Fast and accurate nonenzymatic copying of an RNA-like
synthetic genetic polymer
Shenglong Zhanga,b, J. Craig Blaina,b, Daria Zielinskac, Sergei M. Gryaznovc,1, and Jack W. Szostaka,b,2
a
Howard Hughes Medical Institute and bDepartment of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General
Hospital, Boston, MA 02114; and cDepartment of Nucleic Acid Chemistry, Geron Corporation, Menlo Park, CA 94025
Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved September 14, 2013 (received for review June 28, 2013)
Recent advances suggest that it may be possible to construct
simple artificial cells from two subsystems: a self-replicating cell
membrane and a self-replicating genetic polymer. Although
multiple pathways for the growth and division of model protocell
membranes have been characterized, no self-replicating genetic
material is yet available. Nonenzymatic template-directed synthesis of RNA with activated ribonucleotide monomers has led to the
copying of short RNA templates; however, these reactions are
generally slow (taking days to weeks) and highly error prone. N3′P5′–linked phosphoramidate DNA (3′-NP-DNA) is similar to RNA in
its overall duplex structure, and is attractive as an alternative to
RNA because the high reactivity of its corresponding monomers
allows rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Here we show that both
homopolymeric and mixed-sequence 3′-NP-DNA templates can be
copied into complementary 3′-NP-DNA sequences. G:T and A:C
wobble pairing leads to a high error rate, but the modified nucleoside 2-thiothymidine suppresses wobble pairing. We show that
the 2-thiothymidine modification increases both polymerization
rate and fidelity in the copying of a 3′-NP-DNA template into
a complementary strand of 3′-NP-DNA. Our results suggest that
3′-NP-DNA has the potential to serve as the genetic material of
artificial biological systems.
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origin of life nonenzymatic primer extension
nucleotide modifications mismatch
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T
he phosphoramidate nucleic acids are of particular interest as
potential genetic materials for artificial life-forms because of
their potential for replication by the nonenzymatic polymerization of amino-sugar nucleotides. Because of the greater nucleophilicity of the amino group relative to the 3′-hydroxyl group of
ribo- and deoxyribo-nucleotides, amino-sugar nucleotides exhibit
more rapid spontaneous polymerization. Obviating the requirement
for a polymerase greatly simplifies the task of creating and assembling the components of an artificial cell, and thus of constructing
simple living systems from inanimate materials. We and others have
therefore explored the synthesis of a variety of phosphoramidatelinked nucleic acids, their corresponding amino-sugar monomers,
and the characterization of nonenzymatic template-directed primer
extension reactions in these systems (1–11). Among these systems,
we have examined 2′-amino versions of the acyclic glycerol nucleic
acid (5), 2′-amino-2′,3′-dideoxyribonucleic acid (4, 6), and 3′amino-2′,3′-dideoxyribonucleic acid (7).
The structural simplicity of the acyclic sugar-phosphate nucleic
acid backbones has made them attractive targets for study. Indeed, an acyclic nucleotide consisting of a glycerol-phosphate
backbone linked to a formylated nucleobase (12) was among the
first of such nucleic acids to be chemically synthesized, but incorporation of this nucleotide into oligomers caused a severe loss
of duplex stability. Much later, the glycerol nucleic acids, in
which a glycerophosphate backbone is directly linked to the
nucleobase via the 3′-carbon, were synthesized by Meggers and
coworkers (13), and shown to form an antiparallel, Watson–Crick
double-stranded helix of remarkable thermal stability, despite
the added entropic cost of duplex formation. We subsequently
17732–17737 | PNAS | October 29, 2013 | vol. 110 | no. 44
studied the corresponding phosphoramidate polymer, based on
2′-amino substituted derivative of the glycerol monomers. However, activation of the 1′-phosphate of these monomers led to very
rapid cyclization and the accumulation of inert 1′-2′-cyclic phosphates (5). Indeed, we were only able to demonstrate nonenzymatic
template copying after the synthesis of activated dinucleotides. In
an effort to avoid the problems associated with monomer cyclization, we turned to the 2′-amino-2′,3′-dideoxyribonucleotides,
which polymerize into 2′-5′ linked phosphoramidate DNA (6).
These monomers do not cyclize, as the cyclic sugar keeps the
amine nucleophile away from the activated phosphate electrophile. Moreover, activation of the phosphate with an imidazole
leaving group generated highly reactive monomers that engaged
in rapid primer extension on DNA, RNA, and even 2′-5′ linked
DNA templates. Because this template-copying reaction proceeds independently of divalent cations, which precipitate fatty
acids, we were able to demonstrate template-copying inside model
protocell membranes (4). Although this was an important conceptual advance toward the synthesis of a complete protocell,
subsequent studies of the 2′-5′ NP-DNA system revealed several
limitations. In particular, copying of A and T templates was very
slow; replacement of A with 2,6 diaminopurine and of T with
5-propynyl-T resulted in faster template copying, but at the apparent cost of decreased fidelity (6).
An attractive alternative to 2′-5′ linked phosphoramidate
DNA is the more RNA-like 3′-5′ linked NP-DNA. Structural
studies show that this polymer forms a duplex structure that is
very similar to that of RNA (14). Extensive studies by Richert
Significance
The first cells to have emerged on the early Earth lacked
enzymes, and therefore probably relied on spontaneous reactions to copy their genetic material, commonly thought to have
been RNA. Here we study a close chemical relative of RNA,
known as 3′-phosphoramidate-DNA, in an effort to learn general lessons about nonenzymatic replication. We show that
short regions of this polymer can be copied, but that the error
rate is unacceptably high. We then show that a single atom
change in one of the four nucleotides can restore good copying
accuracy. These advances may help to achieve chemical copying of RNA, and also suggest that 3′-phosphoramidate-DNA
may be a suitable genetic polymer for use in the construction
of simple artificial cells.
Author contributions: S.Z., J.C.B., and J.W.S. designed research; S.Z., J.C.B., D.Z., and
S.M.G. performed research; S.Z. and D.Z. contributed new reagents/analytic tools; S.Z.
and J.C.B. analyzed data; and S.Z. and J.W.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1
Present address: AuraSense Therapeutics, Skokie, IL 60077. Email: gryaznov@astound.
net.
2
To whom correspondence should be addressed. E-mail: szostak@molbio.mgh.harvard.
edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1312329110/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1312329110
Results
3′-NP-DNA Monomers Can Copy Homopolymeric 3′-NP-DNA Templates.
We have recently used activated 3′-amino-2′,3′-dideoxynucleotide
monomers in the nonenzymatic copying of short homopolymeric
DNA, RNA, and locked nucleic acid (LNA) templates (7). The
copying of such templates with 2-methylimidazole-activated monomers was rapid and accurate, with superior copying on RNA and
LNA templates as opposed to DNA templates. These observations
prompted us to investigate the copying of 3′-NP-DNA templates
into complementary 3′-NP-DNA, using 5′-phosphor-(2-methyl)
imidazolides of the 3′-amino-2′,3′-dideoxynucleotides as activated
monomers. Due to the relatively low efficiency of solid-phase synthesis of 3′-NP-DNA, we used chimeric oligonucleotides in which
only the 3′-end of the primer and the 5′-end of the template were
composed of 3′-NP-DNA (Fig. 1 A and B). A DNA primer bearing
a single 3′-amino-2′,3′-dideoxynucleotide at its 3′ terminus was
generated by solid-phase synthesis using 5′-phosphoramidites.
This primer was annealed to an oligonucleotide with a primerbinding DNA region and a seven-nucleotide 3′-NP-DNA region, including a four-nucleotide template region. To minimize the structural discontinuity between the DNA duplex and
the NP-DNA duplex, the two nucleotides in the template oligo-
nucleotide that pair with the last two nucleotides of the primer
were also 3′-NP-DNA. An additional 5′-terminal nucleotide was
added to the template to provide favorable stacking interactions.
