A pre-RNA candidate revisited: Both enantiomers of flexible nucleoside triphosphates are DNA polymerase substrates

Article abstract of DOI:10.1021/ja0770680

One of the earliest proposed pre-RNA candidates is the flexible nucleic acid (FNA) structure based on the mixed acetal aminal of formyl glycerol. FNA achieves dramatic stereochemical simplification by formally deleting the 2′-CHOH group of RNA, thereby re

Full text of DOI:10.1021/ja0770680

Published on Web 12/21/2007
A Pre-RNA Candidate Revisited: Both Enantiomers of Flexible Nucleoside
Triphosphates are DNA Polymerase Substrates
Benjamin D. Heuberger and Christopher Switzer*
Department of Chemistry, UniVersity of CaliforniasRiVerside, RiVerside, California 92521
Received September 12, 2007; E-mail: switzer@ucr.edu
Scheme 1. Synthesis of fNTPsa
Simplified alternatives to natural nucleic acids may have served
in molecular evolution as a bridge between prebiotic chemistry and
the RNA world. While RNA itself currently fulfills both genotypic
and phenotypic roles as a carrier of genetic information and catalyst,
molecular complexity brings into question its suitability as the first
informational molecule. Candidate structures for the latter include
PNA,1 TNA,2 and GNA.3
One of the earliest proposed pre-RNA candidates is the flexible
nucleic acid (FNA) structure based on the mixed acetal aminal of
formyl glycerol.4 FNA achieves dramatic stereochemical simplifica-
tion by formally deleting the 2′-CHOH group of RNA, thereby
replacing the four RNA stereocenters with a single (pro)stereocenter.
This simplicity comes at the price of increased backbone flexibility
a Reaction conditions: (i) trioxane, HCl(g), CH2Cl2, 0°; (ii) N,O-bis-
and possibly less optimal physical properties. Indeed, stability
TMS-acetamide, nucleobase (A, G, C, or T), TMSOTF, CH3CN; (iii) EtOH
or MeOH, cyclohexene, Pd(black), 80°; (iv) [(Bu)4N]5P3O10, CH3CN, room
temperature.
measurements on DNA/FNA chimeric duplexes have shown weak
or no hybridization.5 Yet, an important predicted feature of FNA
is insensitivity to chirality4sin relation to both itself and other
molecules. Despite the documented inability of FNA to form stable
duplexes with DNA, flexible nucleoside triphosphates (fNTPs) are
substrates for DNA polymerases. Consistent with early predictions,
FNA chirality is unimportant as polymerases accept both (S)- and
(R)-fNTP antipodes. These results provide a renewed basis for
positing FNA as a viable informational molecule.
Syntheses of both the (S)- and (R)-enantiomers of all four fNTPs
are shown in Scheme 1. The syntheses began with alcohol 1,
available from commercial (S)- and (R)-glycidyl tosylates.6 Chlo-
romethylation of 1,7 followed by a Vorbruggen reaction8 with
silylated bases, yielded benzyl-protected nucleosides 2. Debenzy-
lation with Pd/cyclohexene7 and then displacement of the tosylate
with butylammonium triphosphate9 gave the triphosphates 4.
Terminating acyclonucleoside triphosphates related to acyclovir
lacking the hydroxyl group required for chain extension have been
employed as an alternative to dideoxynucleotides in the Sanger
DNA sequencing method.10 Due to the efficiency with which 9°N
A485L exo- (Therminator) DNA polymerase has accepted terminat-
ing acyclonucleoside substrates,11 we used it initially in conjunction
with primed template 1 to assess incorporation and extension of
fNTPs. The results, displayed in Figure 1, panel b, show multiple
incorporations of flexible nucleotides in the case of racemic, (R)-,
and (S)-fCTPs. Maximal incorporation is seen with the “non-
natural” configuration found in the (R)-enantiomer, leading mainly
to the incorporation of four nucleotides with longer bands visible,
followed closely in efficiency by the racemic and antipodal
triphosphates. As shown in panel c of Figure 1, a wider screen of
polymerases did not yield results superior to those obtained with
Therminator, although many other polymerases showed activity.
