (PMOs), which are now widely
used antisense agents,[12] the amide-linked morpholino oligo-
mers can potentially be derived through a reductive amina-
tion extension reaction between a 5’-glycinyl monomer or
oligomer and the dialdehyde products formed from period-
ate oxidation of ribonucleosides (Scheme 1a).[13] Whilst a
wide range of morpholines has been prepared, mainly under
anhydrous conditions, from ribose derivatives through peri-
odate oxidation followed by reductive amination, typically
the reported yields are modest.[14] To address this issue, we
explored the oxidation of ribonucleosides and subsequent
reductive amination with benzylamine in aqueous buffer so-
lution under conditions that would be compatible with
DNA-template-directed synthesis. Reactions with equimolar
ratios of dialdehydes and amines give modest yields of prod-
ucts. However, if an excess (5 mol equiv) of dialdehyde is
formed in situ from ribonucleosides (rA, rT, rC and rG)
with NaIO4 and the amine added immediately, along with
NaCNBH3, then N-benzyl morpholine products can be pro-
duced in nearly quantitative yield based on the amine (Sche-
me 2a). In fact, by using this one pot procedure, excellent
yields can be achieved with as little as two molar equivalents
of the dialdehyde relative to benzylamine. Presumably the
low yields previously reported are based on the dialdehyde
and can be accounted by the decomposition, including over-
oxidation, of the reactive intermediates derived from treat-
ment of ribonucleosides with periodate.[15] In light of this ob-
servation the DNA-template-directed morpholino extension
of a synthetic primer was explored, by using an excess of the
ribonucleoside-derived dialdehydes (Scheme 1b).
[a] Dr. N. M. Bell, Dr. R. Wong, Prof. J. Micklefield
School of Chemistry, The University of Manchester
Manchester Interdisciplinary Biocentre
131 Princess Street, Manchester M1 7ND (UK)
Fax : (+44)161 306 4509
E-mail: jason.micklefield@manchester.ac.uk
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902237.
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Chem. Eur. J. 2010, 16, 2026 – 2030

COMMUNICATION
Scheme 2. a) Model periodate oxidation and reductive amination reac-
tions with ribonucleosides 5-methyluridine (rT), adenosine (rA), cytidine
(rC) or guanosine (rG) (1.93 mmol), NaIO4 (3.9 mmol), benzylamine
(0.39 mmol), NaCNBH3 (7.7 mmol), in KH2PO4 buffer (30 mL, 500 mm,
pH 7.0) for 6 h give morpholine products in about 98% yield based on
benzylamine. Reactions are likely to proceed via a dialdehyde, hydrated
cyclic acetal, carbanolamine and other intermediates. b) Possible degra-
dation products resulting from depurination and overoxidation of ribonu-
cleosides. MALDI MS: m/z: calcd for 5: 2317.2; found: 2317.0 [M+H]+;
calcd for 6: 2247.1; found, 2247.3 [M+H]+.
approved the formation of PNA–morpholino extension
products (2a, 2t, 2c and 2g). However, the excess of salt
from the buffer solution and other reagents complicated the
quality of the spectra. To overcome this, the reactions were
repeated with the PNA hybridised to the four templates.
After reductive amination with the complementary nucleo-
side dialdehyde, the resulting primer extension products re-
mained hybridised to the DNA template. The DNA/PNA
complex could then be captured on avidin agarose resin and
the resin washed extensively to remove excess salts and re-
agents. Denaturation of the primer extension products from
the DNA templates, at 858C, resulted in samples that gave
very clean and reproducible MALDI spectra, which were
consistent with calculated masses of the extension products
2a, 2t, 2c and 2g (Figure S1 in the Supporting Information).
By using NH4F for preparing the MALDI matrix it was also
possible to gain reproducible and accurate MALDI spectra
free of Na+ and K+ adducts.[16] Moreover, the intensities of
the [M+H]+ ions in the MALDI MS were proportional to
the ratio of the extension products, presumably because the
protonated product ions have similar global ionisation effi-
ciency.[17]
Scheme 1. a) Retrosynthetic disconnection of morpholino amide oligo-
mers. b) The morpholino primer extension strategy. DNA template 1a,
hybridised with a PNA primer 2, selectively binds complementary ribo-
nucleoside rT, by Watson–Crick base paring. Subsequent periodate oxida-
tion results in the dialdehyde, which reacts with the N-terminal glycine
PNA primer 2 followed by in situ NaCNBH3 reduction to give the mor-
pholine extension product 2t.