Upon the addition of 2-methylimidazole-activated monomers (3′NH2-2-MeImpddNs) to the primer-template complex, the primer
was extended by the spontaneous template-directed synthesis of
3′-NP-DNA. The reaction products were analyzed by PAGE, and
their identity was confirmed by liquid chromatography–mass
spectrometry (LC-MS).
Activated 3′-amino-2′,3′-dideoxynucleotides are just as effective
in copying 3′-NP-DNA templates as they are in copying DNA and
RNA templates in nonenzymatic primer extension reactions. Of
the four 2-methylimidazolide monomers, 3′-NH2-2-MeImpddG
was the most reactive, reaching >92% completion within 5 min
while copying a C4 3′-NP-DNA template (Fig. 1C and SI Appendix, Fig. S1). The C, T, and A monomers also resulted in efficient
copying of their complementary homopolymeric 3′-NP-DNA templates, achieving ≥80% completion in less than an hour (Figs. 1C
and 2). The efficiency of extension on 3′-NP-DNA templates was
comparable to that on RNA templates, and was higher than that on
DNA templates (SI Appendix, Figs. S2–S4). As previously observed
(7), in some cases the kinetics of primer extension are unusual in
that the reaction appears to progress from unreacted primer to fully
extended primer, with the expected intermediates present at low or
undetectable levels. Such kinetics could reflect slow initiation of
primer extension, followed by rapid elongation, possibly due to
a structural discontinuity between the DNA/DNA primer/template
region, and the 3′-NP-DNA portion of the template. As an alternate, monomers may assemble into oligomers on the 3′-NP-DNA
template, but only slowly ligate to the primer. The fact that 3′-NPDNA can serve as a template for its own synthesis inspired us to
look more deeply into the properties of this self-copying reaction.
2-thio-T Replacement of T Enhances Copying of Correct Reactions and
Suppresses Incorrect Copying Reactions. Our previous observation
of polymerization of 3′-NH2-2-MeImpddT on a G4 RNA template
Fig. 1. Nonenzymatic primer extension reactions
on homopolymeric templates. (A) Structure of an
internucleotide linkage in N3′-P5′–linked phosphoramidate DNA (3′-NP-DNA). (B) General primer extension reaction scheme showing a 5′-Cy3–labeled
3′-amino–terminated DNA primer annealed to a
complementary chimeric DNA/3′-NP-DNA template.
The 3′-NH 2-2-MeImpddN monomers extend the
primer by four nucleotides on the complementary
template forming a chimeric DNA/3′-NP-DNA product.
Red lines indicate phosphoramidate bonds. (C) PAGE
analysis of primer extension products on indicated 3′NP-DNA templates. Primer extension reactions contained 0.1 μM Cy3-labeled-3′-amino–terminated DNA
primer, 0.5 μM template, 100 mM Mes-CAPS-Hepes, pH
7.5, and 100 mM 1-(2-hydroxyethyl)imidazole (HEI).
The reactions were initiated by addition of 5.0 mM 3′NH2-2-MeImpddG, 5.0 mM 3′-NH2-2-MeImpddC, and
10.0 mM 3′-NH2-2-MeImpddA, respectively. Arrows indicate the primer and the full-length product (+4).
Zhang et al.
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CHEMISTRY
and coworkers of the extension of primers ending in a 3′-amino
nucleotide show that the template-directed primer extension
reaction can be very fast, and can occur with relatively high
fidelity on both RNA and DNA templates (8, 10). We have recently shown that short homopolymeric RNA and DNA templates can be very rapidly copied by nonenzymatic primer
extension in the presence of 2-methylimidazole-activated 3′amino nucleotides (7). Here we show that these monomers can
also copy 3′-NP-DNA templates, in a true self-copying process. We also examine the fidelity of copying of mixed-sequence
templates and show that wobble base pairing is a major problem,
but that this problem can be largely remedied by replacing T with
2-thiothymidine (2-thio-T).
Fig. 2. Comparison of nonenzymatic primer extension reactions on homopolymeric A4 and G4 templates using T and 2-thio-T monomers. (A) Chemical
structure of monomer 3′-NH2-2-MeImpddsT. (B) PAGE analysis of primer extension products on indicated templates. Primer extension reactions were carried
out as in Fig. 1. The reaction was initiated by addition of 10.0 mM 3′-NH2-2-MeImpddsT or 3′-NH2-2-MeImpddT. Arrows indicate primer and full-length
product. Primer extension is faster using 2-thio-T vs. T on the A4 template, but slower on the G4 template.
suggested that the formation of G:T wobble base pairs would be
a significant problem in the 3′-NP-DNA system (7). Thermodynamic data indicate that 2-thio-U (sU) forms a stronger base pair
with A than with standard U by ∼1 kcal/mol, and the G:sU wobble
pair is slightly destabilized relative to G:U within RNA duplexes
(15). Similarly, in DNA duplexes, the A:sT base pair is stabilized
relative to A:T, and the G: sT wobble pair is significantly destabilized relative to G:T (16, 17). We therefore asked whether
Fig. 3. Faster and more accurate copying of a 3′-NP-DNA template using 2-thio-T in place of T monomer. (A) Scheme for self-copying of a 3′-NP-DNA template
using C and T (or 2-thio-T) monomers. The primer is a 5′-Cy3-DNA-3′-NH2 and the template is a chimeric DNA/3′-NP-DNA (underlined) 5′-CAGAGGACTATCGGC-3′.
(B) PAGE analysis of primer extension products. Primer extension reactions were carried out as in Fig. 1. The reaction was initiated by addition of 5.0 mM 3′-NH2-2MeImpddC and 5.0 mM 3′-NH2-2-MeImpddT/sT as indicated. Arrows indicate primer and full-length product. (C) Scheme for self-copying AGAG 3′-NP-DNA
template with an entirely 3′-NP-DNA primer. (D) High-resolution MS analysis of the full-length extension products from a reaction initiated by the addition of 5.0
mM 3′-NH2-2-MeImpddC and 5.0 mM 3′-NH2-2-MeImpddT. There are three major isomeric families for three different 14-mer full-length N+4 products, corresponding to C3T1, C2T2, and C1T3 respectively. (E) High-resolution MS analysis of the full-length extension products from a reaction initiated by the addition of 5.0
mM 3′-NH2-2-MeImpddC and 5.0 mM 3′-NH2-2-MeImpddsT. The major peak corresponds to the 14-mer full-length N+4 product C2sT2.
17734 | www.pnas.org/cgi/doi/10.1073/pnas.1312329110
Zhang et al.
2-Thio-dT/U Enhances Copying Fidelity When Included in Monomer or
Template Contexts. Encouraged by the excellent rates and yields
for copying homopolymeric templates, we proceeded to examine
the copying of mixed-sequence templates using 3′-amino monomers. We first focused on the extension of a primer over a 5′AGAG-3′ sequence on a 3′-NP-DNA template (Fig. 3). An initial time course using a 5′-Cy3 labeled primer and PAGE to
separate the primer from extended products suggested that the
four-nucleotide template region was rapidly and efficiently copied (about 70% + 4 product after 20 min), using a mixture of 5.0
mM 3′-NH2-2-MeImpddC and 5.0 mM 3′-NH2-2-MeImpddT
monomers. However, in light of the expected high frequency of
G:T mismatches, we conducted a larger scale reaction using an
unlabeled 3′-NP-DNA primer for MS analysis. The correct
product would consist of the primer plus 2 C and 2 T residues;
however, this C2T2 product comprised only 48% of the primer +
4 nucleotide (N+4) products (Fig. 3D and SI Appendix, Fig. S8).
We also detected 6% of a C1T3 product, consisting of the primer
extended by 1 C and 3 T residues caused by a G:T mismatch. We
were surprised to observe 42% of a C3T1 product caused by an
A:C mismatch, as well as 5% of a C4 product resulting from two
A:C mismatches during copying of the AGAG 3′-NP-DNA
template. It is possible that the relatively slow copying of template As by a T monomer allowed time for the insertion of Cs by
wobble pairing. In an effort to reduce the A:C mismatched
products and improve fidelity, we raised the concentration of the
T monomer to 20.0 mM and kept the C monomer at 5.0 mM.
