Hybridization between "six-membered" nucleic acids: RNA as a universal information system

Article abstract of DOI:10.1021/ol016183r

(Matrix Presented) Within the polyA:polyT recognition system, cross-pairing between several nucleic acids with a phosphorylated six-membered carbohydrate (mimic) as repeating unit in the backbone structure has been observed. All investigated nucleic acids (except for β-homo-DNA) hybridize with RNA, leaving RNA as a versatile biopolymer for informational transfer.

Full text of DOI:10.1021/ol016183r

ORGANIC
LETTERS
2001
Vol. 3, No. 26
4129-4132
Hybridization between “Six-Membered”
Nucleic Acids: RNA as a Universal
Information System
Luc Kerremans, Guy Schepers, Jef Rozenski, Roger Busson,
,†
Arthur Van Aerschot, and Piet Herdewijn*
Katholieke UniVersiteit LeuVen, Rega Institute, Minderbroedersstraat 10,
B-3000 LeuVen, Belgium
piet.herdewijn@rega.kuleuVen.ac.be
Received May 28, 2001 (Revised Manuscript Received October 3, 2001)
ABSTRACT
Within the polyA:polyT recognition system, cross-pairing between several nucleic acids with a phosphorylated six-membered carbohydrate
(mimic) as repeating unit in the backbone structure has been observed. All investigated nucleic acids (except for â-homo-DNA) hybridize with
RNA, leaving RNA as a versatile biopolymer for informational transfer.
On one hand, it is now commonly accepted that the RNA-
world preceded the DNA-world. On the other hand, it is hard
to believe that a â-D-ribonucleotide, because of its structural
and stereochemical complexity, was the first nucleotide
formed by self-assembly of organic matter. The â-D-
ribonucleotides are, most likely, themselves the end result
of a stepwise constitutional metamorphosis process of
nucleotide building blocks, finally leading to the selection
of RNA as a biopolymer, which is able to generate life with
a potential to evolve.1,2 This molecular metamorphosis
process has followed the rules of molecular assembly and
reorganization, while retaining informational capacity.1,2 This
hypothetical view on life presumes that informational transfer
between biopolymers to and from RNA (i.e. to DNA) is
easily occurring.
simplest recognition mode (oligoadenylate-oligothymidy-
late).
The modified nucleic acids that have been investigated
on their base pairing properties have mostly a five-membered
phosphorylated carbohydrate (mimic) as repeating unit of
the backbone structure. These oligonucleotides are usually
screened for cross-pairing with RNA (for antisense purposes
or for information exchange with natural nucleic acids) and
for self-hybridization (organization in higher molecular
structures via base pairing). The conformational prerequisites
of oligonucleotides with a five-membered carbohydrate
(mimic) in the backbone structure for hybridization with
RNA have been analyzed.3-4
Extensive information about the selectivity of hybridization
of oligonucleotides with a six-membered carbohydrate
(mimic) is lacking. It has been observed that potentially
natural nucleic acid alternatives of the (6′f 4′) hexopyra-
nosyl (including the â-homo-DNA model) and the (4′ f 2′)
pentopyranosyl families, where base pairing is orthogonal
to that of the natural nucleic acids, do not cross pair with
RNA and DNA.1 In all these cases, the nucleobases are
equatorially positioned on the pyranosyl chains. In cases were
In this study we investigated RNA’s potential for hybrid-
ization with a series of distant related nucleic acids structures,
containing a six-membered phosphorylated carbohydrate
(mimic) backbone. The hybridization is studied within the
† Tel. +32-16-337387. Fax +32-126-337340.
(1) Schoning, K.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.;
Eschenmoser, A. Science 2000, 290, 1347-1351.
(2) Joyce, G. F.; Orgel, L. E. In The RNA World; Gesteland, R. F., Cech,
T. R., Atkins, J. F., Eds.; Cold Spring Harbor Laboratory Press: 1999; pp
49-77.
(3) Herdewijn, P. Liebigs Ann. 1996, 1337-1348.
(4) Herdewijn, P. Biochem. Biophys. Acta 1999, 19, 167-179.
10.1021/ol016183r CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/28/2001