For synthetic expediency a PNA primer with an N-termi-
nal glycinyl group, NH2-Gly-PNA(CCT CAG TG)-CONH2
(2), was prepared by using standard Boc-PNA protocols.
Four DNA-template sequences were also designed: biotin-
5’-GCC GCA CTG AGG BTA GCC-3’ where B is A, T, C
or G (1a, 1t, 1c or 1g). Each template possesses a comple-
mentary antiparallel PNA hybridisation sequence (under-
lined), with a variable base opposite the PNA N-glycinyl ex-
tension site (Scheme 1b). The 5’-biotin label is also included
to aid capture of the template and extension products. Ini-
tially, the non-competitive morpholino extension reactions
with PNA 2 and dialdehydes derived from rC, rT, rA and
rG were investigated, in the absence of a DNA template, by
using the optimised in situ reductive amination procedure
described above. The subsequent MALDI MS experiments
Internally calibrated MALDI MS[17] was therefore adopt-
ed as the method of choice for determination of product
ratios from competitive template extension reactions. Ac-
cordingly, an equimolar mixture of rA, rT, rC and rG was
oxidized with periodate and the mixture was incubated with
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J. Micklefield et al.
porting Information). The low selectivity of template 1c,
combined with greater intensity of side products 5 and 6
(Figure S2 in the Supporting Information), could thus be ex-
plained by the greater propensity of guanosine and/or its ox-
idation products to depurinate and overoxidise during tem-
plating. Nevertheless, by decreasing the amount of NaIO4
used in the initial oxidation to one equivalent relative to the
ribonucleosides it was possible to reduce formation of the
overoxidation side product 6 to a large extent. A series of
experiments were undertaken to optimise conditions with
the problematic template 1c and to further explore how se-
quence selectivity is governed during the morpholino primer
extension reaction. Firstly, equilibration of the ribonucleo-
sides with the primer–template complex (2–1c) prior to oxi-
dation was explored. Accordingly, a mixture of the four ri-
bonucleosides was incubated with the primer–template com-
plex and NaIO4 was added after a period of 0, 2 and 4 h.
MALDI MS spectra, subsequent following reductive amina-
tion, clearly showed that the longer the equilibration time
the greater the selectivity, with the proportion of the com-
plementary product 2g increasing to 55% (Figure S4 in the
Supporting Information). No further increase in selectivity
was observed when equilibration was extended beyond 4 h.
Conversely, if the ribonucleosides are treated with NaIO4
for 2 h prior to incubation with the primer–template com-
plex (2–1c), essentially all selectivity is lost (Figure S4 in the
Supporting Information). Moreover, the side product 5 be-
comes prominent, supporting the idea that ribonucleoside
oxidation products are more prone to depurination.
A second series of experiments were carried out to deter-
mine if selectivity is affected by the length of time that the
dialdehydes are allowed to equilibrate with the primer–tem-
plate complex, prior to reduction with NaCNBH3. In this
case the template 1g was used, which had already been
shown to give very good selectivity for primer extension
product 2c. Accordingly, the ribonucleoside mixture and the
primer–template complex (2–1g) were equilibrated for 4 h,
before the reaction was initiated with NaIO4. The
NaCNBH3 was then added after a period of 0, 2, or 4 h.
From this it was apparent that increasing the time before ad-
dition of NaCNBH3 from 0 to 4 h decreases selectivity for
the 2c product from 82 to 66%, respectively, and also in-
creases the yield of side products 5 and 6 (Figure S5 in the
Supporting Information). Thus, it is clear that template se-
lectivity is governed by non-covalent interaction of the ribo-
nucleosides with the primer–template complex. Further-
more, extended equilibration of the ribonucleoside oxida-
tion products with the primer–template complex prior to
NaCNBH3 reduction of the covalent imine intermediates
serves to reduce selectivity and increases side-product for-
mation. Fully optimised conditions established a full set of
primer extension reactions that were carried out with the
four ribonucleosides on the templates 1g, 1a, 1t and 1c re-
sulting in the formation of the extension products 2c, 2t, 2a
and 2g with selectivities of 95, 86, 70 and 55%, respectively
(Figure 1 and Table 1). Selectivities are high except for ex-
tension with guanosine.