The preference of B family archaeal polymerases11 for chain
terminating acyclonucleotides led us to compare the activities of
Therminator, Vent exo-, and Deep Vent exo- using templates 1-4
and both antipodes of the complete fNTP series, as shown in Figure
2a. Of the three polymerases, Therminator shows notable efficiency
in primer extension with all four fNTPs of both antipodes, incor-
Figure 1. (a) Primer and template sequences; italicized text refers to
template coding regions. (b) Primer extension using template 1 with (()-,
(S)-, and (R)-fCTP as indicated, 2U Therminator DNA polymerase, 1U Tth
pyrophosphatase, 75 °C, 24 h. (c) Primer extension using template 1 with
(S)-fCTP. Reactions were conducted with 1× reaction buffer (as supplied
by manufacturer), 200 µM (S)-fCTP, 1 µL of polymerase as supplied, 74
°C (lanes 2-12), and 37 °C (lanes 13-17), 24 h.
porating up to 7 nt (full-length product) with (S)- and (R)-fATP.
For the series of higher temperature incubations (73 °C, panels
i-iii), Therminator polymerase extends the primer by up to 5 nt
and incorporates monomers in the “natural” (S)-series with a
preferred ordering of A ≈ T > C ≈ G. In contrast, Vent exo- and
Deep Vent exo- polymerases extend a maximum of 2 nt and show
less marked base preferences. At lower incubation temperature (55
°C, panel iv), primer extension by Therminator increases to 7 nt
(full-length product) in the case of fATP, and monomer incorpora-
9
412
J. AM. CHEM. SOC. 2008, 130, 412-413
10.1021/ja0770680 CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. (a) Primer extension with 32P 5′-labeled primer, templates 1-4,
and (S)- and (R)-fNTPs as indicated. In addition to polymerase, all reactions
included 1U Tth pyrophosphatase. Lanes 1 and 10 contain radiolabeled
primer only. (b) MALDI-TOF MS of polymerization products formed under
conditions as in panel (a), inset (i) with (S)-fATP. Calculated mass for
[(N+3) + H+] ) 8519, [(N+4) + H+] ) 8821.
tion efficiency takes on a new ordering of A > T > G > C. For
Therminator, antipodal efficiencies may be summarized as (S)-fG
> (R)-fG, (S)-fA ≈ (R)-fA, (S)-fT ≈ (R)-fT, (S)-fC < (R)-fC at
both incubation temperatures. When integrated over all four
nucleotides, these results indicate that on average antipodal fNTPs
are not distinguished. A mass spectrum of the products from a
preparative polymerization with primed template 3 and (S)-fATP
is given in Figure 2b.
The specificity of fNTP incorporation by Therminator polymerase
was assessed by primer extension experiments with templates 1-4
and mismatched fNTPs (Figure 1S). A comparison was made of
primer extension for pyrimidine-purine mismatches fC:A, fT:G,
fA:C, fG:T and the matched pairs fC:G, fT:A, fA:T, fG:C. The
results show minimal primer extension of mismatched fNTP/
template combinations; primary products in these cases are the result
of incorporating zero or one fNMP.
To further assess fNTP activity and probe the necessity of the
A485L mutation, a series of polymerizations were conducted with
primed template 5 based on progressive replacement of natural
dNTPs by fNTPs using both 9°N A485L exo- (Therminator) and
9°N E143D (9°Nm)12 polymerases (Figure 3). For Therminator,
progressive replacement of dNTPs by fCTP, then fCTP and fTTP,
followed by fCTP, fTTP, and fATP, gave apparent full-length
product in the first two cases, less than full-length product in the
third case at 72 °C, but apparent full-length product for the latter
at 55 °C.13 The corresponding number of FNA incorporations
through the series are 3, 7, and 14 nt. The experiment using 9°Nm
at 55 °C gave similar results to Therminator for lanes with fNTPs
present but dissimilar results in lanes where fNTPs were absent
(i.e., lanes 4, 6, and 8 versus lanes 3, 5, 7, and 9). Evidently, the
3′-5′ exonuclease activity associated with 9°Nm degraded compo-
nents of all reactions incubated for 24 h in the absence of fNTPs
(lane 2 is a positive control), in support of fNMP incorporation in
lanes where the latter are present. It is further clear from these results
that the A485L mutation of 9°N is not essential for activity.
