Oligonucleotide arrays from aldehyde-bearing glass with coated background

Article abstract of DOI:10.1055/s-2002-25758

Presented here is a method for preparing small DNA arrays on aldehyde-bearing glass slides. Immobilization involves reductive amination and employs oligonucleotides with 3′-terminal lysine residues, obtained in high yield from solid phase syntheses. Spot patterns are produced by protecting selected areas of the aldehyde slides with wax, coating the free surface with a methyl triethylene glycol derivative, and removing the wax with dichloromethane. The DNA arrays give better signal to noise ratios in hybridization experiments than slides without passified background.

Full text of DOI:10.1055/s-2002-25758

816
SPECIAL TOPIC
Oligonucleotide Arrays from Aldehyde-Bearing Glass with
Coated Background
O
ligonucleotides A
e
rrays on
A
n
ldehyde
B
ea
d
ring Glass
S
l
r
ide
s
a L. Dombi, Niels Griesang, Clemens Richert*
Department of Chemistry, University of Constance, Fach M 709, 78457 Konstanz, Germany
Fax +49(7531)884573; E-mail: Clemens.Richert@uni-konstanz.de
Received 28 December 2001
Work with modified oligonucleotides as probes, where
the modification is something other than a fluorophore or
the moiety providing the link to the chip surface, has been
limited thus far. Oligomers with a glycine ethylene di-
Abstract: Presented here is a method for preparing small DNA
arrays on aldehyde-bearing glass slides. Immobilization involves
reductive amination and employs oligonucleotides with 3 -terminal
lysine residues, obtained in high yield from solid phase syntheses.
Spot patterns are produced by protecting selected areas of the alde- amine backbone (PNAs) have been employed in microar-
rays.10 Phosphorothioate linked sequences and sequences
hyde slides with wax, coating the free surface with a methyl trieth-
ylene glycol derivative, and removing the wax with
with terminal modifications were tested and the former
dichloromethane. The DNA arrays give better signal to noise ratios
shown to bind their DNA targets with lower affinity than
in hybridization experiments than slides without passified back-
their unmodified counterparts.11 Some modified oligonu-
ground.
cleotides were reported to reduce the number of incorrect
Key words: oligonucleotides, Schiff bases, solid phase synthesis,
results in DNA chip hybridization experiments.12 Struc-
DNA, molecular recognition
tured DNA probes, for whom a three state binding equi-
librium may operate, leading to enhanced selectivity,13
have also been immobilized, though in devices involving
DNA microarrays (‘DNA chips’) have become indispens-
optical fibers14 rather than conventional chips. Finally,
able tools in biomedicine.1,2 Some DNA chips allow the
modified oligonucleotides whose modifications facilitate
detection of up to ~105 different hybridization processes
electrochemical detection have been employed on surfac-
in a single experiment and therefore simultaneous gene
es.15
expression analysis on the level of entire genomes.3 Re-
We and others have an interest in developing modified
sults from these analyses may lead to new targets for drug
development.4 During the preparation of DNA chips,
probes are patterned onto a surface, either by photolitho-
graphic syntheses1a,5 or by immobilizing suitably func-
tionalized pre-synthesized oligonucleotides. Read out
processes after incubation of the probe studded surfaces
with fluorophore labeled or radioactive analyte DNA rely
on the formation of stable Watson–Crick duplexes be-
tween complementary strands.
oligonucleotide probes whose affinity for target strands is
less dependent on their G/C content than that of unmodi-
fied DNA,16,17 and whose ability to bind tightly and selec-
tively at the termini is enhanced.18,19 Since it is not trivial
to design modified oligonucleotides that bind a range of
mixed sequence target strands with similar affinity, and
modifying several nucleotides in a DNA strand can lead to
unexpected cooperativity effects,20 extensive experimen-
tal testing may be required. For this, we became interested
in developing a rugged immobilization methodology that
would allow testing modified oligonucleotides efficiently
at moderate cost. Here we report a method for immobiliz-
ing modified and unmodified oligonucleotides on com-
mercial glass slides, where the oligonucleotides used for
immobilization are prepared entirely on an inexpensive
solid support. Further, we show how surface sites sur-
rounding the DNA strands may be coated such that unspe-
cific adsorption of analyte strands is minimized.
Photolithographic syntheses yield high density chips,
whose false positive rates in routine hybridization experi-
ments can be reduced to less than 2% by using a large
fraction of the spots for probe redundancy and mismatch
control purposes,6 though this level of stringency may
lead to very substantial false negative rates.7 For spotted
microarrays of PCR amplified DNA, with chain lengths
exceeding those that are currently accessible synthetical-
ly, false positive rates (hybridization cross reactivities) are
higher, reaching values of up to 57%.8 Clearly, for appli-
cations such as SNP genotyping9 or sequencing by hybrid-
ization, where high stringency is mandatory, higher
fidelity chips are desirable. These will most probably re-
quire optimization of the chemical structure of the oligo-
nucleotide probes, further optimization of linkers between
surface and probes, and modification of surface sites
where undesirable binding or reactions can occur.
A number of methods have been previously described for
the covalent immobilization of DNA strands on chip sur-
faces. Possibly the simplest of these involve adsorption on
polylysine modified surfaces1c and UV cross linking of
long, otherwise unmodified DNA to surfaces with free
amino groups.21 Other methods employ site selectively
modified DNA and activated surfaces, where the covalent
link does not involve the nucleobases directly. Examples
are surfaces with bromoacetamido moieties reacted with
DNA containing phosphorothioate groups,22,23 epoxide
and isothiocyanate modified surfaces reacted with amino
modified DNA,24,25 semicarbazide derivatized surfaces
Synthesis 2002, No. 6, 29 04 2002. Article Identifier:
1437-210X,E;2002,0,06,0816,0824,ftx,en;C10101SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