Figure 1. MALDI MS spectra of competitive primer extension reactions.
PNA 2 (1.0 nmol) and the biotinylated DNA template 1a, 1t, 1c or 1g
(2.5 nmol) were annealed for 1 h in NaH2PO4 buffer solution (50 mL,
250 mm, pH 7.0). A mixture of the ribonucleosides rA, rT, rC and rG
(0.625 mmol of each) was added and the mixture was equilibrated for 4 h
in NaH2PO4 buffer solution (100 mL, 250 mm, pH 7.0), before addition of
NaIO4 (2.5 mmol) and then NaCNBH3 (2.5 mmol) each separately dis-
solved in NaH2PO4 buffer solution (50 mL, 250 mm, pH 7.0). MALDI
MS: m/z: calcd for 2c: 2428.3; found: 2428.4 [M+H]+; calcd for 2t:
2443.3; found 2443.2 [M+H]+; calcd for 2a: 2452.3; found: 2452.4
[M+H]+; calcd for 2g: 2468.3; found: 2468.5 [M+H]+.
each of the four DNA templates and primer 2. Reductive
amination, work up and MALDI MS analysis, showed that
the extension products 2c, 2t and 2a were clearly the major
products obtained from complementary DNA templates 1g,
1a and 1t, respectively (Figure S2 in the Supporting Infor-
mation). However, only 34% of the 2g extension products
was obtained from the complementary template 1c, with
mismatch products 2c and 2t also present in 27 and 33%,
respectively. It is also evident from the competitive reaction
with the template 1c that two major side products m/z=
2317 and 2247 as well as unreacted primer 2 are present.
The mass of the side product m/z=2317 is consistent with
depurination of guanosine (rG), or its oxidation product, to
generate an oxonium ion 3 that can cyclise to give the 5-
membered acetal 4 that under conditions of reductive ami-
nation results in primer N-capped side product 5 (Sche-
me 2b). The mass of the other side product, m/z=2247, is
consistent with an N-formyl PNA 6, which could be derived
from reaction of 2 with glyoxal or alternative products of
nucleoside overoxidation.[15] Indeed, when PNA primer 2
was reacted with glyoxal, periodate and NaCNBH3 a prod-
uct of m/z=2247 is similarly formed (Figure S3 in the Sup-
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A Morpholino Primer Extension Approach
COMMUNICATION
Table 1. Product ratios for competitive primer extension reactions.
Template
Ribonucleosides[a]
2c/2t/2a/2g
NMPs[b]
Biased NMPs[c]
2cp/2up/2ap/2gp
2cp/2up/2ap/2gp
1g
1a
1t
95:5:0:0
5:86:9:0
16:5:70:10
17:23:5:55
97:0:3:0
94:0:5:0
0:85:14:0
0:0:93:7
7:7:0:86
0:77:19:4
13:13:72:2
18:18:0:64
1c
In addition to depurination, we observed that guanosine
has low solubility and can form hydrogels on oxidation with
NaIO4, presumably due to Na+-mediated G-quadruplex for-
mation.[18] These factors may contribute to lowering the ef-
fective concentration of free guanosine for template-direct-
ed extension. Guanosine 5’-monophosphate (GMP), on the
other hand, is considerably more soluble in aqueous media
and does not form a hydrogel on oxidation with NaIO4.