Under these conditions, we found that no A:C mismatched C4
product could be observed within the limit of detection of the
instrument, and that the A:C mismatched product C3T1 was indeed reduced to 26% (SI Appendix, Fig. S8), but the C1T3
product caused by a G:T mismatch increased to 16%. Overall the
correct C2T2 product was 58% of the +4 material, only a slight
enhancement of fidelity as a result of changing the monomer C/T
ratio. These results indicate that the fidelity of template copying
with the canonical C and T monomers is unacceptably low in
nonenzymatic copying of 3′-NP-DNA templates.
In an effort to improve the fidelity of copying of the 5′-AGAG
template, we used the activated 2-thio-T monomer 3′-NH2-2MeImpddsT. Indeed, the copying of the 5′-AGAG 3′-NP-DNA
template was significantly faster and more efficient (about 90% +
4 product after 10 min) using 5.0 mM 3′-NH2-2-MeImpddC and
5.0 mM of the 2-thio monomer 3′-NH2-2-MeImpddsT when
compared to the normal T monomer (Fig. 3B). Most importantly, MS analysis revealed that the fidelity was enhanced dramatically with the 2-thio-T monomer. We observed mainly the
correct C2sT2 product (82%) by MS analysis of the full-length
N+4 products (Fig. 3E and SI Appendix, Fig. S8), corresponding
to an average fidelity of over 95% per position. The C3sT1 product
resulting from an A:C mismatch was reduced to 12%, compared
with 42% when the T monomer was used. We did not observe
Zhang et al.
any C4 product within the limit of detection of the instrument.
To further confirm that the product was the expected primer–
CsTCsT product (and not an isomeric mismatched product), we
sequenced the 3′-NP-DNA product by partial acid hydrolysis of
the N3′-P5′ phosphoramidate bonds (18) followed by LC-MS
analysis of the resulting sequence ladder. The sequencing data
confirmed that the product was the expected primer–CsTCsT
product (SI Appendix, Table S1), showing directly that the two
monomers were added to the primer faithfully while copying the
3′-NP-DNA template. A similarly high proportion (85%) of
correct N+4 products was also observed when a mixture of 5.0
mM 3′-NH2-2-MeImpddsT and 5.0 mM 3′-NH2-2-MeImpddC
was used to copy a 5′-AGAG RNA template, and the proportion
of correct product was greater than 95% when copying a DNA
template under the same conditions (SI Appendix, Fig. S8).
The enhanced fidelity that we observed with the 2-thio-T
monomer led us to examine the copying of templates containing
T and 2-thio-T residues. We first examined the fidelity of nonenzymatic copying of a 5′-TCTC 3′-NP-DNA template using an
optimized monomer ratio of 1.0 mM 3′-NH2-2-MeImpddG and
10.0 mM 3′-NH2-2-MeImpddA. LC-MS analysis of the products
of primer extension showed that the expected A2G2 product
accounted for 43% of the N+4 product (SI Appendix, Fig. S9), while
the mismatched products A4, A3G1, A1G3, and G4 accounted for 1%,
26%, 27%, and 2%, respectively. Similarly poor fidelity was also
observed using DNA and RNA templates with the same sequences
(SI Appendix, Fig. S9). Unfortunately, synthetic problems in the
synthesis of the 3′-amino-2-thiothymidine-5′-O-phosphoramidite
(SI Appendix) have so far made it impossible to replace T with 2thio-T in 3′-NP-DNA oligonucleotides. We therefore compared
the fidelity of 3′-NP-DNA synthesis in the copying of RNA templates containing either U or 2-thio-U.
We compared primer extension on 5′-UCUC and 5′-sUCsUC
templates, using a mixture of 5.0 mM 3′-NH2-2-MeImpddG and
10.0 mM 3′-NH2-2-MeImpddA monomers (Fig. 4). We observed
90% full-length product at 1 h on the 2-thio-U–containing
template, compared with 84% full-length product on the native
RNA template over the course of 3 h (Fig. 4B). As expected, LCMS studies of the primer +4 product from the normal RNA 5′UCUC template revealed four major isomeric families of fulllength products, corresponding to the desired A2G2, as well as
A3G1, A1G3, and G4 (Fig. 4C). The correct A2G2 product
accounted for 45% of the full-length N+4 products (SI Appendix,
Fig. S9). A larger portion of N+4 products in the copying reaction
resulted from G:T mismatches, with 41% A1G3 and 8% G4, consistent with the high levels of G:T wobble pairing previously observed in nonenzymatic primer extension experiments (7, 19). We
also detected 5% A3G1, presumably due to A:C mismatch pairing.
The fidelity of template copying improved dramatically on the
2-thio-U containing RNA template (Fig. 4D). When a mixture of
5.0 mM 3′-NH2-2-MeImpddG and 15.0 mM 3′-NH2-2-MeImpddA
was used to copy a 5′-sUCsUC RNA template, the proportion of
correct full-length N+4 products was 92% (SI Appendix, Fig. S9),
corresponding to a per-site average fidelity of ∼98%. To confirm
that the major product was the Watson–Crick GAGA product, we
sequenced the 3′-NP-DNA products as previously described. LCMS sequencing confirmed that the A2G2 product had the correct
primer-GAGA sequence (SI Appendix, Table S2). The similarity of
3′-NP-DNA and RNA templates leads us to expect that similarly
accurate copying would be observed with 3′-NP-DNA templates
containing 2-thio-T in place of T.
The replication of arbitrary sequences of a nucleic acid
requires accurate copying of mixed-sequence templates in the
presence of all four different monomers (i.e., A, C, G, and T/sT).
Therefore, we used first three, and then four different monomers in copying reactions to assess fidelity in this more
complex situation. When we added 1.25 mM of the G monomer
to 5.0 mM of the C and T monomers to copy the 5′-AGAG
PNAS | October 29, 2013 | vol. 110 | no. 44 | 17735
CHEMISTRY
activated 2-thio-T monomer (3′-NH2-2-MeImpddsT, a compound not previously used in primer extension reactions) might
allow more rapid and efficient copying of A residues in 3′-NPDNA templates as a result of the higher stability of the sT:A base
pair, and reducing misincorporation due to G:T wobble pairing in
nonenzymatic template-directed primer extension reactions. It is
remarkable that the copying of the A4 3′-NP-DNA template (Fig. 2)
with the 2-thio-T monomer 3′-NH2-2-MeImpddsT was almost
complete in 5 min, compared with 1 h using 3′-NH2-2-MeImpddT.
Similar increases in the rate of primer extension were seen at
shorter times and with lower monomer concentrations (SI Appendix,
Figs. S5–S7). We observed extensive copying of a G4 3′-NP-DNA
template using the normal T monomer 3′-NH2-2-MeImpddT.
It was gratifying that this incorrect copying reaction was almost completely suppressed when we used the 2-thio monomer
3′-NH2-2-MeImpddsT.
Fig. 4. Enhanced copying fidelity of an RNA template containing 2-thioU in place of U. (A) Scheme for nonenzymatic primer extension on UCUC (or sUCsUC)
RNA templates. Red segment indicates newly formed phosphoramidate bonds. (B) PAGE analysis of primer extension products. Primer extension reactions
were carried out as in Fig. 1. The reaction was initiated by addition of 5.0 mM 3′-NH2-2-MeImpddG and 10.0 mM 3′-NH2-2-MeImpddA. Arrows indicate primer
and full-length product. (C) High-resolution MS analysis of the full-length extension products from a reaction of 100 pmol primer extended on a UCUC RNA
template for 12 h followed by ethanol precipitation. There are four distinct major families of isotopic peaks for four different 14-mer full-length N+4
products, corresponding to A3G1, A2G2, A1G3, and G4, respectively. (D) High-resolution MS analysis of the full-length extension products from copying
a sUCsUC RNA template. The major peak corresponds to the N+4 full-length product A2G2.
