Xylonucleic acid: Synthesis, structure, and orthogonal pairing properties

Article abstract of DOI:10.1093/nar/gkv719

There is a common interest for studying xeno-nucleic acid systems in the fields of synthetic biology and the origin of life, in particular, those with an engineered backbone and possessing novel properties. Along this line, we have investigated xylonuclei

Full text of DOI:10.1093/nar/gkv719

Published online 14 July 2015
Nucleic Acids Research, 2015, Vol. 43, No. 15 7189–7200
doi: 10.1093/nar/gkv719
Xylonucleic acid: synthesis, structure, and orthogonal
pairing properties
Mohitosh Maiti1, Munmun Maiti1, Christine Knies2, Shrinivas Dumbre1, Eveline Lescrinier1,
Helmut Rosemeyer2, Arnout Ceulemans3 and Piet Herdewijn1,*
1Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000
Leuven, Belgium, 2Organic Materials Chemistry and Bioorganic Chemistry, Institute of Chemistry of New Materials,
University of Osnabru¨ck, Barbarastrasse. 7, D-49069 Osnabru¨ck, Germany and 3Department of Chemistry, KU
Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium
Received May 19, 2015; Revised June 29, 2015; Accepted July 01, 2015
ABSTRACT
bone i.e. deoxy-D-ribose for DNA and D-ribose for RNA
(Figure 1), thereby remarkably influencing their structural
properties and biological functions (1). Recently, a number
of sugar-modified nucleic acid variants has been revealed as
new genetic polymers, (2) some of them are endowed with
catalytic activity (for e.g. FANA and HNA) (3). The struc-
ture of these artificial nucleic acids, however, mimics natural
nucleic acid helicity (4). In the context of studies on chemi-
cal etiology of nucleic acid structure (5–7) and the origin of
life, alternative nucleic acids with more deviating backbone
structures have been investigated. Some of them are having
different double helical geometry than that of DNA/RNA,
(5–8) others with a similar geometry, and thus shown as po-
tential direct RNA precursors (9–11) (like TNA and PNA)
that might have been evolved into the existing nucleic acid.
A relatively much less explored area in alternative nucleic
acid engineering is the search and study of a complemen-
tary pair of synthetic nucleic acids with novel backbone and
helical geometries which can support life i.e. perform cataly-
sis like RNA and store genetic information like DNA. This
might assist in the design principles of an orthogonal nu-
cleic acid pair for application in xenobiology (orthogonal
life) (12–14). Likewise, it may also expand our knowledge
in the context of prebiotic origin and evolution of nucleic
acids.
In this context, we have studied the physicochemical
properties of a nucleic acid containing D-xylose (wood
sugar), a prebiotic pentofuranosyl sugar. When compared
with ribose, the xylose sugar has an inverted 3ꢀ-carbon cen-
ter; hence xylose nucleic acid (XyloNA) and its cousin de-
oxyxylonucleic acid (dXyloNA) possess a diastereoisomeric
backbone (Figure 1) in comparison with the natural RNA
and DNA respectively. Previously, dXyloNA (15–18) and
locked XyloNA (19–22) were studied in the perspective of
potential therapeutic applications, but without success. Re-
cently, we have shown the potential heterogeneous heli-
cal structure and conformational malleability of dXyloNA
(23,24). In the present study, we describe the systematic ex-
There is a common interest for studying xeno-nucleic
acid systems in the fields of synthetic biology and the
origin of life, in particular, those with an engineered
backbone and possessing novel properties. Along
this line, we have investigated xylonucleic acid (Xy-
loNA) containing a potentially prebiotic xylose sugar
(a 3ꢀ-epimer of ribose) in its backbone. Herein, we
report for the first time the synthesis of four Xy-
loNA nucleotide building blocks and the assembly
of XyloNA oligonucleotides containing all the natu-
ral nucleobases. A detailed investigation of pairing
and structural properties of XyloNAs in comparison
to DNA/RNA has been performed by thermal UV-
melting, CD, and solution state NMR spectroscopic
studies. XyloNA has been shown to be an orthogo-
nal self-pairing system which adopts a slightly right-
handed extended helical geometry. Our study on one
hand, provides understanding for superior structure-
function (-pairing) properties of DNA/RNA over Xy-
loNA for selection as an informational polymer in the
prebiotic context, while on the other hand, finds po-
tential of XyloNA as an orthogonal genetic system
for application in synthetic biology.
INTRODUCTION
DNA and RNA are the two monopolistic biopolymers that
are capable of storage and dissemination of genetic infor-
mation in the extant biology. While the nucleobases of DNA
and RNA are at the very core of the helical structure pro-
viding crucial stabilization by Watson–Crick type hydro-
gen bonding and stacking interactions, the unique sugar-
phosphate backbone is significantly providing an optimal
structural and dynamic framework. These two natural nu-
cleic acid versions differ in the sugar moiety in their back-
*To whom correspondence should be addressed. Tel: +32 16 337387; Fax: +32 16 337340; Email: Piet.Herdewijn@rega.kuleuven.be
ꢁ
C
The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
journals.permissions@oup.com

7190 Nucleic Acids Research, 2015, Vol. 43, No. 15
TLC. Flash silica column chromatography was performed
on silica gel 60 A, 40–60 ␮M (Acros Organics). NMR spec-
tra were recorded on Bruker Avance II spectrometers; 300
MHz with a 5mm broad band probe, 500 MHz with a TXI-
HCP Z gradient probe, or on a 600 MHz NMR equipped
with a 5 mm TCI-HCN Z gradient cryo-probe. The spectra
were processed with Bruker Topspin 2.1 software. Chem-
ical shifts (δ) were reported in parts per million (ppm).
1
The H and 13C chemical shifts were referenced relative
to TMS peak (δ = 0.00 ppm). 31P NMR chemical shifts
were referenced to an external 85% H3PO4 standard (δ =
1
0.00 ppm). H and 13C resonance assignments were made
by using 2D correlation experiments (COSY, gHSQC and
gHMBC). High resolution mass spectra were acquired on
a quadrupole orthogonal acceleration time-of-flight mass
spectrometer (Synapt G2 HDMS, Waters, Milford, MA,
USA).
Synthetic procedures and the characterization data for
the intermediates of XyloNA building blocks synthesis are
provided in the supplementary information.
General procedure for the phosphitylation reaction
Tritilated nucleoside (1 mmol) was coevaporated with dry
THF (3×) and further dried under high vacuum for 1 h. Un-
der an argon atmosphere, the compound was dissolved in
anhydrous CH2Cl2 (10 ml) and N,N-diisopropyl ethylamine
(5 mmol) was added. To this solution, 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite (2.5 mmol) was added
dropwise and the reaction mixture was stirred at room tem-
perature for 4 h. Methanol (0.5 ml) was added dropwise to
the reaction mixture at 0◦C and the solution was stirred for
another 15 min at room temperature. Then volatile organic
solvents were removed under reduced pressure at room tem-
perature and the crude product was purified by flash column
chromatography.
Figure 1. Chemical structure of the sugar-phosphate backbone in XyloNA
(dXyloNA) compared with RNA (DNA). Sequence and base-pairing are
shown for a fully-modified self-complementary XyloNA duplex (ON-1)
investigated (by CD and NMR) in this study. Types of sugar puckering
are indicated as exist and characterized by NMR spectroscopy in different
natural and xylose-based nucleic acid systems.
1-{2ꢀ-O-Acetyl-3ꢀ-O-[2-cyanoethoxy(diisopropyla
mino)phosphanyl]-5ꢀ-O-(4,4ꢀ-dimethoxytrityl)-␤-D-
xylofuranosyl}uracil (6)
ploration on self-pairing versus cross-pairing ability of Xy-
loNA and its helical structural characterization by circu-
lar dichroism (CD) and solution NMR spectroscopy that
illuminate on the inherent structural properties of xylonu-
cleic acid backbone (Figure 1). In particular, the effect of 2ꢀ-
hydroxyl groups is shown in tuning the structural dynamics
in comparison with the previously studied dXyloNA (24)
system.
Tritilated nucleoside 5 (1.3 g, 2.2 mmol) was converted
into the corresponding amidite by following the aforemen-
tioned general procedure for phosphitylation reaction. The
crude product was purified by flash column chromatogra-
phy using solvent system 25–75% EtOAc in hexane con-
taining 0.5% triethylamine (Rf = 0.49, 0.64 for 50% EtOAc
in CH2Cl2). Compound 6 was isolated as white foam con-
taining a mixture of two diastereomers in 87% yield (1.54
1
g). H NMR (500 MHz, acetone-d6): δ 10.06 (br s, H3),
MATERIALS AND METHODS
7.63, 7.58 (d, J = 8.2 Hz, 1H, H6), 7.54–6.87 (m, 13H, Ar-
H), 6.00, 5.99 (s, 1H, H1ꢀ), 5.54, 5.50 (d, J = 8.2 Hz, 1H,
H5), 5.39, 5.23 (s, 1H, H2ꢀ), 4.53–4.49 (m, 1H, H4ꢀ), 4.43–
4.40 (m, 1H, H3ꢀ), 3.94–3.70 (m, 2H, OCH2CH2CN), 3.80,
3.79 (s, 6H, 2 OCH3), 3.69–3.31 (m, 2H, H5ꢀ,5ꢀꢀ), 3.54–3.43
(m, 2H, 2×CH(CH3)2), 2.73–2.56 (m, 2H, OCH2CH2CN),
2.12 (s, 3H, COCH3), 1.15, 1.13, 1.02, 0.97 (d, 12H, J =
5.0, 4.7, 6.7, 6.7 Hz, 2×CH(CH3)2); 13C NMR (125 MHz,
acetone-d6): δ 169.9, 169.8 (COCH3), 163.4, 163.3 (C4),
159.7 (Ar-C), 151.1 (C2), 146.0 (Ar-C), 141.0, 140.8 (C6),
136.9–127.6 (Ar-C), 118.8, 118.7 (CN), 114.0 (Ar-C), 102.3,
General
For all reactions, analytical grade solvents were used. All
moisture sensitive reactions were carried out under argon or
nitrogen atmosphere in oven dried glasswares (120◦C). 1,2-
O-Isopropylidene-␣-D-xylofuranose was purchased from
Acros Organics. Dry acetonitrile and 1,2-dichloroethane
were obtained by distillation over CaH2. All other dry
solvents and reagents were purchased from commercial
sources. Pre-coated aluminum sheets (Fluka) were used for