Therefore primer extension reactions were repeated by
using the ribonucleoside 5’-monophosphates (adenosine 5’-
monophosphate (AMP), uridine 5’-monophosphate (UMP),
cytosine 5’-monophosphate (CMP) and GMP). Under iden-
tical conditions the extension reactions with ribonucleoside
5’-monophosphates gave selectivities of 97, 77, 72 and 64%
for the primer extension products 2cp, 2up, 2ap and 2gp, re-
spectively (Figure S6 in the Supporting Information and
Table 1). Notably, the selectivity for the template 1c, al-
though still lower than the other templates, increases from
55 for guanosine to 64% for GMP. The 5’-phosphoryl group
presumably prevents formation of side product 5. However,
depurination and further oxidation still resulted in the side
product 6, which is most evident for reactions with the tem-
plate 1c. To further explore if degradation of the GMP oxi-
dation products results in a lower effective concentration of
the GMP-derived dialdehyde, the concentrations of the ribo-
nucleoside 5’-monophosphates was biased from equimolar
to 1.0:0.8:0.8:1.2 (AMP/UMP/CMP/GMP), in favour of
GMP. This led to an increase in selectivity of 94, 85, 93 and
86% for the primer extension products 2cp, 2up, 2ap and
2gp, respectively (Figure 2 and Table 1). This means for the
first time template 1c shows selectivity that is on par with
the other templates. Indeed, these selectivities compare well
with the best selectivities observed with similar non-enzy-
matic primer extension reactions by using standard 5’-acti-
vated nucleotides,[6,7] which had been developed and opti-
mised over 30 years since Orgel first introduced this chemis-
try.[1] In addition, high selectivities are obtained with the
Figure 2. MALDI MS spectra of competitive primer extension reactions
with biased ribonucleoside 5’-monophosphates. PNA 2 (1.0 nmol), biotin-
ylated the DNA template 1a, 1t, 1c or 1g (2.5 nmol) were annealed for
1 h in NaH2PO4 buffer solution (50 mL, 250 mm, pH 7.0). AMP
(0.625 mmol), UMP (0.50 mmol), CMP (0.50 mmol) and GMP (0.75 mmol)
were added and the mixture was equilibrated for 4 h in NaH2PO4 buffer
solution (100 mL, 250 mm, pH 7.0), before addition of NaIO4 (2.38 mmol)
and then NaCNBH3 (2.5 mmol) each separately dissolved in NaH2PO4
buffer solution (50 mL, 250 mm, pH 7.0). MALDI MS m/z: calcd for 2cp:
2508.3; found: 2508.0 [M+H]+; calcd for 2up: 2509.3; found: 2509.1
[M+H]+; 2ap: 2532.3; found: 2532.5 [M+H]+; 2gp: 2548.3; found: 2548.7
[M+H]+.
try.[19] However, this proved not to be the case as extended
equilibration of the dialdehydes with the template–primer
complex actually reduces selectivity. Instead, extending the
equilibration time of the ribonucleosides with the template–
primer complex prior to oxidation, was shown to increase
selectivity in accordance with the Watson–Crick base-pairing
rules. Presumably the subsequent oxidation of the ribonu-
cleoside results in the formation of stable covalent adducts
with the N-terminal amino group of the PNA primer, that
do not exchange significantly before in situ reduction. From
a practical perspective this approach could be further devel-
oped for use in the detection of single-nucleotide poly-
morphisms (SNPs). Finally, research in prebiotic chemistry
has focused on alternative genetic systems, which may have
acted as templates for the first synthesis of todayꢁs genetic
material.[4,20] In contrast, the work described in this paper
seeks to address the problem of how natures genetic infor-
mation could be transcribed into the sequence of non-natu-
ral or abiological polymers.[21]
morpholino approach without the aid of
a “helper”
strand.[6,7]
In summary, we have developed a new non-enzymatic
DNA-template-directed synthesis approach that delivers
morpholino nucleoside extension of a synthetic PNA primer
with high sequence selectivity that is similar to those ob-
served with the well established prebiotic chemistry inspired
approaches.[6,7] Initially, we envisaged that exchange of the
ribonucleoside dialdehydes with the N-terminal amino
group of the PNA primer would control selectivity following
the principles of covalent dynamic combinatorial chemis-
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J. Micklefield et al.
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Acknowledgements
EPSRC are acknowledged for financial support (grant EP/D053951) and
for PhD studentships awarded to N.M.B & R.W.
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Keywords: DNA · morpholino · non-enzymatic · PNA ·
ribonucleosides · primer extension
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Received: August 11, 2009
Published online: January 19, 2010
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