3′-NP-DNA template, only 38% of the full-length N+4 product
had the expected C2T2 composition. The majority of N+4 products
in the copying reaction resulted from G:T mismatches, with 49%
C1T3 and 7% T4. We also detected 7% C3T1 caused by an A:C
mismatch (SI Appendix, Fig. S8). The fidelity was enhanced significantly when the T monomer was replaced with sT. The expected
C2sT2 product accounted for 79% of the full-length N+4 products,
and the G:T mismatched product C1sT3 was reduced to only 9%,
although the A:C mismatched products increased slightly (2% C4
and 11% C3sT1) (SI Appendix, Fig. S8). The product mixture was
even more complicated when we used 5.0 mM A, C, and T, and
1.25 mM G monomers. The expected C2T2 product accounted only
for 25% of the full-length N+4 products (SI Appendix, Fig. S8). We
observed largely G:T mismatch products (56% C1T3 and 8% T4),
and 6% C3T1 and 1% C4 resulting from A:C mismatch pairing as
well as 4% A1C2T1. As above, the fidelity was greatly enhanced
when we replaced T with sT under the same conditions (SI Appendix, Fig. S8). We observed 74% of the expected C2sT2 product,
and G:T mismatched products were reduced to only 9% of C1sT3;
a small increase of A:C mismatched products was also observed
(3% C4 and 15% C3sT1). These results indicate that even in the
presence of all four monomers, reasonable fidelity can be achieved
in the nonenzymatic copying of mixed templates, as long as T is
replaced by 2-thio-T.
Discussion
In addition to suppressing G:T mismatches, we were initially
surprised to find that 2-thio-T also reduced A:C mismatches. We
attribute the suppression of A:C mismatch products to stronger A:
sT base pairing, so that 2-thio-T outcompetes C for binding to A.
This is consistent with our observations on the copying of homopolymer templates, where under the same conditions the reactivity
17736 | www.pnas.org/cgi/doi/10.1073/pnas.1312329110
of the 2-thio-T monomer on A4 templates was much faster than
that of the normal T monomer, and was similar to that of the C
monomer on G4 templates (SI Appendix, Figs. S5–S7). Replacement of T with 2-thio-T also enhanced both rate and yield in
replicating the 5′-AGAG 3′-NP-DNA template. Thus, the increased fidelity of nonenzymatic copying of 3′-NP-DNA with 2thio-T is most likely due to the combination of decreased G:sT
wobble pairing, and stronger A:sT Watson–Crick pairing, which
decreases the opportunity for A:C mismatches to form.
Our results suggest that the 2-thio modification of U (or T)
might have played a role in primordial RNA replication processes. It is interesting that 2-thio-U and 2-seleno-U (20) are
found in the third (wobble) position of the anticodons of
specific tRNAs, where they may contribute to enhanced affinity and specificity of the codon–anticodon interaction (21,
22). The modulation of base-pair affinity and specificity by
simple chemical modifications may have been particularly
important during the emergence of the RNA world, before the
evolution of polymerase-catalyzed replication. It is an intriguing speculation that the continued use of these modifications in extant biochemistry may be a relic of the chemical
origins of life (23).
The fast and accurate nonenzymatic copying of short mixedsequence 3′-NP-DNA templates suggests that 3′-NP-DNA may
be a suitable genetic polymer for use in the construction of artificial cellular systems. Assessing the genomic potential of 3′-NPDNA will require studies of the copying of longer 3′-NP-DNA
templates with varied sequences. Such studies are currently limited by the difficulty of solid-phase synthesis of 3′-NP-DNA,
which is expensive and inefficient. However, the nonenzymatic
synthesis of 3′-NP-DNA by copying RNA or LNA templates
may provide an alternative route to the synthesis of 3′-NP-DNA
Zhang et al.
Materials and Methods
Synthesis of Monomers and 3′-NP-DNA Primer and Templates. Here, 3′-NH2-2MeImpddNs (A, C, G, and T) were synthesized as previously described (7). The
synthesis of 3′-NH2-2-MeImpddsT is described in SI Appendix. The 3′-NP-DNA
primers and templates (29, 30) were synthesized at Geron Corp., and were
purified by reverse-phase HPLC (Agilent 1100 series LC) on a 50 × 4.6-mm
C18 column (XTerra) at 25 °C.
Nonenzymatic Primer Extension Reactions. Template copying reactions contained 0.1-μM 5′-Cy3/carboxytetramethylrhodamine (TAMRA)-labeled 3′amino–terminated primer, 0.5-μM template oligonucleotide, 150 mM NaCl,
100 mM HEI, 100 mM 2-(N-morpholino)ethanesulfonic acid/N-cyclohexyl-3aminopropanesulfonic acid/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(MES-CAPS-Hepes), pH 7.5, and 3′-NH2-2-MeImpddNs at indicated concentrations. Reactions were initiated by addition of activated monomers and
incubated at 4 °C. Aliquots were removed and stopped at indicated time
points by addition of three volumes of formamide and heating to 95 °C for
5 min, followed immediately by ethanol precipitation on dry ice. Stopped
reactions were resuspended in 8.0 M urea and heated to 95 °C for 5 min.
Samples were analyzed by electrophoresis on 7.0 M urea, 17% (wt/vol) polyacrylamide sequencing gels. Reaction products were visualized by fluorescence
imaging on a Typhoon 9410 PhosphorImager using the Cy3 fluorophore filter
set. Product quantification and analysis was performed using ImageQuant TL
software (GE Healthcare Life Sciences).
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LC-MS Studies of Fidelity in Nonenzymatic Primer Extension Reactions. Primer
extension products analyzed by LC-MS were prepared by extending 40–100
pmol of 3′-amino–terminated DNA primer at 4 °C for 12 h on complementary templates in similar conditions to those previously described. Crude
samples were injected for LC-MS analysis after ethanol precipitation. LC-MS
measurements were performed using an Agilent 6520 Q-TOF mass analyzer
and 1200 series HPLC with a Waters XBridge C18 column (3.5 μm, 1 × 100
mm). Mobile phase A was aqueous 200 mM hexafluoroisopropanol and 3
mM triethylamine at pH 7.0, and mobile phase B was methanol. The HPLC
method for 35 μL of a 2.5 μM solution was a linear increase of 5–20% B over
30 min at 0.1 mL/min, with the column heated to 60 °C. Sample elution was
monitored by absorbance at 260 nm and the eluate was passed directly to an
electrospray ionization source with 325 °C drying nitrogen gas flowing at 8.0
L/min, a nebulizer pressure of 30 pounds per square inch gauge and a capillary voltage of 3500 V. Agilent MassHunter Qualitative Analysis software
was used to analyze Q-TOF–derived MS data. The relative quantities of
different product species were quantified for fidelity measurements by integrating the extracted ion current peak for a 0.3 mass-to-charge ratio (m/z)
range centered on the −3 charge state of the 13C2 isotopes (for the 3′-NPDNA primer) or a 0.26 m/z range centered on the −4 charge state of the 13C3
isotopes (for the TAMRA-labeled DNA primer). The narrow ranges were used
to minimize overlap between peaks; however, the relative values obtained
were very similar when larger ranges were used.
Acidic Hydrolysis of 3′-NP-DNA for LC-MS Sequencing. The 3′-NP-DNA primer
extension products were prepared for sequence analysis from a reaction of
200 pmol 3′-amino–terminated primer extended on mixed templates for
12 h followed by ethanol precipitation. To the crude 3′-NP-DNA product
(8 μL, 25 μM) in a 0.2 mL Eppendorf tube was added 2 μL 25% acetic acid. The
solution was vortexed, and heated in a water bath at 40 °C for 30 min. The
resultant partially degraded products were subjected to LC-MS analysis after
ethanol precipitation.
ACKNOWLEDGMENTS. We thank members of our laboratory for helpful
discussions and comments on the manuscript. This research was funded in
part by Grant CHE-0809413 from the National Science Foundation. J.W.S. is
an Investigator of the Howard Hughes Medical Institute.
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Res 30(17):3739–3747.
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21. Caton-Williams J, Huang Z (2008) Biochemistry of selenium-derivatized naturally occurring and unnatural nucleic acids. Chem Biodivers 5(3):396–407.