Nucleic Acids Research, 2015, Vol. 43, No. 15 7191
102.2 (C5), 90.1, 90.0 (C1ꢀ), 87.3, 87.2 (tert C of DMTr),
83.6, 83.3 (d, JCP = 3.5, 9.0 Hz, C4ꢀ), 81.4, 81.3 (d, JCP
= 3.0, 0.0 Hz, C2ꢀ), 77.3, 75.9 (d, JCP = 18.3, 12.7 Hz,
C3ꢀ), 63.7, 63.5 (C5ꢀ), 59.8, 59.5 (d, JCP = 22.3, 21.2 Hz,
6.9 Hz, 2H, o H-Bz), 7.66–7.63 (m, 1H, p H-Bz), 7.57–
7.53 (m, 2H, m H-Bz), 7.57–6.85 (m, 13H, Ar-H of DMTr),
6.40, 6.39 (s, 1H, H1ꢀ), 5.82, 5.68 (s, 1H, H2ꢀ), 4.70–4.69
(m, 1H, H4ꢀ), 4.59–4.53 (m, 1H, H3ꢀ), 3.79, 3.78, 3.77 (s,
6H, 2 OCH3), 3.74–3.54 (m, 2H, OCH2CH2CN), 3.74–3.35
(m, 2H, H5ꢀ,5ꢀꢀ), 3.42–3.35 (m, 2H, 2×CH(CH3)2), 2.65–
2.52 (m, 2H, OCH2CH2CN), 2.18, 2.17 (s, 3H, COCH3),
1.11, 1.08, 0.95, 0.92 (d, 12H, J = 6.9, 7.2, 6.9, 6.7 Hz,
2×CH(CH3)2); 13C NMR (125 MHz, acetone-d6): ␦ 169.9
(COCH3), 159.7 (Ar-C), 152.8 (C4, C2), 151.2 (C6), 146.1
(Ar-C), 142.5, 142.4 (C8), 136.9–127.6 (Ar-C), 125.9, 125.7
(C5), 118.8, 118.7 (CN), 113.9 (Ar-C), 89.1, 88.7 (C1ꢀ), 87.3,
87.2 (tert C of DMTr), 84.0, 83.9 (d, JCP = 3.4, 0.0 Hz, C4ꢀ),
OCH2CH2CN), 55.6, 55.5 (OCH3), 43.9, 43.8 (d, JCP
=
4.0, 3.8 Hz, CH(CH3)2), 25.1, 24.9, 24.7 (d, JCP = 8.6,
7.4, 7.0 Hz, CH(CH3)2), 20.8, 20.7 (COCH3), 20.5, 20.4 (d,
JCP = 7.6, 7.7 Hz, OCH2CH2CN); 31P NMR (202 MHz,
acetone-d6): δ 152.71, 148.94; HRMS (ESI+), calcd for
C41H50N4O10P [M+H]+ 789.3259, found 789.3268.
1-{2ꢀ-O-Acetyl-3ꢀ-O-[2-cyanoethoxy(diisopropylamino)pho
sphanyl]-5ꢀ-O-(4,4ꢀ-dimethoxytrityl)-␤-D-xylofuranosyl}-4-
N-benzoylcytosine (10)
81.3, 81.2 (d, JCP = 2.5, 0.0 Hz, C2ꢀ), 77.3, 76.0 (d, JCP
=
=
18.1, 12.5 Hz, C3ꢀ), 64.2, 64.0 (C5ꢀ), 59.8, 59.5 (d, JCP
21.9, 20.8 Hz, OCH2CH2CN), 55.6 (OCH3), 43.9, 43.8 (d,
JCP = 12.5, 12.1 Hz, CH(CH3)2), 25.1, 24.9, 24.7, 24.6 (d,
JCP = 8.0, 7.5, 8.2, 8.0 Hz, CH(CH3)2), 20.8, 20.7 (COCH3),
20.5, 20.4 (d, JCP = 7.5 Hz, OCH2CH2CN); 31P NMR (202
MHz, acetone-d6): ␦ 152.14, 148.98; HRMS (ESI+), calcd
for C49H54N7O9PNa [M+Na]+ 938.3613, found 938.3616.
Tritilated nucleoside 9 (1.35 g, 2.0 mmol) was converted into
the corresponding amidite by following the general proce-
dure for phosphitylation. The crude product was purified by
flash column chromatography using solvent system 20–40%
acetone in hexane containing 0.5% triethylamine (Rf = 0.58,
0.66 for 60% acetone in hexane). Compound 10 was isolated
as white foam containing a mixture of two diastereoisomers
in 81% yield (1.4 g). 1H NMR (500 MHz, acetone-d6): ␦9.78
(br s, NH), 8.16, 8.15 (d, J = 6.9, 6.5 Hz, 2H, o H-Bz), 7.97,
7.96 (d, J = 7.4 Hz, 1H, H6), 7.68–7.64 (m, 1H, p H-Bz),
7.58–7.56 (m, 2H, m H-Bz), 7.44–7.33 (d, J = 7.4 Hz, 1H,
H5), 7.58–6.90 (m, 13H, Ar-H of DMTr), 5.94, 5.91 (s, 1H,
H1ꢀ), 5.53, 5.34 (s, 1H, H2ꢀ), 4.72–4.65 (m, 1H, H4ꢀ), 4.39–
4.37 (m, 1H, H3ꢀ), 3.82, 3.81 (s, 6H, 2 OCH3), 3.82–3.54 (m,
2H, OCH2CH2CN), 3.82–3.33 (m, 2H, H5ꢀ,5ꢀꢀ), 3.40–3.33
(m, 2H, 2×CH(CH3)2), 2.71–2.56 (m, 2H, OCH2CH2CN),
2.15 (s, 3H, COCH3), 1.09, 1.04, 0.92 (d, 12H, J = 6.8 Hz,
2×CH(CH3)2); 13C NMR (125 MHz, acetone-d6): ␦ 169.8,
169.7 (COCH3), 164.0, 163.9 (C4), 159.8 (Ar-C), 155.2 (C2),
146.1 (Ar-C), 145.2 (C6), 136.8–127.7 (Ar-C), 118.8, 118.7
(CN), 114.0, 113.9 (Ar-C), 96.3 (C5), 92.1, 91.9 (C1ꢀ), 87.3,
87.2 (tert C of DMTr), 84.9, 84.8 (d, JCP = 4.5 Hz, C4ꢀ),
80.8, 80.2 (d, JCP = 3.3, 0.0 Hz, C2ꢀ), 77.0, 75.8 (d, JCP
= 19.3, 11.6 Hz, C3ꢀ), 63.9, 63.7 (C5ꢀ), 59.9, 59.5 (d, JCP
= 23.1, 22.9 Hz, OCH2CH2CN), 55.6 (OCH3), 43.9, 43.8
(d, JCP = 9.4, 8.8 Hz, CH(CH3)2), 25.1, 24.9, 24.6 (d, JCP
= 8.2, 7.3, 4.9 Hz, CH(CH3)2), 20.8, 20.7 (COCH3), 20.6,
20.2 (d, JCP = 7.5, 8.2 Hz, OCH2CH2CN); 31P NMR (202
MHz, acetone-d6): ␦ 152.80, 148.52; HRMS (ESI+), calcd
for C48H55N5O10P [M+H]+ 892.3681, found 892.3669.
9-{2ꢀ-O-Acetyl-3ꢀ-O-[2-cyanoethoxy(diisopropylamino)pho
sphanyl]-5ꢀ-O-(4,4ꢀ-dimethoxytrityl)-␤-D-xylofuranosyl}-2-
N-acetylguanine (20)
Tritilated nucleoside 19 (0.87 g, 1.3 mmol) was converted
into the corresponding amidite by following the general
procedure for phosphitylation. The crude product was pu-
rified by flash column chromatography using solvent sys-
tem 25–60% acetone in hexane containing 0.5% triethy-
lamine (Rf = 0.60, 0.60 for 60% acetone in hexane). Com-
pound 20 was isolated as white foam containing a mix-
ture of two diastereoisomers in 88% yield (1.0 g). 1H NMR
(500 MHz, acetone-d6): δ 12.04 (br s, 1H, H1), 10.54 (br
s, 1H, NHAc), 7.92, 7.89 (s, 1H, H8), 7.52–6.84 (m, 13H,
Ar-H), 6.06, 6.04 (s, 1H, H1ꢀ), 5.62, 5.47 (s, 1H, H2ꢀ), 4.58
(m, 1H, H4ꢀ), 4.53–4.50 (m, 1H, H3ꢀ), 3.79, 3.78 (s, 6H, 2
OCH3), 3.85–3.63 (m, 2H, OCH2CH2CN), 3.69–3.29 (m,
2H, H5ꢀ,5ꢀꢀ), 3.47–3.40 (m, 2H, 2×CH(CH3)2), 2.71–2.51
(m, 2H, OCH2CH2CN), 2.31 (s, 3H, NHCOCH3), 2.15 (s,
3H, OCOCH3), 1.13, 1.10, 0.97, 0.95 (d, 12H, J = 6.8, 6.8,
5.2, 5.4 Hz, 2×CH(CH3)2); 13C NMR (125 MHz, acetone-
d6): δ 174.0, 173.9 (NHCOCH3), 169.9, 169.8 (OCOCH3),
159.7 (Ar-C), 155.8, 155.7 (C6), 149.2, 149.1 (C4, C2),
146.1, 146.0 (Ar-C), 137.9, 137.8 (C8), 136.9–127.6 (Ar-C),
121.9, 121.7 (C5), 118.8, 118.7 (CN), 113.9 (Ar-C), 88.6,
9-{2ꢀ-O-Acetyl-3ꢀ-O-[2-cyanoethoxy(diisopropylamino)pho
sphanyl]-5ꢀ-O-(4,4ꢀ-dimethoxytrityl)-␤-D-xylofuranosyl}-6-
N-benzoyladenine (14)
88.3 (C1ꢀ), 87.2, 87.2 (tert C of DMTr), 83.9, 83.7 (d, JCP
=
3.0, 9.0 Hz, C4ꢀ), 81.8, 81.7 (d, JCP = 3.1, 0.0 Hz, C2ꢀ), 77.6,
76.2 (d, JCP = 18.8, 11.3 Hz, C3ꢀ), 64.1, 64.0 (C5ꢀ), 59.7,
59.4 (d, JCP = 22.5, 21.0 Hz, OCH2CH2CN), 55.5 (OCH3),
43.9, 43.8 (d, JCP = 12.4, 12.1 Hz, CH(CH3)2), 25.1, 24.9,
24.7, 24.6 (d, JCP = 8.3, 7.3, 6.9, 9.0 Hz, CH(CH3)2), 24.2
Tritilated nucleoside 13 (2.28 g, 3.2 mmol) was converted
into the corresponding amidite by following the general
procedure for phosphitylation. The crude product was pu-
rified by flash column chromatography using solvent sys-
tem 45–85% EtOAc in hexane containing 0.5% triethy-
lamine (Rf = 0.42, 0.54 for 90% EtOAc in hexane). Com-
pound 14 was isolated as white foam containing a mixture
(NHCOCH3), 20.9, 20.8 (OCOCH3), 20.5, 20.4 (d, JCP
=
7.7 Hz, OCH2CH2CN); 31P NMR (202 MHz, acetone-d6):
δ 152.68, 148.48; HRMS (ESI+), calcd for C44H53N7O10P
[M+H]+ 870.3586, found 870.3594.
1
of two diastereoisomers in 84% yield (2.46 g). H NMR
Synthetic procedure and characterization data for Xy-
loNA oligonucleotides, experimental procedures for ther-
mal (UV-) melting, circular dichroism (CD) and NMR
(500 MHz, acetone-d6): δ 10.00 (br s, NH), 8.67, 8.65 (s,
1H, H2), 8.30, 8.29 (s, 1H, H8), 8.14, 8.12 (d, J = 6.2,