Table 1. Melting Points and Hyperchromicity (%) As Determined at 260 nm in 0.1 M NaCl Phosphate Buffer pH 7.5 with for Each
Oligonucleotide at a Concentration of 4 µMa
a All oligonucleotides are 13-mers. NS: complex not stable enough to be detected at 20 °C. (1) Melting point determined in 1 M NaCl (the Tm at 0.1 M
NaCl is approximately 20 °C). (2) Tm of dissociation is 36 °C; Tm of association is 33 °C.
the nucleobases are axially oriented on the six-membered
ring, which is for example the case for hexitol-based nucleic
acids,5 base pairing with RNA and DNA is observed.
ing dideoxy nucleic acid analogues, we avoid that interpreta-
tion of the results is hampered by these “secondary” effects.
In the oligothymidylate series, â-homo-DNA, R-homo-
DNA, â-HNA, and CNA were selected (Table 1). These
oligonucleotides were available from previous research
projects.7,10-12 In the oligoadenylate series, â-homo-DNA is
removed because of its very strong self-pairing properties,13
and R-HNA and CeNA are added (Table 1).14 The synthesis
of the R-HNA phosphoramidite is given in Scheme 1. This
amidite was used for the synthesis of a R-HNA 13-mer from
which a correct MS analysis was obtained. The synthesis of
the nucleoside itself was previously described by following
a different scheme.15
The base-pairing landscape of these artificial oligonucle-
otides is given in Figure 1. All oligonucleotides are used as
13-mers. Several of the associations show low hyperchro-
micity, suggesting the absence of intensive stacking interac-
tions (Table 1). This low hyperchromicity is observed for
the hybrids formed between â-homo-DNA or R-HNA and
all other six-membered nucleic acids (except for the â-homo-
With equatorially oriented bases, quasilinear duplexes are
formed.6 With axially oriented bases, duplexes are formed
that are similar in structure to natural nucleic acids helices.5
However, recently we observed that intermediate structures
may be formed upon hybridization of R-homo-DNA with
RNA.7 In this case, hybrids are formed between nucleic acids
having on the one strand equatorially oriented bases (R-
homo-DNA) and on the other strand pseudoaxially oriented
bases (RNA).
For further investigation of the hybridization potential of
DNA with a six-membered ring in the backbone structure,
we selected several dideoxy nucleic acids as examples. The
selection of dideoxy nucleic acids is based on the observation
that the presence of free hydroxyl groups on the sugar moiety
of the nucleic acids may weaken base pairing due to steric
hindrance, as was demonstrated for â-D-glucopyranosyl
nucleic acids.8 The hydroxyl groups may, likewise, be
involved in intramolecular interactions and induce a con-
formational preorganization maladjusted for base pairing, as
was demonstrated with D-mannitol nucleic acids.9 By select-
(10) Augustyns, K.; Van Aerschot, A.; Urbanke, C.; Herdewijn, P. Bull.
Soc. Chim. Belg. 1991, 101, 119-129
(11) Van Aerschot, A.; Verheggen, I.; Hendrix, C.; Herdewijn, P. Angew.
Chem., Int. Ed. Engl. 1995, 34, 1338-1485.
(12) Maurinsh, Y.; Rosemeyer, H.; Esnouf., R.; Medvedovici, A.; Wang,
J.; Ceulemans, G.; Lescrinier, E.; Hendrix, C.; Busson, R.; Sandra, P.; Seela,
F.; Van Aerschot, A; Herdewijn, P. Chem. Eur. J. 1999, 5, 2139-2150.
(13) Hunziker, J.; Roth, H.-J.; Bo¨hringer, M.; Giger, A.; Diederichsen,
U.; Go¨bel, M.; Krishnan, R.; Jaun, B.; Leumann, C.; Eschenmoser, A. HelV.
Chim. Acta 1993, 76, 259-352
(5) Lescrinier, E.; Esnouf, R.; Schraml, J.; Busson, R.; Heus, A.; Hilbers,
C. W.; Herdewijn, P. Chem. Biol. 2000, 7, 719-731.
(6) Schlo¨nvoght, I.; Pitsch, S.; Lesueur, C.; Eschenmoser, A.; Jaun, B.;
Wolf, R. M. HelV. Chim. Acta 1996, 79, 2316-2345.
(7) Froeyen, M.; Lescrinier, E.; Kerremans, L.; Rosemeyer, H.; Seela,
F.; Verbeure, B.; Lagoja, I.; Rozenski, J.; Van Aerschot, A.; Busson, R.;
Herdewijn, P. Eur. J. Chem. In press.
(8) Eschenmoser, A. Science 1999, 284, 2118-2124.
(9) Hossain, N.; Wroblowski, B.; Van Aerschot, A.; Rozenski, J.; De
Bruyn, A.; Herdewijn, P. J. Org. Chem. 1998, 63, 1574-1582.
(14) Wang, J.; Verbeure, B.; Luyten, I.; Lescrinier, E.; Froeyen, M.;
Hendrix, C.; Rosemeyer, H.; Seela, F.; Van Aerschot, A.; Herdewijn P. J.
Am. Chem. Soc. 2000, 122, 8595-8602.
(15) Hossain,N.; Rozenski, J.; De Clercq, E.; Herdewijn, P. J. Org. Chem.
1997, 62, 2442-2447.
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Org. Lett., Vol. 3, No. 26, 2001