22. Ajitkumar P, Cherayil JD (1988) Thionucleosides in transfer ribonucleic acid: Diversity,
structure, biosynthesis, and function. Microbiol Rev 52(1):103–113.
23. Szostak JW (2012) The eightfold path to non-enzymatic RNA replication. J Syst Chem
3:2.
24. Gryaznov S, Chen JK (1994) Oligodeoxyribonucleotide N3′->P5′ phosphoramidates:
Synthesis and hybridization properties. J Am Chem Soc 116(7):3143–3144.
25. Gryaznov SM, et al. (1995) Oligonucleotide N3′—>P5′ phosphoramidates. Proc Natl
Acad Sci USA 92(13):5798–5802.
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27. Powner MW, Sutherland JD, Szostak JW (2010) Chemoselective multicomponent onepot assembly of purine precursors in water. J Am Chem Soc 132(46):16677–16688.
28. Mansy SS, Szostak JW (2009) Reconstructing the emergence of cellular life through
the synthesis of model protocells. Cold Spring Harb Symp Quant Biol 74:47–54.
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N3′->P5′ phosphoramidate building blocks. Tetrahedron Lett 47:4495–4499.
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PNAS | October 29, 2013 | vol. 110 | no. 44 | 17737
CHEMISTRY
oligonucleotides if this can be carried out with sufficient fidelity
and on an appropriate scale. A potential barrier to 3′-NP-DNA
replication is the difficulty of strand separation due to the high
Tm of duplex 3′-NP-DNA (24–26), and the additional duplex
stabilization expected from 2-thio-T substitution. This problem
may be ameliorated by moderate concentrations of formamide
or urea (27), or alternatively, a heterogeneous backbone of N3′P5′ and N2′-P5′ linkages could be used (6). The acid lability of 3′NP-DNA would constrain protocells based on this genetic
polymer to neutral or moderately alkaline environments. If
complete cycles of 3′-NP-DNA replication can be achieved, it
may be possible to assemble simple protocells (4, 28) within
which genomic information resides in a nonbiological polymer.
Supporting Information Appendix
For manuscript titled “Fast and accurate non-enzymatic copying of an RNA-like
synthetic genetic polymer”
SI Text
General information for reagents and instrumentation. All solvents and reagents were
reagent grade, purchased commercially, and used without further purification unless
specified. All chemicals were purchased from Sigma-Aldrich unless otherwise indicated.
Oligonucleotides used as primers or templates were synthesized on an Expedite nucleic
acid synthesizer (Applied BioSystems) or purchased from IDT (Coralville, IA) unless
otherwise indicated. All the Nuclear Magnetic Resonance (NMR) spectra were recorded
on a Varian NMR spectrometer (Oxford AS-400). Chemical shifts are reported as parts
per million (ppm) using tetramethylsilane (TMS) as internal standard or by reference to
proton resonances resulting from incomplete deuteration of the NMR solvent. Data were
reported as follows: (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd =
doublet of doublets, J = coupling constant in Hz, integration). Proton-decoupled 13C
NMR (100 MHz) spectra were reported in ppm from CDCl3, CD3OD, or DMSO-d6
(77.0, 49.0, or 39.5 ppm, respectively). Proton-decoupled 31P NMR (161.8 MHz) spectra
were reported in ppm using phosphate buffer as reference. Electrospray mass spectra
were recorded on a Bruker Daltonics Esquire 6000 ESI-MS. LC-MS studies of
oligonucleotides were carried out on Agilent 6520 Q-TOF LC/MS system. 5'-O(dimethoxytrityl)-N3/O4-(toluoyl)-2-thiothymidine was purchased from Berry &
Associates (Dexter, MI). The activated phosphor-2-methylimidazole nucleotide
monomers were purified by reverse-phase preparative HPLC (Varian ProStar Preparative
LC) on a Prep-C18 column (Varian Dynamax 250 × 21.4 mm) equilibrated with 25 mM
triethylammonium bicarbonate, pH 8.0 and eluted with a linear acetonitrile gradient (060%).
Synthesis of 3′-amino-3′-deoxy-2-thiothymidine-5′-phosphor-2-methylimidazolide
(3′-NH2-2-MeImpddsT).
O
N
S
DMTrO
N
N
55 oC
1h
S
OH
O
DMTrO
2
O
NH
N
5
NH3
MeOH
NH
Ms-Cl
pyridine
0 oC-r.t.
4h
N
DMTrO
S
OMs
O
LiN3
DMF
90 oC
2h
O2N
O
N3
O
O
O
1
O
O
NH
N
S
r.t.
4h
OH
O
O
N
DMTrO
O
DMTrO
O
DIAD
PPh3
4-Nitrobenzoic
acid
THF
O
S
i)
POCl3
Proton
S spondge
PO(MeO)3
4
O
NH
Dichloroacetic
acid
DCM
r.t.
1h
3
N
HO
O
N3
6
O
NH
N
O
N
ii)
2-MeImidazole
N
P
O
O
ON3
7
S
i)
PPh3
Pyridine
ii)
NH4OH
NH
N
O
N
N
P
O
O
ONH2
8
S
1-[5′-O-(Dimethoxytrityl)-N3/O4-(toluoyl)-3′-O-(4-nitrobenzoyloxy)-β-D-threopentofuranosyl]-2-thiothymine (Compound 2)
The preparation of compound 2 was adapted from a previously reported procedure (1-3),
with minor modifications as follows.
To a solution of 5'-O-(dimethoxytrityl)-N3/O4-(toluoyl)-2-thiothymidine 1 (From Berry &
Associates, Inc.) (100 mg; 0.147 mmol) in anhydrous THF (2.0 ml) were added
triphenylphosphine (58 mg; 0.221 mmol) and diisopropyl azodicarboxylate (DIAD) (45
mg, 44 µl, 0.221 mmol) at room temperature. After 20 min, 4-nitrobenzoic acid (37 mg,
0.221 mmol) was added to the reaction mixture and the reaction mixture was stirred
further for 4 h. The solvent was removed under vacuum and the residue was purified by
flash column chromatography over silica gel using methanol-dichloromethane (1%-10%)
as the eluent to afford 2 (103 mg, 85%) as a yellow foam. 1H NMR δ (400 MHz, CDCl3):
8.20 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.69 (s, 1H), 7.68-7.63 (m, 5H), 7.567.52 (m, 4H), 7.47-7.43 (m, 6H), 7.38 (dd, J = 7.6 Hz, 5.6 Hz, 1H), 6.84 (m, 1H), 6.74
(m, 2H), 5.79-5.76 (m, 1H), 4.60-4.53 (m, 1H), 3.75 (s, 6H), 3.68-3.62 (m, 1H), 3.573.53 (m, 1H), 2.99-2.93 (m, 1H), 2.46-2.44 (m, 1H), 2.40 (s, 3H), 2.03 (s, 3H); ESI-MS
calcd for C46H41N3NaO10S+ [M+Na]+: 850.2, found: 850.0.
1-[5′-O-(Dimethoxytrityl)-3′-OH-β-D-threo-pentofuranosyl]-2-thiothymine (Compound
3).
A suspension of 2 (146 mg, 0.177 mmol) in methanolic ammonia (10 ml) was stirred for
1 h at 55 oC. The homogeneous solution was concentrated under vacuum and the residue
was purified by flash column chromatography over silica gel using methanol-chloroform
(5%-10%) as the eluent to afford 3 (80 mg; 81% yield) as a white foam. 1H NMR δ (400
MHz, CDCl3): 7.73 (s, 1H), 7.67 (d, J = 6.8 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.33 (d, J
= 8.0 Hz, 2H), 7.26 (dd, J = 6.4 Hz, 6.8 Hz, 1H), 7.18 (d, J = 7.6 Hz, 2H), 6.81 (d, J = 8.8
Hz, 4H), 6.73 (dd, J = 1.4 Hz, 6.8 Hz, 1H), 4.37-4.35 (m, 1H), 4.12-4.09 (m, 1H), 3.74 (s,
6H), 3.65-3.61 (m, 1H), 3.49-3.45 (m, 1H), 2.56-2.54 (m, 1H), 2.35 (s, 3H), 2.20-2.17
(m, 1H); ESI-HRMS calcd for C31H33N2O6S+ [M+H]+: 561.2054, found: 561.2060.