7192 Nucleic Acids Research, 2015, Vol. 43, No. 15
spectroscopic studies are provided in the supplementary in-
formation.
tural restraints. After the torsion angle molecular dynam-
ics round, the majority of structures were converged to very
similar structures with similar total energies and with no
violations of the NOE and dihedral restraints. The 20 low-
est energy structures were used for further refinement dur-
ing a ‘gentle molecular dynamics’ round by using the X-
PLOR NIH script refine gentle.inp. The final refinement
was started with 20 ps constant-temperature molecular dy-
namics simulation at 300 K (20 000 steps of 0.001 ps) which
gradually introduces the van der Waals radii, and was fol-
lowed by a 200-step conjugate-gradient energy minimiza-
tion on an average structure of the last 10 of 20 ps molecu-
lar dynamics simulation. Torsion angles and helical param-
eters of the obtained solution structures were determined
using CURVES 5.3 (28). Finally, visual representations of
the molecules were obtained by using the UCSF Chimera
1.10rc program.
NMR-derived structural restraints
Distance restraints were derived from NOESY spectra
recorded with 50, 100, 150 and 300 ms mixing times. By
using the computer-aided resonance assignment (CARA)
program, (25) the cross-peaks in NOESY spectra were as-
signed and the cross-peak volumes were obtained by inte-
gration. Based on the NOE build-up curves, inter-proton
distances were calculated. The calibration of NOE cross-
relaxation rates was performed against the average of all
the isolated H5-H6 cross-peaks as a reference with distance
˚
2.45 A. Upper and lower bounds were set to 20% of the
calculated distances in the structure calculation. Hydrogen-
bonding restraints were used as distance restraints in all
structure calculation.
All xylose sugar rings were restrained to North (N)-
type sugar puckering in structure calculations [dihedral re-
straints applied: H1ꢀ-C1ꢀ-C2ꢀ-H2ꢀ (99.2 20◦), H2ꢀ-C2ꢀ-C3ꢀ-
O3ꢀ (35 25◦), O3ꢀ-C3ꢀ-C4ꢀ-H4ꢀ (−157 25◦)].
RESULTS
Synthesis of XyloNA building blocks
Backbone torsion angles ␤, ␥ and ⑀ were restrained as
follows. The ␤ torsion angles (C4ꢀ-C5ꢀ-O5ꢀ-P5ꢀ) were re-
strained to trans regions in the structure calculations as de-
In the chemistry part, we have set out to implement an easy
synthetic route (Scheme 1) for the XyloNA building blocks.
The commercially available 1,2-acetonide of xylofuranose
was benzylated to have compound 1. The subsequent steps
to obtain protected glycone (2) are acid-catalyzed acetonide
deprotection and acetylation. Since the one-pot transfor-
mation solely produced a ring-opened byproduct 2b, we
have followed a two-step procedure to synthesize diacetate
2 in a reasonably good yield. Although the formation of
the 1-fluoroacetate side product 2a could not be completely
avoided in the latter procedure, its formation was reduced to
some extent by decreasing the percentage of trifluoroacetic
acid (TFA). Fluoroacetate sugar 2a could not afford the de-
sired nucleoside 3 in a classical glycosylation reaction since
it formed several decomposition products as observed by
TLC. While standard Vorbru¨ggen-type glycosylation reac-
tion was performed for pyrimidine nucleosides (3 and 7),
for purines non-standard conditions were necessary. Thus,
the reaction of N-benzoyladenine with the protected sugar
2 was performed in the presence of SnCl4 without silyla-
tion at room temperature, where the desired N9-β-isomer
(11) was majorly formed as a kinetically controlled prod-
uct. Nonetheless, depending on the reaction time (<2 h) a
minor amount of N9-α-isomer (11a in SI) was formed and
separated from the N9-β-stereoisomer 11 by column chro-
matography. For guanine, N2-acetyl-O6-diphenylcarbamoyl
(DPC) protection (15 in SI) was employed for the regiose-
lective glycosylation at the N9 position, (29) since glycosy-
lation without DPC protection (15a in SI) resulted in the
formation of an inseparable (1:1) mixture of N7 (17a in SI)
and N9 (17) regioisomers. Then the DPC protecting group
was selectively removed by treating with 90% aqueous TFA
as it was no longer necessary and thought to procure com-
plication in the subsequent steps. The removal of the ben-
zyl (-Bn) groups was found to be difficult due to the slug-
gish reaction rate under different hydrogenation conditions,
with potential problems like reduction of the pyrimidine
ring and solvolysis of the acetyl and benzoyl groups were
additionally encountered. These problems were efficiently
termined by experimental vicinal hetero-nuclear phospho-
3
rus proton (3JP5 -H5 , JP5 -H5 ) couplings (Supplementary
ꢀ
ꢀ
ꢀ
ꢀꢀ
Table S1, in the supporting information) derived from the 31
P
coupled and decoupled DQF-COSY spectrum and based
on a standard Karplus plot (26). For purine and pyrim-
idine nucleotides of XyloNA1 duplex, the β torsion an-
gle regions 180
75◦ and 200
40◦ were restrained re-
spectively. The γ torsion angles (C3ꢀ-C4ꢀ-C5ꢀ-O5ꢀ) were re-
strained from gauche – to anticlinal – region in the struc-
ture calculations as determined by vicinal proton-proton
3
(3JH4 -H5 , JH4 -H5 ) couplings (Supplementary Table S1) de-
rived from the 31P decoupled DQF-COSY spectrum (26).
For purine and pyrimidine nucleotides of XyloNA1 duplex,
the ␥ torsion angles were restrained to regions 210 35◦ and
270 45◦ respectively. The ⑀ torsion angles (P3ꢀ-O3ꢀ-C3ꢀ-
C4ꢀ) were restrained to the trans region (⑀ = 180 60◦) in all
the residues of XyloNA1 based on the observed cross-peaks
ꢀ
ꢀ
ꢀ
ꢀꢀ
for long range phosphorus proton couplings (4JH4 -P3 ) in
1H-31P HETCOR spectra (Supplementary Figure S1, in the
supporting information), indicative for a W-shaped confor-
mation of the P3ꢀ-O3ꢀ-C3ꢀ-C4ꢀ- H4ꢀ fragment in the back-
bone.
ꢀ
ꢀ
No torsion angle restraints were applied on the backbone
dihedral angles α, and ζ.
Structure calculations
All structure calculations were performed using the X-
PLOR NIH program (27). The standard topology and pa-
rameter file (topallhdg.dna, parallhdg.dna) for DNA were
adapted for Xylose residue by introducing inversion in the
3ꢀ-chiral improper angle. The torsion-angle molecular dy-
namics protocol used was largely identical to that proposed
for the DNA duplex. A set of 100 structures was generated
by torsion-angle molecular dynamics, starting from two ex-
tended strands with random structures by using all struc-