In summary, both enantiomers of fNTPs are substrates for
polymerases. Moreover, the resulting syntactic (S)- or (R)-FNA
oligomers interact sufficiently with a complementary DNA template
to enable serial chain extension. These results are consistent with
earlier nonenzymatic template-directed synthesis of RNA on atactic
FNA.14 The inconsequence of FNA chirality likely derives from a
combination of backbone flexibility and single stereogenic center
Figure 3. Primer extension on template 5 with progressive replacement
of the dNTP pool with fNTPs ((R)-fCTP, (S)-fTTP, and (S)-fATP), as indi-
cated. In addition to polymerase, all reactions included 1U Tth pyrophos-
phatase.
with the result being functionally equivalent conformational space
shared by the two possible configurations. Intriguingly, this may
include the unnatural antipode adopting the equivalent of an
R-anomeric configuration.4,15 The absence of enantiomer cross-
inhibition16 seen with FNA may have implications for the pre-RNA
world and molecular evolution. Future work will be directed toward
derivation of mutant polymerases with enhanced activity to support
in vitro evolution17 of functional FNAs.
Acknowledgment. Support for this work from the National
Aeronautics and Space Administration is gratefully acknowledged.
Supporting Information Available: Experimental procedures,
including spectroscopic information. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
254, 1497-1500. (b) Nelson, K. E.; Levy, M.; Miller, S. L. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 3868-3871.
(2) Schoning, K. J.; Scholtz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.;
Eschenmoser, A. Science 2000, 290, 1347-1351.
(3) Zhang, L. L.; Peritz, A.; Meggers, E. J. Am. Chem. Soc. 2005, 127, 4174-
4175.
(4) Joyce, G. F.; Schwartz, A. W.; Miller, S. L.; Orgel, L. E. Proc. Natl.
Acad. Sci. U.S.A. 1987, 84, 4398-4402.
(5) (a) Schneider, K. C.; Benner, S. A. J. Am. Chem. Soc. 1990, 112, 453-
455. (b) Merle, L.; Spach, G.; Merle, Y.; Sagi, J.; Szemzo, A. Origins
Life EVol. Biosphere 1993, 23, 91-103. (c) Merle, Y.; Bonneil, E.; Merle,
L.; Sagi, J.; Szemzo, A. Int. J. Biol. Macromol. 1995, 17, 239-246.
(6) Byun, H.-S.; Bittman, R. Tetrahedron Lett. 1989, 30, 2751-2754.
(7) Ogilvie, K. K.; Nguyen-Ba, N.; Gillen, M. F.; Radatus, B. K.; Cheriyan,
U. O.; Hanna, H. R. Can. J. Chem. 1984, 62, 241-252.
(8) Niedballa, U.; Vorbru¨ggen, H. J. Org. Chem. 1974, 39, 3654-3660.
(9) Dixit, V. M.; Poulter, C. D. Tetrahedron Lett. 1984, 25, 4055-4058.
(10) (a) Trainor, G. L. U.S. Patent No. 5,558,991, 1996. (b) Sanger, F.; Nicklen,
S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5463-5467.
(11) Gardner, A. F.; Jack, W. E. Nucleic Acids Res. 2002, 30, 605-613.
(12) Southworth, M. W.; Kong, H.; Kucera, R. B.; Ware, J.; Jannasch, H. W.;
Perler, F. B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5281-5285.
(13) FNA is anticipated to have faster gel mobility compared to DNA and,
correspondingly, a shorter apparent length.
(14) Chaput, J.; Switzer, C. J. Mol. EVol. 2000, 51, 464-470.
(15) Assignment of S and R configurations’ parity values of +1/-1 allows
application of Westhof’s rules: Westhof, E. Nature 1992, 358, 459-460.
(16) Joyce, G. F.; Visser, G. M.; VanBoeckel, C. A. A.; VanBoom, J. H.;
Orgel, L. E.; VanWestrenen, J. Nature 1984, 310, 602-604.
(17) Wilson, D. S.; Szostak, J. W. Annu. ReV. Biochem. 1999, 68, 611-647.
JA0770680
9
J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008 413