1-[5′-O-(Dimethoxytrityl)-3′-O-mesyl-β-D-threo-pentofuranosyl]-2-thiothymine
(Compound 4).
To a solution of 3 (80 mg; 0.143 mmol) in anhydrous pyridine (1.0 ml) were added MsCl
(20 mg; 13 µl; 0.171 mmol) dropwise at room temperature. The reaction mixture was
quenched with MeOH after stirring for 4 h and concentrated. The residue was dissolved
in CH2Cl2 (100 ml) and washed with water (2×50 ml). The organic layer was dried
(Na2SO4), concentrated under vacuum, and the residue was purified by flash column
chromatography over silica gel using ethyl acetate-hexane (10%-50%) as the eluent to
afford 4 (81mg; 89% yield) as a white foam. 1H NMR δ (400 MHz, CDCl3): 7.45 (s, 1H),
7.43 (d, J = 6.8 Hz, 2H), 7.35-7.29 (m, 5H), 7.25 (d, J = 6.4 Hz, 2H), 6.86 (d, J = 8.8 Hz,
4H), 6.75 (dd, J = 2.0 Hz, 5.6 Hz, 1H), 5.25-5.23 (m, 1H), 4.32-4.28 (m, 1H), 3.79 (s,
6H), 3.68-3.65 (m, 1H), 3.44-3.39 (m, 1H), 2.91-2.85 (m, 1H), 2.77 (s, 3H), 2.61-2.56
(m, 1H), 1.83 (s, 3H); ESI-HRMS calcd for C32H35N2O8S2+ [M+H]+: 639.1829, found:
639.1832.
S2
3′-Azido-5′-O-(dimethoxytrityl)-3′-deoxy-2-thiothymidine (Compound 5).
To a solution of 4 (80 mg, 0.125 mmol) in anhydrous DMF (1.0 ml) was added lithium
azide (31 mg, 0.626 mmol). The reaction mixture was heated at 90 °C until all starting
materials were consumed. The solvent was then evaporated under vacuum and the
residue was purified by flash column chromatography over silica gel using ethyl acetatehexane (10%-60%) as the eluent to afford 5 (62 mg; 85% yield) as a white foam. 1H
NMR δ (400 MHz, CDCl3): 7.85 (s, 1H), 7.64 (m, 1H), 7.51-7.42 (m, 1H), 7.38-7.31 (m,
2H), 7.31-7.19 (m, 5H), 6.83-6.80 (m, 4H), 6.79 (m, 1H), 4.33-4.29 (m, 1H), 3.99-3.94
(m, 1H), 3.76 (s, 6H), 3.61-3.57 (m, 1H), 3.33-3.29 (m, 1H), 2.68-2.60 (m, 1H), 2.462.39 (m, 1H), 1.46 (s, 3H); ESI-HRMS calcd for C31H31N5NaO5S+ [M+Na]+: 608.1938,
found: 608.1949.
3′-Azido-3′-deoxy-2-thiothymidine (Compound 6).
To a solution of 5 (62 mg; 0.106 mmol) in anhydrous dichloromethane (2.0 ml) was
dropwise added 0.1 ml 5% dichloroacetic acid at room temperature in two portions. After
stirring at room temperature for 30 min, the resulting red reaction mixture was
concentrated under reduced pressure and partitioned between H2O and CHCl3. The
organic layer was separated and dried over Na2SO4. After concentration, the residue was
purified by flash column chromatography over silica gel methanol-chloroform (2%-10%)
as the eluent to afford 6 (27 mg; 91% yield) as a white foam. 1H NMR δ (400 MHz,
CD3OD): 8.17 (s, 1H), 6.91 (dd, J = 5.6 Hz, 6.0 Hz, 1H), 4.37-4.32 (m, 1H), 3.97-3.94
(m, 1H), 3.92-3.91 (m, 1H), 3.80-3.79 (m, 1H), 2.59-2.43 (m, 1H), 2.41-2.34 (m, 1H),
1.92 (s, 3H); ESI-HRMS calcd for C10H14N5O3S+ [M+H]+: 284.0812, found: 284.0819.
3′-Azido-3′-deoxy-2-thiothymidine-5′-phosphor-2-methylimidazole (Compound 7).
Compound 6 (20 mg; 0.071 mmol) and proton sponge (18 mg; 0.085 mmol) were dried in
a vacuum desiccator over P2O5 overnight before dissolving in trimethyl phosphate (1.0
ml). Then freshly distilled POCl3 (8.0 µl; 0.29 mmol) was added dropwise at 0 °C. After
stirring at 0°C for 1.0 h, 2-methylimidazole (30 mg; 0.353 mmol) was then added at 0 °C.
After stirring for additional 4 h at room temperature, the reaction mixture was partitioned
between H2O and CH2Cl2. The crude product was further purified to afford 7 by reversephase preparative HPLC (Varian ProStar Preparative LC) on a Prep-C18 column (Varian
Dynamax 250 × 21.4 mm) equilibrated with 25 mM triethylammonium bicarbonate, pH
8.0 and eluted with an acetonitrile linear gradient (0-60%). 1H NMR δ (400 MHz,
CD3CN): 7.96 (s, 1H), 7.16 (s, 1H), 6.81(dd, J = 6.0 Hz, 6.4 Hz, 1H), 6.71 (s, 1H), 4.284.23 (m, 1H), 3.97-3.93 (m, 1H), 3.96-3.93 (m, 1H), 3.87 (m, 1H), 2.40-2.28 (m, 2H),
2.54 (s, 3H), 1.94 (s, 3H); 31P NMR δ (168.1 MHz, CD3CN): -10.67; ESI-MS calcd for
C14H17N7O5PS- [M-]: 426.08, found: 426.0.
3′-Amino-3′-deoxy-2-thiothymidine-5′-phosphor-2-methylimidazolide
(3′-NH2-2MeImpddsT 8).
To a solution of 7 (10 mg; 0.02 mmol) in a mixture solution of pyridine (1 ml) and 30%
ammonium hydroxide solution (1.0 ml) was added triphenylphosphine (12 mg; 0.05
mmol) at room temperature. The reaction was then stirred for 5 h at room temperature.
The resulting mixture was concentrated under vacuum and the residue was diluted with 1
S3
ml of DMSO for NaClO4 precipitation as previously described (4). The crude product
was further purified by reverse-phase preparative HPLC as previously described to afford
8. 1H NMR δ (400 MHz, D2O): 7.69 (s, 1H), 7.18 (s, 1H), 6.88 (dd, J = 4.8 Hz, 5.6 Hz,
1H), 6.83 (s, 1H), 4.16-4.12 (m, 1H), 4.08-3.95 (m, 2H), 3.66-3.61 (m, 1H), 2.45-2.34
(m, 1H), 2.46 (s, 3H), 1.87 (s, 3H); 31P NMR δ (168.1 MHz, D2O): -10.41; ESI-HRMS
calcd for C14H19N5O5PS- [M-H]-: 400.0850, found: 400.0862.
Efforts to synthesize 3′-NH-trityl-2-thiothymidine-5′-O-phosphoramidite.
The 5′-O-phosphoramidite of 3′-NH-trityl-2-thiothymidine is the protected monomer
required for the solid phase synthesis of 3′-NP-DNA oligomers containing 2-thio
substituted T. Because the route that we employed to generate the 5′-phosphor-(2methyl)-imidazolide is too inefficient to yield the amounts of the amidite required for
solid phase synthesis, we sought a more efficient synthesis starting with 3′-NH-tritylthymidine. We converted this to the 5′-iodo derivative, and then obtained the 2-5’anhydro cyclic derivative in good yield. Unfortunately, all efforts to convert this
compound to 3′-NH-trityl-2-thiothymidine by nucleophilic attack of attack of H2S on the
C2 position failed. We obtained a complex reaction mixture of at least five (detectable by
TLC and RP HPLC) products, with no major reaction product in the mixture. The
products were separated and isolated using high-resolution silica gel column
chromatography. None of the isolated compounds contained the 2-thiothymine
heterocyclic base (characteristic UV Abs. max. at ~280nm), as judged by UV spectral
analysis, c.f. with commercially available sample of 2-thiothymidine. We are currently
investigating alternative synthetic strategies in search of an efficient route to the desired
phosphoramidite.