Nucleic Acids Research, 2015, Vol. 43, No. 15 7193
Scheme 1. Schematic representation for the synthesis of four XyloNA nucleotide building blocks. Reagents and conditions: (a) BnBr, NaH, dry THF, 0◦C–
RT, 16 h; (b) AcOH, Ac2O, cat. H2SO4/CF3SO3H, 0◦C–RT, 2 h; (c) 60% TFA in H2O, 0◦C–RT, 4 h; (d) Ac2O, dry pyridine, 0◦C–RT, 4 h; (e) for 3: uracil,
BSA, CH3CN, 80◦C, 1 h, then sugar 2, SnCl4, 50◦C, 2 h; (f) for 7: N4-benzoylcytosine, BSA, CH3CN, 80◦C, 45 min, then 2, SnCl4, 80◦C, 1.5 h; (g) for 11:
N6-benzoyladenine, 2, SnCl4, CH3CN, RT, 2 h; (h) for 16: N2-acetyl-O6-DPC guanine (15), BSA, DCE, 80◦C, 30 min, then 2 in dry toluene, TMSOTf,
80◦C, 1 h; (i) 90% TFA in H2O, RT, 2 h; (j) for 4: method A-Pd/C 10%, MeOH, H2, RT, 20 h or method B- BCl3, dry CH2Cl2, –78◦C to –10◦C, 3 h, then
EtOH, –78/–40◦C to RT, 30 min, 55%; for 8 and 12: method B, quenching with a mixture of EtOH and Et3N; (k) for 18: Pd/C 10%, Pd(OH)2/C 20%,
H2O, MeOH, H2, RT, 72 h; (l) DMTr-Cl, dry pyridine, RT, 16 h; (m) NC(CH2)2OP(Cl)N(iPr)2, DIPEA, dry CH2Cl2, RT, 4 h.
addressed by using the Lewis-acid (BCl3) mediated deben-
zylation reaction. The debenzylated xylonucleosides were
tritylated and phosphitylated to the corresponding amidites
(6, 10, 14 and 20) using the standard reaction conditions
(Scheme 1). Three fully modified oligonucleotide sequences
(Table 1, XyloNA1–3) containing ␤-D-xylofuranosyl nu-
cleotides were assembled on the solid support using synthe-
sized phosphoramidites.
system. A non self-complementary fully-modified XyloNA
oligonucleotide with mixed sequence context (Table 1, Xy-
loNA2) was additionally synthesized and investigated for its
potential to cross-pair with the natural DNA/RNA com-
plements. These studies highlight that the XyloNA system
could neither pair with DNA nor with RNA (Table 1 and
Figure 2B, C), and thus demonstrating autonomous self-
pairing property of XyloNA.
In order to investigate if XyloNA is universally orthog-
onal or not in pairing with other DNA/RNA sequence,
we have also constructed a homo-pyrimidine sequence (Xy-
loNA3, xU12) and evaluated its cross pairing ability with
natural DNA (dA12) and RNA (rA12) complements. As
shown in Figure 3, xU12 is indeed capable of pairing with
both dA12 and rA12 strands (Table 1). In both the cases
triplex formation could be confirmed based on the follow-
ing observations (Figure 3): (i) a strong hysteresis effect
(ꢀTm = 13◦C between heating and cooling curves) was
observed (Figure 3) in normal melting buffer, in particu-
lar with rA12, which can be considered unusual for a du-
plex; (ii) these structures are highly stabilized in a Mg2+
buffer without any significant hysteresis effect; since the ex-
tent of cationic stabilization is usually more pronounced
in a triplex than a duplex; (iii) moreover, a temperature-
dependent hypochromism was observed in 284 and 295 nm
(Figure 3) which represents the melting of Hoogsteen paired
third-strand from a triplex structure (30–32). Melting tem-
peratures of both the strands of these triplexes are identical
and represent a single transition at 260 nm while melting of
the third strand can only be visible at 284/295 nm. Melt-
Thermal (UV-) melting study
The fully-modified self-complementary XyloNA oligonu-
cleotide (ON-1, Figure 1) was initially investigated for its
capability in forming a duplex structure by thermal (UV-
) melting study. This data indicates that XyloNA is able
to form a self-paired duplex with a melting temperature
(Tm) of 67◦C and is associated with a typical sigmoidal
cooperative melting profile (Figure 2A). In order to com-
pare the thermal stability of duplexes possessing different
backbones with the same sequence context (Table 1), we
have synthesized and analyzed ON-1 to ON-4. The relative
thermal stability of different duplexes decreased in the or-
der (Table 1, ON1–4) of XyloNA:XyloNAꢂdXyloNA:dXyl
oNAꢂRNA:RNAꢂDNA:DNA. Notably, the xylose-based
backbone modification (XyloNA/dXyloNA) significantly
increased the duplex stability by 1.6–2.2-fold in compari-
son with the corresponding ribose-based DNA/RNA pair.
In particular, the effect of the 2ꢀ-hydroxyl moiety is instru-
mental in increasing the duplex stability of XyloNA versus
dXyloNA, as also seen in the case of natural RNA vs. DNA