SPECIAL TOPIC
Oligonucleotides Arrays on Aldehyde Bearing Glass Slides
817
reacted with benzaldehyde modified DNA,26 and mercap- been used previously for immobilizing oligonucleotides
topropylsilane modified surfaces allowed to form disul- on surfaces,11 though not via reductive amination, and not
fides with disulfide bearing DNA in disulfide exchange with lysine residues as linkers.
reactions.27 A number of these and related attachment reg-
The synthesis of the strands to be immobilized started
iments rely on bifunctional linkers.28 Some involve self
from long chain alkylamine controlled pore glass (LCAA
assembled monolayers, on silica29 or gold substrates.30 To
CPG) and involved intermediate 1, whose preparation fol-
improve accessibility of the DNA strands, spacers2b,31 and
lows a method for the preparation of 3 ,5 -dipeptidyloligo-
dendrimer linkers32 have been employed.
nucleotides,37 except that a single -hydroxylauric acid
Several immobilization methods for oligonucleotides residue is employed between the controlled pore glass and
were recently compared, while using the ability to bind the L-lysine residue (Scheme 1). The linker includes a 2,2-
analyte strands as the basis for evaluation.33 The authors dimethylhydroxypropionic acid (DP) moiety, which is
found DNA microarrays prepared via reductive amination known to reduce side reactions originating from 3 -ap-
from aldehyde modified glass slides and DNA with amino pendages.37 Standard DNA synthesis on support 1 yielded
groups (‘aminated DNA’) to have the most favorable intermediates 2, whose deprotection with saturated aque-
properties.33 Another study tested for the ability to per- ous ammonia gave lysine bearing oligonucleotides (3) in
form minisequencing reactions with immobilized primers high purity, as determined by MALDI–TOF mass spec-
and found that disulfide linkages between DNA and the trometry of crude products (see Figure S4, Supporting In-
surface gave the best results.34 A third comparative study formation, for an example).
focused on solid phase DNA amplification as the read out
technique and recommended 5 -thiol-modified oligonu-
Several oligonucleotides of general structure 3 were im-
mobilized on aldehyde bearing glass microscope slides,
cleotides and attachment to amino silanized glass slides
following the protocol recommended by Zammatteo and
via a heterobifunctional cross linker as the method of
collaborators.33 When solutions of fluorophore bearing
choice.35
DNA were spotted onto the resulting DNA bearing spots,
We were interested in a technique allowing immobiliza- it was found that one step reductive amination with
tion of pre-synthesized oligonucleotides via the 3'-termi- NaCNBH3 (3 to 4, Scheme 1), rather than the recom-
nus. Immobilization of pre-synthesized strands was mended two step procedure (Schiff base formation, fol-
preferred over in situ synthesis on the chip surface, since lowed by reduction with NaBH4)33 gave the strongest
this allows characterization and purification of the fluorescence signal upon hybridization. However, when
strands, a feature considered critical when developing several DNA spots were incubated collectively, with ex-
new chemistries. Further, it was desirable to use the 3 -, posure of the non DNA bearing background surface be-
rather than the 5 -terminus for immobilization, since a tween the spots to the fluorophore bearing target DNA,
number of modifications to the 5 -terminus that enhance strong background signals were observed, sometimes to
target affinity and base pairing selectivity had already the extent that the background fluorescence was stronger
been developed.19,36 These would be non trivial to incor- than that from the DNA bearing spots. Unacceptably
porate in the linker required for anchoring the probe on the strong background signals of up to 100 intensity units on
chip. It was also desirable to employ a slide surface that a scale with a maximum of 200 units were also detected
was not electrostatically attractive for DNA, to prevent when the remaining aldehyde groups of the surface were
that the DNA lies flat on the chip, which would block oth- reduced with NaBH4 prior to hybridization,33 indicating
er immobilization sites and would reduce the accessibility that the signals were the result of adsorption, not reactions
for hybridization. Finally, we were interested in develop- with the aldehyde groups. Both the cyanine dye Cy3 and
ing a methodology that was not only versatile and provid- fluorescein as the fluorophore gave this result, and neither
ed good hybridization results, but was also affordable. of the numerous washing protocols tested, including those
Many of the techniques routinely employed for preparing recommended in reference 33 alleviated the problem. To
DNA arrays require pre-modified CPG, non nucleosidic further exclude side reactions between nucleobases and
phosphoramidites, or very expensive slides as starting ma- aldehyde groups, the reactivity of mixed sequence DNA
terials.
oligomers of general structure 3 towards aldehydes was
tested in solution. Monitoring of the conversion under the
chosen reductive amination conditions with several
equivalents of benzaldehyde by MALDI–TOF mass spec-
trometry showed that the reactivity was similar to that ob-
served for amino terminal, but otherwise protected DNA
strands,36a indicating that the exocyclic amino groups of
the nucleobases were not susceptible to Schiff base forma-
tion.
For our present work, hybridization with fluorophore la-
beled target(s) was the required application for DNA mi-
croarrays. We therefore used aldehyde slides and the
conditions recommended in the recent comparative study
by Zammatteo and collaborators33 as a starting point for
our work. Unfortunately, this publication does not give
the structure of the appendage to the oligonucleotides that
provides the amino groups for immobilization on the alde-
hyde bearing slides. We decided to choose oligonucle- The problem of unspecific background adsorption of tar-
otides with an L-lysine residue at the 3'-terminus37 as get strands on surfaces has been known since the early
amino modified DNA strands for immobilization. Oligo- days of DNA microarrays. A detailed study published in
nucleotides with amino groups at their 3'-termini have 1994 reported that efforts to reduce or eliminate this prob-
Synthesis 2002, No. 6, 816–824 ISSN 0039-7881 © Thieme Stuttgart · New York