S4
References:
1.
2.
3.
4.
Jain ML & Bruice TC (2006) Solid-phase synthesis of positively charged
deoxynucleic guanidine (DNG) oligonucleotide incorporating 7-deazaguanine
bases. Bioorg Med Chem 14(21):7333-7346.
Zhang S, Zhang N, Blain JC, & Szostak JW (2013) Synthesis of N3'-P5'-linked
phosphoramidate DNA by nonenzymatic template-directed primer extension. J
Am Chem Soc 135(2):924-932.
Eisenhuth R & Richert C (2009) Convenient syntheses of 3'-amino-2',3'dideoxynucleosides,
their
5'-monophosphates,
and
3'-aminoterminal
oligodeoxynucleotide primers. J Org Chem 74(1):26-37.
Zhang N, Zhang S, & Szostak JW (2012) Activated ribonucleotides undergo a
sugar pucker switch upon binding to a single-stranded RNA template. J Am Chem
Soc 134(8):3691-3694.
S5
SI Figures
A
B
Fig. S1. Comparison of non-enzymatic primer extension reaction using 3′-NH2-2MeImpddG as a monomer on DNA, RNA and 3′-NP-DNA templates. (A) Reaction
scheme for non-enzymatic primer extension reaction. Red segments indicate
phosphoramidate bonds. (B) PAGE analysis of primer-extension products on indicated
templates. Primer extension reactions were carried out as previously described, and the
reaction was initiated by addition of 5.0 mM 3′-NH2-2-MeImpddG. Arrows indicate
primer and full-length product.
A
B
Fig. S2. Comparison of non-enzymatic primer extension reaction using 3′-NH2-2MeImpddC as a monomer on DNA, RNA and 3′-NP-DNA templates. (A) Reaction
scheme for non-enzymatic primer extension reaction. Red segments indicate
phosphoramidate bonds. (B) PAGE analysis of primer extension products on indicated
templates. Primer extension reactions were carried out as previously described, and the
reaction was initiated by addition of 5.0 mM 3′-NH2-2-MeImpddC. Arrows indicate
primer and full-length product.
S6
A
B
Fig. S3. Comparison of non-enzymatic primer extension reaction using 3′-NH2-2MeImpddT as a monomer on DNA, RNA and 3′-NP-DNA templates. (A) Reaction
scheme for non-enzymatic primer extension reaction. Red segments indicate
phosphoramidate bonds. (B) PAGE analysis of primer extension products on indicated
templates. Primer extension reactions were carried out as previously described, and the
reaction was initiated by addition of 10.0 mM 3′-NH2-2-MeImpddT. Arrows indicate
primer and full-length product.
A
B
Fig. S4. Comparison of non-enzymatic primer extension reaction using 3′-NH2-2MeImpddA as a monomer on DNA, 3′-NP-DNA and LNA templates. (A) Reaction
scheme for non-enzymatic primer extension reaction. Red segments indicate
phosphoramidate bonds. (B) PAGE analysis of primer-extension products on indicated
templates. Primer-extension reactions were carried out as previously described, and the
reaction was initiated by addition of 10.0 mM 3′-NH2-2-MeImpddA. Arrows indicate
primer and full-length product.
S7
A) Monomer concentration: 1.0 mM
B) Monomer concentration: 2.5 mM
Fig. S5. Non-enzymatic primer-extension reactions on different 3′-NP-DNA templates
with their complementary 3′-NH2-2-MeImpddN monomers. PAGE analysis of primerextension products on indicated templates. Primer-extension reactions were carried out as
previously described, and the reaction was initiated by addition of 1.0 mM (A) and 2.5
mM (B) 3′-NH2-2-MeImpddN as indicated. Arrows indicate primer and full-length
product.
Fig. S6. Non-enzymatic primer-extension reactions on different DNA templates with
their complementary 3′-NH2-2-MeImpddN monomers. PAGE analysis of primerextension products on indicated templates. Primer-extension reactions were carried out as
previously described, and the reaction was initiated by addition of 1.0 mM 3′-NH2-2MeImpddN as indicated. Arrows indicate primer and full-length product.
S8
A) Monomer concentration: 5.0 mM
B) Monomer concentration: 2.5 mM
C) Monomer concentration: 1.0 mM
Fig. S7. Effect of monomer concentration on non-enzymatic primer extension reactions
on RNA templates. PAGE analysis of primer extension products on indicated templates.
Primer extension reactions were carried out as previously described, and the reaction was
initiated by addition of 5.0 mM (A), 2.5 mM (B) and 1.0 mM (C) 3′-NH2-2-MeImpddN
as indicated. Arrows indicate primer and full-length product.
S9
1.0
1.0
Fraction N+4 Product
0.8
0.8
0.09
0.04
0.08
0.09
0.06
0.07
0.16
0.06
0.16
0.15
0.11
0.15
0.02
0.49
0.03
0.51
0.12
0.46
0.27
0.26
C3Tx
Tx4
C2TA
0.6
0.6
0.06
0.79
0.74
C4
CTx3
0.42
0.56
0.4
0.4
C2Tx2
0.07
0.05
1.00
0.85
0.23
0.06
0.01
0.48
0.2
0.2
0.04
0.53
0.58
0.82
0.48
0.38
0.25
0.26
5
6
7
8
9
NPDNA
NPDNA
RNA
RNA
NPDNA
NPDNA
NPDNA
0
10
0
2.5
0
0
5
0
5
0
0
2.5
0
10
0
0
5
0
20
0
0
5
0
5
0
0
5
0
0
5
DNA
0
5
0
0
5
RNA
12
4
NPDNA
1.25
5
0
5
0
11
1.25
5
0
0
5
10
1.25
5
5
5
0
3
1
Template: NPDNA
2
0
0.0
[Monomer] (mM) G 1.25
C
5
A
5
T
0
sT 5
0
5
0
0
5
NPDNA
Fig. S8. Comparison of fidelity of full-length N+4 products from copying 5′-AGAG-3′
templates.
NP-DNA:
a
chimeric
DNA/3′-NP-DNA
oligonucleotide
5′CAGAGGACTATCGGC-3′ (3′-NP-DNA underlined).
S10
1.0
1.0
0.02
0.27
Fraction N+4 Product
0.8
0.8
0.04
0.24
0.04
0.13
0.08
0.33
0.16
0.4
0.4
0.2
0.2
0.26
0.41
A2G2
A4
A3G
AG3
G4
0.32
0.35
0.01
0.43
0.16
0.07
0.10
0.40
0.6
0.6
0.08
0.40
0.92
0.08
0.05
0.77
0.74
0.45
0.40
0.29
1
2
3
4
5
6
7
8
0
0.0
NPDNA
DNA
RNA
RNA
RNA
RNA
RNA
RNA
Sequence: TCTC
TCTC
UCUC
Primer: NPDNA
NPDNA
NPDNA
[Monomer] (mM) G
A
Template:
1
10
1
10
1
10
5
15
5
15
5
20
5
10
5
10
UCUC sUCsUC sUCsUC UCUC sUCsUC
NPDNA
NPDNA
NPDNA
3'-NH2DNA
3'-NH2DNA
Fig. S9. Comparison on fidelity of full-length N+4 products from copying a template
region of 5′-TCTC/UCUC/sUCsUC. 1) NP-DNA primer: an entirely 3′-NP-DNA
oligonucleotide (linked by N3′-P5′-phosphoramidate bonds); 2) NP-DNA TCTC
template: a chimeric DNA/3′-NP-DNA oligonucleotide 5′-ATCTCGACTATCGGC-3′
(3′-NP-DNA underlined).