7194 Nucleic Acids Research, 2015, Vol. 43, No. 15
Table 1. Thermal melting data for the evaluation of self-pairing and cross-pairing abilities of XyloNA/dXyloNA vs. natural DNA/RNA systems
Oligo. no.
ON-1
Type of pairing
Sequence
Tm (◦C)
XyloNA1:XyloNA1
5ꢀ-x(GUGUACAC)-3ꢀ
3ꢀ-(CACAUGUG)x-5ꢀ
5ꢀ-dx(GTGTACAC)-3ꢀ
3ꢀ-(CACATGTG)dx-5ꢀ
5ꢀ-r(GUGUACAC)-3ꢀ
3ꢀ-(CACAUGUG)r-5ꢀ
5ꢀ-d(GTGTACAC)-3ꢀ
3ꢀ-(CACATGTG)d-5ꢀ
5ꢀ-x(UGCUACGC)-3ꢀ
3ꢀ-(ACGAUGCG)r-5ꢀ
5ꢀ-x(UGCUACGC)-3ꢀ
3ꢀ-(ACGATGCG)d-5ꢀ
5ꢀ-r(UGCUACGC)-3ꢀ
3ꢀ-(ACGAUGCG)r-5ꢀ
5ꢀ-r(UGCUACGC)-3ꢀ
3ꢀ-(ACGATGCG)d-5ꢀ
xU12:rA12
67
ON-2a
ON-3
ON-4a
ON-5
ON-6
ON-7
ON-8
dXyloNA:dXyloNA
RNA:RNA
57
41
DNA:DNA
26
XyloNA2:RNA
XyloNA2:DNA
RNA:RNA
n.o.
n.o.
50
RNA:DNA
n.c.
ON-9
ON-10
XyloNA3:RNA
XyloNA3:DNA
37–50
35–38
xU12:dA12
Melting points were determined in a buffer solution (pH 7.5) containing 0.1 M NaCl, 20 mM KH2PO4, and 0.1 mM Na2EDTA. For the self-complementary
sequences (ON-1 to ON-4) 8 ␮M and for other sequences (ON-5 to ON-10) 4 ␮M concentration of each strand have been used.
[a] Ref. (24); n.o., no cross-pairing observed; n.c., Tm could not be calculated due to an unfavorable melting profile. Melting profiles for ON-3, ON-7, and
ON-8 are provided in the SI (Supplementary Figure S2–S4).
ing curves for 1:0.5, 1:1, and 1:2 (dA12/rA12:xU12) mix-
ing stoichiometries are appeared to be very similar (data
not shown). In all these cases, a triplex is formed and when
heating, it is directly converted to single strands. Therefore,
a stable duplex could not be identified and this observa-
tion is in contrary with the corresponding natural triplexes
(30). However, these results are in agreement with the previ-
ously reported data for analogous study on dXyloNA sys-
tem. Initially, it was assumed that homo-pyrimidine dXy-
loNA (dxT12) forms a duplex with the complementary nat-
ural DNA/RNA; (15,33) however, later on Wengel et al.
(20) confirmed it as a triplex. Additionally, Gottikh et al. re-
ported that pyrimidine dXyloNA sequences are in general
prone to form triplexes with natural purine DNA/RNA se-
quences having more or less similar stability to that of the
natural counterparts (34–36). Notably, no duplex formation
was observed in all of these reported studies.
type helix), and might facilitate in the future cataloging of
non-natural nucleic acid conformation by CD spectroscopy.
Unlike dXyloNA duplex (24), the CD spectra of XyloNA
in saline buffer (Supplementary Figure S5) appeared to be
identical with the spectra in pure water. This suggests that
the presence of the 2ꢀ-hydroxyl group in the XyloNA back-
bone has resulted in a reduced conformational flexibility
compared to the dXyloNA system. In our previous study,
the latter has been identified to be conformationally mal-
leable since it exists in two different helical conformations
in saline aqueous/buffer solution (24).
Structure elucidation by solution state NMR spectroscopy
To examine the nature of the helical structure adopted
by the dsXyloNA (ON-1), we have undertaken solution
state NMR structural analysis. Appearance of a single set
of resonances in the NMR spectra suggests the forma-
tion of a unique type of duplex conformation (Figure 5A
and B). The duplex forms Watson–Crick type base-pairing
with anti-parallel strand orientation as evidenced by the
hydrogen-bonded imino-proton signals in the NMR spec-
trum recorded in H2O (Figure 5A and Supplementary Fig-
ure S6). All the xylose sugar rings are in N-type puckering
as suggested by the appearance of singlet anomeric-proton
peaks in the 1H NMR spectrum (Figure 5B). Unlike native
DNA/RNA double helices, unique diagonal cross-strand
(n+1 to n-1) NOE interactions between anomeric (H1ꢀ) pro-
tons were observed in XyloNA self-duplex, as depicted in
Figure 5C. Although less pronounced, a classical NOE-
walk (Figure 6) between aromatic and anomeric protons
was observed, that is generally more prominent in the natu-
ral DNA/RNA helices. During simulations, the structures
were converged (structure statistics in SI, Supplementary
Table S2) to a family of structures (Figure 7B-D and E) with
similar geometries and energies. The aforementioned diag-
onal NOE distance restraints were also reflected in the final
structure as depicted in Figure 7A. Diagnostic helical pa-
Circular dichroism (CD) study
To gain insights into the helical conformation and dynam-
ics of double-stranded (ds) XyloNA (ON-1), we have per-
formed circular dichroism (CD) studies. The CD spectra of
XyloNA appear to be different from the known CD spec-
tra of various helical nucleic acid structures i.e. A-, B- and
Z-type helices, (1) as shown in Figure 4. However, the CD
spectrum of XyloNA was found to be very similar to that of
our previously studied analogous dXyloNA system in wa-
ter (24). A characteristic positive Cotton (effect) band at
256 nm was observed in the CD spectrum, which showed
a gradual melting effect with increasing temperature (Fig-
ure 4, bottom). These observations suggest that the Xy-
loNA duplex has formed a slightly right-handed extended
ladder-like structure, as characterized by NMR (described
in the following section), and is in line with our previous
observation for dXyloNA (24). The repeated appearance
of this type of CD spectrum can be considered as a finger-
print for the right-handed extended duplex structure (RE-

Nucleic Acids Research, 2015, Vol. 43, No. 15 7195
with largely reduced helicity of XyloNA compared with
DNA/RNA might be the reasons for its inability in cross-
pairing with the DNA/RNA complements (37). A compar-
ison of the torsion angles (Table 2) between XyloNA and
DNA/RNA suggests a cooperative adjustment in different
dihedral angles across the sugar-phosphate backbone. The
chiral inversion in the 3ꢀ-carbon center of the sugar ring in
XyloNA with respect to DNA/RNA primarily changed the
endo-cyclic torsion angle ␦ to synclinal− from gauche+ or
anticlinal+. This leads to a chain of inversions in α, γ and ζ
torsion angles from an almost mirror-image domain (oppo-
site in sign and magnitude, shown in Table 2) while retaining
both the torsion angles ␤ and ⑀ in the anti domain. These
distinctive features allow a rationalization of the structural
orthogonality of XyloNA in comparison with the natural
DNA/RNA.
A
B
Temperature (°C)
0.75
0.7
0.65
0.6
0.55
0.5
DISCUSSION
As outlined above, our endeavor in synthesizing and char-
acterizing a xeno-nucleic acid analogue by appending the
potentially prebiotic xylose sugar in the backbone has re-
sulted in the creation of a novel xylonucleic acid (XyloNA)
system with interesting structural and biophysical proper-
ties. Like RNA, XyloNA could have been formed and ex-
isted in the prebiotic era since xylose has shown to be a
product in the formose reaction (38), the most plausible
prebiotic route of sugar synthesis. This suggests a possi-
bility that XyloNA/dXyloNA could have been selected by
natural evolution as a genetic information system. There
are, however, several structure-function properties of the
xylose-based information system (as revealed by our stud-
ies) that make it less suitable for this purpose. Although
XyloNA/dXyloNA could form base-paired duplexes, it
lacks the very helical nature and thus the compactness pos-
sessed by the natural helices. Moreover, XyloNA/dXyloNA
systems, by virtue of their predominant interstrand stack-
ing property, are thermodynamically more stable than the
natural ones. The higher duplex stability might be a plausi-
ble disadvantage since it would be rather difficult to unwind
the helices for accessing the genetic information in the pre-
biotic and/or biotic polymerization. Previously it was em-
phasized that natural DNA/RNA structures are selected
based on the principle of optimization rather than maxi-
mization of duplex stability and conformational flexibility
(5). The highly inclined bases along with reduced helical-
ity in XyloNA/dXyloNA strand make them ineligible to
cross-pair with the natural nucleic acids in the general se-
quence context, as evidenced by the melting studies (Ta-
ble 1). Since unable to cross-pair, it lacks communication
with the RNA world, and therefore XyloNA could not be
considered as a direct prebiotic RNA progenitor (39). These
unfavorable properties together might have contributed and
favored the selection of RNA rather than XyloNA as an
informational polymer in the prebiotic nucleic acid evolu-
tion. Besides structure-pairing properties, there is a prebi-
otic generational issue for any proposed primordial RNA
analogues. Conformational rigidity and altered helical ge-
ometry of XyloNA in comparison with RNA may poten-
tially influence the prebiotic polymerization fidelity, linkage
regio-chemistry (3ꢀ-5ꢀ versus 2ꢀ-5ꢀ isomers) and differential
20
30
40
50
60
70
80
Temperature (°C)
0.75
0.7
C
0.65
0.6
0.55
0.5
10
20
30
60
70
80
Tem4p0eratu5r0e (°C)
Figure 2. Thermal (UV-) melting profile of (A) XyloNA1 self-duplex (ON-
1), sigmoidal up- and down-curves are shown along with first derivative
plots for Tm determination; (B) XyloNA2:RNA hybrid (ON-5) and (C)
XyloNA2:DNA hybrid (ON-6), up- and down-curves are shown; no sig-
moidal behavior was observed and thus Tm could not be determined.
rameters and backbone dihedral angles were calculated and
compared (Table 2) with the natural DNA/RNA helices.
Atomic coordinates for 20 refined structures, structural re-
straints, and assigned chemical shifts have been deposited
in the RCSB Protein Data Bank with PDB ID 2N4J. Un-
like the natural B-DNA and A-RNA helices, XyloNA du-
plex like dXyloNA (24) has adopted a slightly right-handed
extended ladder-like structure (Figure 7B–D) with signifi-
cantly impaired helicity. This has been reflected by the rel-
atively low helical twist (10.7◦ versus 32.7◦) and high base-
pair rise (5.1 versus 2.8 ). Both the major and minor grooves
are widened to an extent that they are almost indistinguish-
able (Figure 7B and D). Nucleobases are highly inclined
with respect to the backbone, as shown by a high inclination
value (Table 2). Thus, it is predominantly forming a zipper
like interstrand stacking interaction to provide duplex sta-
bilization, in contrary to a dominant intrastrand stacking
as found in native DNA/RNA helices. This interstrand in-
teraction could be a cause for higher thermodynamic sta-
bility in XyloNA. High and dissimilar inclination together