Scheme 1. Synthesis of the Phosphoramidite of
1,5-Anhydro-2,3-dideoxy-2-(N6-benzoyladenin-9-yl)-D-glucitol
(the Building Block for R-HNA-synthesis)a
Figure 2. Thermal dissociation curves (temperature versus absor-
bance at 260 nm) in 0.1 M NaCl of RNA-A with RNA-U (g),
CNA-T (4), R-homo-T (0), and â-HNA-T (O).
a (i) pNO2BzOH, Ph3P, DEAD, THF; NH3 MeOH, 85%; (ii)
pTosCl, pyridine; 80% HOAc, 80 °C, 52%; (iii) PivCl, pyridine,
85%;(iv) adenine, LiH, 12-crown-4, DMF, 60 °C, 25%; (v)
NaOH 1N, dioxane, 60 °C, 55%; (vi) TMSiCl, pyridine, BzCl,
NH4OH, 80%; (vii) MMTrCl, pyridine, 75%, (viii) (iPr)2NP(Cl)
(OCH2CH2CN); (iPr)2NEt, CH2Cl2, 85%.
hydroxymethyl group. R-HNA, â-HNA, CNA, and CeNA
have a 1,4 relationship between both functionalities. Except
for â-homo-T:CNA, â-homo-T:RNA, and R-homo-T:R-
HNA, stable associations are formed; the distance between
the base moiety and the hydroxymethyl group cannot be
considered as a decisive factor influencing base pairing.
â-Homo-DNA and R-HNA are all-equatorial systems. In
â-HNA, the base is axially oriented and the two other
substituents are equatorially oriented. In R-homo-DNA (when
pairing with RNA), the base moiety is equatorially oriented
and the hydroxyl and hydroxymethyl groups are axially
oriented.7 The situation is less clear for CNA (both might
be possible)12 and for CeNA (that is a more flexible
system).14 As most of these nucleic acids do cross pair, base
orientation seems, likewise, not to be a decisive factor for
hybridization. The strongest pairing, however, is observed
for complexes involving â-HNA.
T:R-HNA-A association itself).16 This was, likewise, ob-
served for â-homo-A self-association (5-10% hyperchro-
micity).13 Low hyperchromicity is observed when one of the
pairing partners is an all-equatorial system (i.e. the three
substituents on the six-membered ring are equatorially
oriented). High hyperchromicity values are indicative for
triple helix formation as was observed for CNA(A):RNA-
(U) and CNA:CNA.12
â-Homo-DNA, R-homo-DNA, and RNA have a 1,3
relationship between the base moiety and the phosphorylated
All the six-membered oligonucleotides (except for â-homo-
DNA) do hybridize with RNA. RNA seems to be an
oligonucleotide system of excellent adaptability when con-
sidering cross-pairing with other informational pairing
systems. The observation that â-homo-DNA does not
hybridize with RNA seems to be an exception in the pairing
behavior of “six-membered” artificial nucleic acids. DNA
is much less versatile in its pairing behavior than RNA. No
hybridization of DNA (oligo dT) at 0.1 M NaCl was
observed with R-homo-DNA or R-HNA, and no hybridiza-
tion of DNA (oligo dA) at 0.1 M NaCl was observed with
â-homo-DNA and R-homo-DNA (data not shown). RNA,
the bioisosteric cyclohexene congener, and its conforma-
tionally restricted artificial congener â-HNA are the most
universal pairing partners, the latter giving the most stable
complexes (Figures 2 and 3 and Supporting Information).We
realize that polyA:polyT is a weak base pairing system and
Figure 1. Melting points as determined in 0.1 M NaCl, phosphate
buffer pH 7.5. The base-pairing landscape shows that the strongest
pairing occurs for complexes involving â-HNA.
(16) The probability that R-HNA might pair with â-homo-DNA was also
predicted by A. Eschenmoser and communicated to Piet Herdewijn.
Org. Lett., Vol. 3, No. 26, 2001
4131