S11
SI Tables
Table S1. LC-MS sequencing of the crude product from non-enzymatic primer extension
on a GAGA 3′-NP-DNA template.
Peak
Calculated
monoisotopic
mass
Observed
monoisotopic
mass
Error
(ppm)
1
2
3
4
5
4230.9111
3911.8719
3623.8096
3304.7704
3016.7080
4230.8962
3911.8594
3623.7942
3304.7619
3016.7002
3.52
3.20
4.24
2.57
2.60
Observed
mass
difference
between
ladder
oligos
319.0368
288.0652
319.0323
288.0617
Ladder sequencea,b
Ladder
oligo
length
5′-GCCGATAGTCCsTCsT-3′
5′-GCCGATAGTCCsTC-3′
5′-GCCGATAGTCCsT-3′
5′-GCCGATAGTCC-3′
5′-GCCGATAGTC-3′
14
13
12
11
10
a. All sequences are linked with N3′-P5′ phosphoramidate bonds
b. 3′-end is a NH2 group
Table S2. LC-MS Sequencing of the crude product from non-enzymatic primer extension
on a 20-mer sUCsUC RNA template.
Peak
1
2
3
4
5
Calculated
isotopic
mass with 3
13
C
6536.3229
6224.2493
5896.1808
5584.1072
5256.0387
Observed
isotopic
mass with 3
13
C
6536.3393
6224.2364
5896.1870
5584.0953
5256.0634
Error
(ppm)
-2.51
2.07
-1.05
2.13
-4.70
Observed mass
difference
between ladder
oligos
312.1029
328.0494
312.0917
328.0319
Ladder
sequencea,b
N-GAGA-3′
N-GAG-3′
N-GA-3′
N-G-3′
N
a. 3′-end of all oligos are NH2-terminated
b. N = Primer (5′-TAMRA-GCG TAG ACT GAC TGG-3′)
S12
Ladder
oligo
length
19
18
17
16
15
Note
Full-length
Primer
Note
Full-length
Primer
8.207
8.186
7.809
7.788
7.692
7.671
7.662
7.644
7.641
7.561
7.542
7.527
7.524
7.481
7.475
7.462
7.456
7.445
7.443
7.438
7.385
7.368
7.283
7.260
7.239
7.231
7.218
6.854
6.748
6.733
6.726
5.251
5.235
5.219
4.988
4.972
4.957
4.143
4.126
4.108
4.090
3.796
3.754
3.748
3.646
3.555
2.409
2.373
2.039
1.805
1.779
1.647
1.434
1.418
1.320
1.304
1.268
1.262
1.253
1.236
1.204
1.157
1.142
1H-­‐NMR for compound 2, Solvent: CDCl
O
S
DMTrO
10ppm
-1 ppm-25.015
910
89
78
67
3 O
N
N
O
O
O
O2N
56
45
34
23
12
01
-10
1.226
1.032
1.014
0.997
1.734
2.617
2.599
2.581
2.563
2.348
2.209
2.172
4.366
4.109
4.102
3.741
3.629
3.615
3.490
3.478
3.464
3 7.732
7.684
7.664
7.505
7.487
7.445
7.426
7.339
7.318
7.279
7.261
7.242
7.207
7.189
7.169
6.817
6.795
6.733
6.715
1H-­‐NMR for compound 3, Solvent: CDCl
O
NH
N
DMTrO
S
OH
O
2.11 9.26
2.11
9.26 1.11.1
1.05
1.051.011.01
1.13 3.87
1.133.87 4.24.2
10ppm-25.015910
-1
89
78
1
67
56
3.03
3.03
15.84 1.05
5.841.05
45
34
23
12
01
-10
DMTrO
-1
7.691
7.671
7.661
7.642
7.540
7.521
7.471
7.448
7.428
7.374
7.346
7.339
7.324
7.317
7.306
7.287
7.258
7.242
7.223
6.857
6.836
6.752
6.738
6.734
5.244
5.234
5.225
4.315
4.301
4.293
4.279
3.788
3.682
3.667
3.657
3.641
3.472
3.432
3.418
3.407
3.393
3.003
2.909
2.892
2.878
2.870
2.852
2.838
2.769
2.594
2.554
1.830
1.781
1.480
1.256
1.234
10.068
1H-­‐NMR for compound 4, Solvent: CDCl
N
1.29
1.29
11ppm-25.015
1011
910
89
78
3 O
NH
S
OMs
O
0.842 8.37
0.842
8.37
1.04
1.04
1.06
1.06 1.331.33
1.15 1.32
1.151.32
4.764.76
4.674.67
1
67
1
56
6.76 1.16
6.76
1.16 3.013.01
45
34
3.61
3.61
23
12
01
-10
N
DMTrO
ppm-25.015
10ppm
-1
910
89
78
67
4.313
4.297
3.969
3.962
3.955
3.755
3.605
3.581
3.578
3.319
3.296
3.292
2.916
2.843
2.669
2.655
2.637
2.620
2.604
2.457
2.440
2.424
2.408
1.895
1.761
1.465
1.224
7.985
7.847
7.637
7.441
7.423
7.378
7.360
7.335
7.314
7.267
7.260
7.246
7.226
7.208
7.190
6.826
6.806
6.795
6.780
1H-­‐NMR for compound 5, Solvent: CDCl
3 O
NH
S
O
N3
0.7390.739
2.582.58
5.985.98
0.974 0.974
1.231.23
1 1.48
1
7.99
1.48
7.99
56
45
34
1.551.55
23
3.02
3.02
1.06
1.066.92 6.92
1.041.04 1.311.31
12
01
-10
N
HO
1
ppm-16.035
10ppm
-1
910
89
78
4.900
4.376
4.359
4.345
4.016
4.009
4.002
3.995
3.987
3.979
3.956
3.949
3.845
3.839
3.815
3.808
3.360
3.356
3.353
3.349
3.042
2.909
2.634
2.615
2.599
2.584
2.440
2.426
2.422
2.407
2.392
2.388
1.966
1.926
1.331
6.923
6.908
6.894
8.170
1H-­‐NMR for compound 6, Solvent: CD
3OD O
NH
S
O
N3
1.13 1.13
1.971.97
1
67
56
45
3.1
1.121.123.1
1.051.05
1.031.03
1.19
1.19
34
23
12
01
-10
N
O
N
-1
P
ON
O
1.1 1.1
ppm-25.015
10ppm
910
89
78
4.258
4.243
3.969
3.944
3.933
3.855
3.849
3.839
3.834
2.730
2.712
2.693
2.530
2.495
2.478
2.464
2.444
2.428
2.396
2.378
2.361
2.348
2.330
2.313
2.296
2.283
1.940
1.934
1.928
1.922
1.249
1.148
0.951
0.947
0.939
0.931
0.924
0.851
0.833
0.815
7.160
6.822
6.807
6.792
6.713
7.959
1H-­‐NMR for compound 7, Solvent: CD
3CN O
NH
S
O
N3
1.08 1.08
1.031.03
1.021.02
67
56
45
3.6
3.493.49
3.6
2.672.67
1.56
1.56
34
23
12
01
-10
N
O
N
P
O-
ppm-10.782
10ppm
0
N
O
910
89
1.01 1.01
78
4.820
4.815
4.131
4.130
4.128
4.119
4.118
3.995
3.631
3.630
3.535
3.508
2.475
2.473
2.456
2.450
2.430
2.386
2.385
2.376
2.373
2.372
2.368
2.366
2.355
2.353
2.150
1.893
1.879
1.877
1.862
1.274
1.256
1.251
1.239
7.175
7.088
6.888
6.876
6.871
6.830
7.690
1H-­‐NMR for compound 8, Solvent: D
2O O
NH
S
O
NH2
0.853 0.853
1.481.48 2.012.01
1.981.98
1.261.26
1.241.24
67
56
45
4.014.01
2.96
2.96
34
23
12
01