7196 Nucleic Acids Research, 2015, Vol. 43, No. 15
A dA12 : xU12
B rA12 : xU12
In Mg2+-buffer
1.4
1.3
1.2
1.1
1
1.5
1.4
In Mg2+-buffer
1.3
1.2
1.1
1
0.9
10 20 30 40 50 60 70 80
10 20 30 40 50 60 70 80
Temperature (°C)
Temperature (°C)
0.3
0.27
0.24
0.21
0.18
0.15
0.3
0.27
0.24
0.21
0.18
0.15
10 20 30 40 50 60 70 80
10 20 30 40 50 60 70 80
Temperature (°C)
Temperature (°C)
0.012
0.011
0.01
0.01
0.009
0.008
0.007
0.006
0.005
0.004
0.003
0.009
0.008
0.007
0.006
10 20 30 40 50 60 70 80
10 20 30 40 50 60 70 80
Temperature (°C)
Temperature (°C)
Figure 3. Thermal (UV-) melting profile for (A) pairing of XyloNA3 (xU12) with dA12; (B) pairing of XyloNA3 (xU12) with rA12. Sigmoidal melting
curves are shown in different wavelengths (260, 284 and 295 nm). Heating- and cooling-curves are highlighted in red and blue respectively. Melting exper-
iments were generally performed in 20 mM KH2PO4 buffer (pH 7.5) containing 0.1 M NaCl, and 0.1 mM Na2EDTA or in a Mg2+-buffer, as indicated
above, containing 10 mM Tris·HCl (pH 7.5) and 50 mM MgCl2. Melting curves at 295 nm are from a faster heating-cooling experiments. Data presented
here are only for a 1:1 mixing stoichiometry with 4 ␮M concentration of individual strand.
Table 2. Torsion angles and key helical parameters of natural B-DNA and A-RNA duplexes are in comparison with the synthetic XyloNA and dXyloNA
duplex structures derived by NMR structural analysisa
B-DNA
A-RNA
dXyloNA
XyloNA
Torsion angles [◦]
α
β
γ
− 47
− 146
36
− 62
180
47
53
− 156
− 122
− 64
− 25
146
75
− 161
18
14
8 (A/G)
4 (C/T)
47
20
14
25 (A/G)
9 (C/U)
− 130
− 100
− 91
− 21
132
86
− 165
δ
ꢁ
ζ
χ
156
155
83
− 152
− 73
3
6
13
12
6
16
21
17
− 95
− 98
− 166
Helical parameters
Twist [◦]
36.0
− 5.9
0
32.7
15.8
− 30
2.8
2.7
− 52.2
0.5
0.4
10.7
− 45. 2 0.9
1.7
Inclination [◦]
Inclination-␩ [◦]
51
41
0.05
b
˚
Rise [A]
3.4
5.8
0.06
5.1
aStandard values for DNA, RNA and parameters for dXyloNA are from (24). Average and standard deviations ( ) were calculated for the XyloNA duplex
from the output data generated by using the CURVES program for an ensemble of 20 refined NMR structures. Backbone-base inclination angles (␩b) were
calculated by using the ‘Inclination’ program, (37).

Nucleic Acids Research, 2015, Vol. 43, No. 15 7197
20.0
15.0
10.0
5.0
dXyloNA
XyloNA
A-RNA
0.0
220
240
B-DNA
260
280
300
320
340
-5.0
-10.0
Z-DNA
Wavelength/nm
16.0
12.0
8.0
10 °C
80 °C
4.0
XyloNA
0.0
220
240
260
280
300
320
Figure 5. (A) Hydrogen-bonded imino-proton peaks of the XyloNA du-
plex (ON-1) representing Watson–Crick type base-pairing; (B) Appear-
ance of eight singlet anomeric-proton peaks (G1 to C8) in the 1H NMR
spectrum suggesting N-type puckering for the xylose sugar in XyloNA;
(C) a unique diagonal cross-strand NOE interaction(s) between H1ꢀ pro-
tons observed (boxed and indicated by red arrow) in the 2D NOESY spec-
trum of XyloNA duplex. All the above spectra were recorded in a 600 MHz
spectrometer with a 1.5 mM XyloNA (ON-1) sample (pH 7.2) at 10◦C.
-4.0
Wavelength/nm
Figure 4. Top: Circular dichroism (CD) spectra of XyloNA and dXyloNA
duplexes in water (12 ␮M, pH 7.0) at 10◦C. For comparison, the CD spec-
tra of the right-handed B-DNA, A-RNA and the left-handed Z-DNA
duplexes are shown and illustrated. Bottom: Temperature dependent CD
spectra of the XyloNA duplex showing a typical cooperative melting pro-
file.
C8
chemical stability (40) of the competing regioisomers that
can form, as shown previously in the case of polymerization
of different natural and non-natural systems (41–44). In the
prebiotic synthesis of nucleotide building blocks, there can
also be a different outcome with a xylose nucleoside con-
taining 2ꢀ, 3ꢀ-trans diol functionality than a ribonucleoside
with 2ꢀ,3ꢀ-cis diol functionality (22,45). These aspects re-
main to be studied to realize the full prebiotic potential of
XyloNA.
C6
U4
U2
H6/H8
G3
Previously, six-membered sugar-modified homo-
DNA/pyranosyl-RNA (p-RNA) (8) and acyclic glycol
nucleic acid (GNA) (46) have also adopted a similar
extended ladder-like structure. Interestingly, we could here
demonstrate that an extended helical structure could also
be derived by introducing a pentofuranosyl (Xylo) sugar.
Xylose nucleic acid (XyloNA) is found to be conforma-
tionally less flexible (as seen in RNA compared to DNA),
due to the 2ꢀ-hydroxyl effect, than the previously studied
analogous dXyloNA counterpart (24). In contrary, the
structural compactness and flexibility of XyloNA and
dXyloNA are not as favorable as in the case of natural
(DNA/RNA) systems. DNA (usually B-form) and RNA
(A-form) can shape into different types of helical con-
A7
G1
A5
Figure 6. Expansion of the NOESY spectrum (recorded with 300 ms
mixing time, in a 600 MHz spectrometer) of XyloNA (ON-1: 5ꢀ-
G1UGUACAC8–3ꢀ) duplex (1.5 mM, pH 7.2) in D2O at 10◦C, show-
ing NOE walk between H6/H8-H1ꢀ intra-residue and H1ꢀ(n)-H6/H8(n+1)
inter-residue protons.