tions of nucleic acids is more extended than what is presently
described (natural nucleic acids helices and ladder-like
structures). Indications for this are the different UV-spectra
obtained for some of the complexes (data not shown).
In conclusion, these data confirm that the structural and
conformational properties of the carbohydrate moiety have
an important influence on hybridization strength. Extensive
cross-pairing between oligonucleotides with a six-membered
carbohydrate moiety within the polyA:polyT mode is oc-
curring. Although none of the artificial systems studied can
be considered to be a potentially natural nucleic acid
alternative, the observation that RNA (and its conformational
rigid analogues that mimic A-form nucleic acid duplexes)
is an universal pairing partner suggests that molecular
evolution to and from RNA is easily possible and that the
A-form duplex is probably the most stable helical association
form in the nucleic acid field.
Figure 3. Thermal dissociation curves (temperature versus absor-
bance at 260 nm) in 0.1 M NaCl of RNA-U with RNA-A (g),
CNA-A (4), R-homo-A (0), â-HNA-A (O), and CeNA-A ()) and
with R-HNA-A (at 1.0 M NaCl) (3).
Acknowledgment. The authors thank K. U. Leuven
(GOA 97/11) and FWO for financial support. We are
indebted to Chantal Biernaux for editorial help.
Supporting Information Available: Tm curves of â-HNA-
A, CNA-A, and CeNA-A with RNA-U, CNA-T, R-homo-
T, and â-HNA-T. Experimental section for the synthesis of
protected 1,5-anhydro-2,3-dideoxy-2-(N6-benzoyl-adenin-9-
yl)-D-glucitol. This material is available free of charge via
the Internet at http://pubs.acs.org.
that the pairing landscape may be different with heterobasic
systems. However, within this simple system, base pairing
is possible between most six-membered oligomers, whether
orthogonal to each other or not. The structure of most of
these complexes has not been determined yet, but on the
basis of experiments with R-homo-DNA,7 these results
suggest that the universe of stable (supra)molecular associa-
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Org. Lett., Vol. 3, No. 26, 2001