7198 Nucleic Acids Research, 2015, Vol. 43, No. 15
Figure 7. Structural representations of the XyloNA duplex as determined by NMR structural analysis, (A) a close view of the minimum energy closest-
to-average structure showing base stacking interactions and the proximity of the diagonal anomeric protons as observed in the NOESY spectra, (B) view
from the minor groove, (C) side view with highlighted backbone, (D) view from the major groove, and (E) an overlay of 20 refined structures representing
convergence in the structure calculation.
formations by adopting different sugar pucker (1) (as
shown in Figure 1), as well as by essentially changing the
important endo-cyclic δ-torsion angle in the backbone. A
similar structural polymorphism could not be observed in
the xylose-based nucleic acids due to an identical δ value
and sugar pucker for both XyloNA and dXyloNA, and
thereby limiting their conformational diversity. Torsion
angle δ is found here to be crucial in determining the helical
twist and backbone flexibility versus rigidity as previously
suggested for DNA/RNA (47). Chiral inversion in the C3ꢀ
of furanose sugar in XyloNA/dXyloNA is constraining the
sugar conformation and ␦ torsion angle in comparison with
DNA/RNA, as a result forming a ladder-like structure with
increased duplex stability and to some extent rigidifying
the backbone conformation (16). Although dXyloNA was
previously shown to be structurally somewhat malleable in
its duplex conformation than natural DNA duplex (23,24).
In DNA/RNA, duplex stability and backbone flexibility
seem to have optimally selected and supported by their
very unique double helical structures. Considering the fa-
vorable properties that are required for an efficient storage
and transfer of genetic information, natural DNA/RNA
structure is unique and superior to that of xylose-based
(XyloNA/dXyloNA) nucleic acids.
the phosphodiester moiety inaccessible by the neighboring
2ꢀ-hydroxyl nucleophile. By virtue of its unique structure,
it could demonstrate novel orthogonal (inability in pairing
with DNA/RNA) self-pairing properties. Triplex formation
with purine DNA/RNA (as shown in melting study) is a
special case with pyrimidine XyloNA/dXyloNA sequences.
From an applicability point of view, if XyloNA/dXyloNA
is not a universal orthogonal system, most importantly
XyloNA/dXyloNA (as shown for XyloNA2) should be an
orthogonal information system in a mixed sequence context
which is mostly the case with natural unbiased genomic se-
quences. In general, xylose or deoxyxylose nucleic acid by
virtue of their inability to cross talk with the natural nucleic
acids could in principle be used for construction of an or-
thogonal episome for exploration in synthetic biology (12–
14). Along this line, the main bottleneck for implementing
such a XyloNA-based technology is the discovery of a Xy-
loNA polymerase which can efficiently polymerize XyloNA
nucleotide precursors. In our previous study, dXylo nucle-
oside triphosphates were shown to be partially recognized
by some mutant DNA polymerases, like for example Taq
mutant M1, for up to four nucleotides incorporation (24).
Recently, polymerases have been evolved that can copy or
transfer the genetic information from the non-natural nu-
cleic acid backbones, those mimicking the natural helical
structure (2,3). Polymerase evolution for XyloNA like de-
viant nucleic acid system poses a major challenge in the
hands of evolutionary molecular biologist.
Although xylonucleic acid could not supersede the struc-
ture and pairing properties of natural nucleic acids, its back-
bone could support essential self-pairing ability to form
double helix with slightly right-handed extended conforma-
tion. It is anticipated that XyloNA should be chemically
and enzymatically more stable than RNA due to an inverted
C-3ꢀ orientation of the phosphate linkage, and thus making

Nucleic Acids Research, 2015, Vol. 43, No. 15 7199
4. Egli,M. and Herdewijn,P. (eds). (2012) Chemistry and Biology of
Artificial Nucleic Acids. VHCA, Zurich & WILEY-VCH Verlag,
Weinheim.
5. Eschenmoser,A. (1999) Chemical etiology of nucleic acid structure.
Science, 284, 2118–2124.
6. Beier,M., Reck,F., Wagner,T., Krishnamurthy,R. and
Eschenmoser,A. (1999) Chemical etiology of nucleic acid structure:
comparing pentopyranosyl-(2ꢀ–>4ꢀ) oligonucleotides with RNA.
Science, 283, 699–703.
7. Schoning,K., Scholz,P., Guntha,S., Wu,X., Krishnamurthy,R. and
Eschenmoser,A. (2000) Chemical etiology of nucleic acid structure:
the ␣-threofuranosyl-(3ꢀ–>2ꢀ) oligonucleotide system. Science, 290,
1347–1351.
8. Ebert,M.O. and Jaun,B. (2010) Oligonucleotides with sugars other
than ribo- and 2ꢀ-deoxyribofuranose in the backbone: the solution
structures determined by NMR in the context of the ‘Etiology of
nucleic acids’ project of Albert Eschenmoser. Chem. Biodivers., 7,
2103–2128.
9. Yu,H., Zhang,S. and Chaput,J.C. (2012) Darwinian evolution of an
alternative genetic system provides support for TNA as an RNA
progenitor. Nat. Chem., 4, 183–187.
10. Egholm,M., Buchardt,O., Christensen,L., Behrens,C., Freier,S.M.,
Driver,D.A., Berg,R.H., Kim,S.K., Norden,B. and Nielsen,P.E.
(1993) PNA hybridizes to complementary oligonucleotides obeying
the Watson-Crick hydrogen-bonding rules. Nature, 365, 566–568.
11. Nelson,K.E., Levy,M. and Miller,S.L. (2000) Peptide nucleic acids
rather than RNA may have been the first genetic molecule. Proc.
Natl. Acad. Sci. U.S.A., 97, 3868–3871.
12. Herdewijn,P. and Marliere,P. (2009) Toward safe genetically modified
organisms through the chemical diversification of nucleic acids.
Chem. Biodivers., 6, 791–808.
CONCLUSION
Studies on artificial nucleic acid analogues from the struc-
tural neighborhood of DNA/RNA are inspired by motiva-
tions like gaining knowledge on the structural uniqueness
of DNA/RNA, and understanding the primordial ances-
tors that are lost in the evolutionary past. Among them,
those with novel properties could be useful in synthetic bi-
ology. Herein, we have investigated such an isomeric RNA
analogue, XyloNA derived from prebiotic xylose sugar. A
convergent synthetic route was implemented to access Xy-
loNA building blocks. Structural and pairing properties
of the synthesized XyloNAs are elucidated by employing
biophysical techniques. The helical form of DNA/RNA is
found to be structurally unique in a sense that they pos-
sess optimal duplex stability and conformational flexibility
in comparison with that of XyloNA. Unlike DNA/RNA,
XyloNA adopts an altered extended helical geometry, and
thus unable to cross-pair with the unbiased sequences of
DNA/RNA. Based on the preliminary structure-function
understanding available so far, it seems that RNA may have
gained an evolutionary advantage over XyloNA. On the
other hand, the autonomous pairing potential of XyloNA
could further be exploited as an orthogonal information
system in synthetic biology.
13. Marliere,P. (2009) The farther, the safer: a manifesto for securely
navigating synthetic species away from the old living world. Syst.
Synth. Biol., 3, 77–84.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
14. Schmidt,M. (2010) Xenobiology: a new form of life as the ultimate
biosafety tool. BioEssays, 32, 322–331.
15. Rosemeyer,H. and Seela,F. (1991)
1-(2ꢀ-Deoxy-␤-D-xylofuranosyl)thymine Building Blocks for
Solid-Phase Synthesis and Properties of
ACKNOWLEDGEMENTS
Oligo(2ꢀ-Deoxyxylonucleotides). Helv. Chim. Acta, 74, 748–760.
16. Rosemeyer,H. and Seela,F. (1995) Xylose-DNA:
Sequence-Dependent Duplex Stability as Function of Backbone
Configuration. Nucleosides Nucleotides, 14, 1041–1045.
17. Seela,F., Heckel,M. and Rosemeyer,H. (1996) Xylose-DNA
Containing the Four Natural Bases. Helv. Chim. Acta, 79, 1451–1461.
18. Schoppe,A., Hinz,H.J., Rosemeyer,H. and Seela,F. (1996)
Xylose-DNA: comparison of the thermodynamic stability of
oligo(2ꢀ-deoxyxylonucleotide) and oligo(2ꢀ-deoxyribonucleotide)
duplexes. Eur. J. Biochem., 239, 33–41.
19. Rajwanshi,V.K., Hakansson,A.E., Dahl,B.M. and Wengel,J. (1999)
LNA stereoisomers: xylo-LNA (␤-D-xylo configured locked nucleic
acid) and ␣-L-LNA (␣-L-ribo configured locked nucleic acid). Chem.
Commun., 1395–1396.
20. Poopeiko,N.E., Juhl,M., Vester,B., Sorensen,M.D. and Wengel,J.
(2003) Xylo-Configured oligonucleotides (XNA, xylo nucleic acid):
synthesis of conformationally restricted derivatives and hybridization
towards DNA and RNA complements. Bioorg. Med. Chem. Lett., 13,
2285–2290.
Dr Mohitosh Maiti is a postdoctoral fellow (1200113N) of
FWO Vlaanderen, Belgium. Technical support by Mr Guy
Schepers is gratefully acknowledged. We thank Dr Puneet
Srivastava for scientific discussion and Chantal Biernaux
for secretarial assistance. This article is dedicated to Profes-
sor A. Eschenmoser on the occasion of his 90th birthday.
FUNDING
European Research Council under the European Union’s
Seventh Framework Programme [FP7/2007–2013] /ERC
Grant agreement [ERC-2012-ADG 20120216/320683];
FWO Vlaanderen, Belgium (G078014N to P.H., O6260
to MoM); Mass spectrometry was made possible by the
support of the Hercules Foundation of the Flemish Gov-
ernment [20100225-7]. Funding for open access charge:
FWO Vlaanderen, Belgium [O6260 of MoM].
21. Ravindra Babu,B., Raunak, Poopeiko,N.E., Juhl,M., Bond,A.D.,
Parmar,V.S. and Wengel,J. (2005) XNA (xylo Nucleic Acid): A
Summary and New Derivatives. Eur. J. Org. Chem., 2005, 2297–2321.
22. Seela,F., Wo¨rner,K. and Rosemeyer,H. (1994)
Conflict of interest statement. None declared.
1-(2ꢀ-Deoxy-␤-D-xylofuranosyl)cytosine: Base pairing of
oligonucleotides with a configurationally altered sugar-phosphate
backbone. Helv. Chim. Acta, 77, 883–896.
REFERENCES
1. Bloomfield,V.A., Crothers,D.M. and Tinoco,I. (2000) Nucleic Acids:
Structures, Properties, and Functions. University Science Books.
2. Pinheiro,V.B., Taylor,A.I., Cozens,C., Abramov,M., Renders,M.,
Zhang,S., Chaput,J.C., Wengel,J., Peak-Chew,S.Y., McLaughlin,S.H.
et al. (2012) Synthetic genetic polymers capable of heredity and
evolution. Science, 336, 341–344.
3. Taylor,A.I., Pinheiro,V.B., Smola,M.J., Morgunov,A.S.,
Peak-Chew,S., Cozens,C., Weeks,K.M., Herdewijn,P. and Holliger,P.
(2015) Catalysts from synthetic genetic polymers. Nature, 518,
427–430.
23. Ramaswamy,A., Froeyen,M., Herdewijn,P. and Ceulemans,A. (2010)
Helical structure of xylose-DNA. J. Am. Chem. Soc., 132, 587–595.
24. Maiti,M., Siegmund,V., Abramov,M., Lescrinier,E., Rosemeyer,H.,
Froeyen,M., Ramaswamy,A., Ceulemans,A., Marx,A. and
Herdewijn,P. (2012) Solution structure and conformational dynamics
of deoxyxylonucleic acids (dXNA): an orthogonal nucleic acid
candidate. Chem. Eur. J., 18, 869–879.
25. Keller,R. (2004) The Computer Aided Resonance Assignment Tutorial.
1st ed. Cantina, Goldau.

7200 Nucleic Acids Research, 2015, Vol. 43, No. 15
26. Wijmenga,S.S. and van Buuren,B.N.M. (1998) The use of NMR
methods for conformational studies of nucleic acids. Prog. Nucl.
Magn. Reson. Spectrosc., 32, 287–387.
27. Schwieters,C.D., Kuszewski,J.J., Tjandra,N. and Clore,G.M. (2003)
The Xplor-NIH NMR molecular structure determination package. J.
Magn. Reson., 160, 65–73.
28. Ravishanker,G., Swaminathan,S., Beveridge,D.L., Lavery,R. and
Sklenar,H. (1989) Conformational and helicoidal analysis of 30 PS of
molecular dynamics on the d(CGCGAATTCGCG) double helix:
‘curves’, dials and windows. J. Biomol. Struct. Dyn., 6, 669–699.
29. Robins,M.J., Zou,R., Guo,Z. and Wnuk,S.F. (1996) Nucleic acid
related compounds. 93. A solution for the historic problem of
regioselective sugar-base coupling to produce 9-glycosylguanines or
7-glycosylguanines. J. Org. Chem., 61, 9207–9212.
37. Pallan,P.S., Lubini,P., Bolli,M. and Egli,M. (2007) Backbone-base
inclination as a fundamental determinant of nucleic acid self- and
cross-pairing. Nucleic Acids Res., 35, 6611–6624.
38. Mu¨ller,D., Pitsch,S., Kittaka,A., Wagner,E., Wintner,C.E.,
Eschenmoser,A. and Ohlofjgewidmet,G. (1990) Chemie von
␣-Aminonitrilen. Aldomerisierung von Glycolaldehyd-phosphat zu
racemischen Hexose-2,4,6-triphosphaten und (in Gegenwart von
Formaldehyd) racemischen Pentose-2,4-diphosphaten:
rac-Allose-2,4,6-triphosphat und rac-Ribose-2,4-diphosphat sind die
Reaktionshauptprodukte. Helv. Chim. Acta, 73, 1410–1468.
39. Orgel,L.E. (2004) Prebiotic chemistry and the origin of the RNA
world. Crit. Rev. Biochem. Mol. Biol., 39, 99–123.
40. Usher,D.A. (1972) RNA double helix and the evolution of the 3ꢀ,5ꢀ
linkage. Nat. New Biol., 235, 207–208.
30. Pilch,D.S., Levenson,C. and Shafer,R.H. (1990) Structural analysis of
the (dA)10.2(dT)10 triple helix. Proc. Natl. Acad. Sci. U.S.A., 87,
1942–1946.
31. Mergny,J.L., Li,J., Lacroix,L., Amrane,S. and Chaires,J.B. (2005)
Thermal difference spectra: a specific signature for nucleic acid
structures. Nucleic Acids Res., 33, e138.
32. Miyoshi,D., Nakamura,K., Tateishi-Karimata,H., Ohmichi,T. and
Sugimoto,N. (2009) Hydration of Watson-Crick base pairs and
dehydration of Hoogsteen base pairs inducing structural
polymorphism under molecular crowding conditions. J. Am. Chem.
Soc., 131, 3522–3531.
33. Alekseev,Y.I., Gottikh,M.B., Romanova,E.A. and Shabarova,Z.A.
(1996) Interaction of Escherichia coli RNase H with modified
oligonucleotide duplexes .II. Duplexes formed by oligonucleotides
consisting of (1-␤-D-2ꢀ-deoxy-threo-pentofuranosyl)thymine
residues. Mol. Biol., 30, 206–208.
41. Rohatgi,R., Bartel,D.P. and Szostak,J.W. (1996) Nonenzymatic,
template-directed ligation of oligoribonucleotides is highly
regioselective for the formation of 3ꢀ-5ꢀ phosphodiester bonds. J. Am.
Chem. Soc., 118, 3340–3344.
42. Kozlov,I.A., Politis,P.K., Van Aerschot,A., Busson,R., Herdewijn,P.
and Orgel,L.E. (1999) Nonenzymatic synthesis of RNA and DNA
oligomers on hexitol nucleic acid templates: the importance of the A
structure. J. Am. Chem. Soc., 121, 2653–2656.
43. Schrum,J.P., Ricardo,A., Krishnamurthy,M., Blain,J.C. and
Szostak,J.W. (2009) Efficient and rapid template-directed nucleic acid
copying using
2ꢀ-amino-2ꢀ,3ꢀ-dideoxyribonucleoside-5ꢀ-phosphorimidazolide
monomers. J. Am. Chem. Soc., 131, 14560–14570.
44. Schrum,J.P., Zhu,T.F. and Szostak,J.W. (2010) The origins of cellular
life. Cold Spring Harb. Perspect. Biol., 2, a002212.
45. Banfalvi,G. (2006) Why ribose was selected as the sugar component
of nucleic acids. DNA Cell Biol., 25, 189–196.
46. Meggers,E. and Zhang,L. (2010) Synthesis and properties of the
simplified nucleic acid glycol nucleic acid. Acc. Chem. Res., 43,
1092–1102.
34. Gottikh,M.B., Alekseev,Y.I., Perminov,A.V., Pinskaya,M.D. and
Shabarova,Z.A. (1999) Oligodeoxyxylonucleotides Form Stable
Triplexes with Single-Stranded DNA. Nucleosides Nucleotides, 18,
1625–1627.
47. Eschenmoser,A. and Dobler,M. (1992) Warum Pentose- und nicht
Hexose-Nucleinsa¨uren?? Teil I. Einleitung und Problemstellung,
Konformationsanalyse fu¨r Oligonucleotid-Ketten aus
2ꢀ,3ꢀ-Dideoxyglucopyranosyl-Bausteinen (‘Homo-DNS’) sowie
Betrachtungen zur Konformation von A- und B-DNS. Helv. Chim.
Acta, 75, 218–259.
35. Ivanov,S.A., Alekseev,Y.I. and Gottikh,M.B. (2002) Pyrimidine
oligodeoxyxylonucleotides form triplexes with purine DNA in neutral
medium. Mol. Biol., 36, 160–170.
36. Ivanov,S., Alekseev,Y., Bertrand,J.R., Malvy,C. and Gottikh,M.B.
(2003) Formation of stable triplexes between purine RNA and
pyrimidine oligodeoxyxylonucleotides. Nucleic Acids Res., 31,
4256–4263.