818
K. L. Dombi et al.
SPECIAL TOPIC
HO
BPG
BPG
HO
B = nucleobase, PG =protecting group
B
B
n
BPG
n
DNA synthesis
NH4OH
P=O
B
CNC2H4
O
OH
O
PO2
O
TFA
TFA
N
H
N
O
O
H
NH
NH
O
H
2N
O
O
NH
O
O
3
O
(CH2
CO
)
11
(CH2
CO
)
11
O
2
1
NH
NH
NH2
CPG
aldehyde
slides,
O
O
NaCNBH3,
PBS
O
NH
O
O
O
HO
HO
HO
H
H
H
B
B
B
NaCNBH3,
PBS
B
n
B
n
B
n
spotting area
B
B
B
12
PO2
O
PO2
O
PO2
O
O
O
O
O
6, NaCNBH3,
O
O
O
PBS
O
O
NH
NH
4
NH
O
O
O
O
O
O
O
O
O
O
O
O
NH
O
NH
NH
NH
NH
NH
NH
O
O
O
O
H
H
H
H
H
Scheme 1
lem by using non specific blocking DNA or various deter- than the known methods for the preparation of 6.42,43 Al-
gents were unsuccessful.25 For ‘aminated’ surfaces and cohol 5 was converted to phthalimide 742 in a Mitsunobu
PCR amplified DNA, some improvement may be reaction, and 7 was converted to 6 via hydrazinolysis.
achieved with betaine as an additive to the spotting solu- Both reactions proceeded uneventfully, though distilla-
tion,38 but no general solution for aldehyde- or alcohol- tion of 6 lowered the yield for the second step to 70%. Ex-
bearing surfaces has emerged. We therefore decided to ploratory experiments showed that undistilled 6, obtained
develop a ‘passifying’ or coating approach to decreasing by our procedure in 84%, gave coating results that are in-
the non specific adsorption on the slide surface. It has distinguishable from those obtained with distilled 6.
been shown that poly- and oligoethyleneglycol chains can
With passifying amine 6 in hand, a method was developed
prevent non specific adsorption of biomacromolecules.39
for coating the background surface of the slides prior to
Further, oligoethylene chains have been used in the con-
the immobilization of the DNA probe strands. Paraffin
struction of linkers between DNA and surface.2b,40 An al-
wax was chosen as a protecting agent for the spots where
ternative approach, the use of fluorinated compounds to
DNA was to be placed later. This led to the procedure
shown in Scheme 3. Melted wax was applied to slide 8 via
a multichannel pipette, using an inexpensive drill press for
positioning the pipette, producing ‘spot protected’ 9. The
create non adsorptive areas surrounding DNA spots,41 was
not pursued due to higher costs and possible difficulties of
working with solvophobic surfaces.
To ensure a system that is chemically well defined, we remaining exposed aldehyde groups of 9 were then react-
chose oligoethylene chains that are available as single ed with 6 in a reductive amination to give 10. The protec-
compounds, rather than the longer chains that are pro- tive wax layers were then removed by dissolving the wax
duced by polymerization and have to be employed as mix- in dichloromethane, which gave slide 11, whose aldehyde
tures with a distribution of chain lengths. Triethyl- bearing spots were then used for immobilizing lysine
eneglycol monomethylether (5) is available at low cost modified DNA strands of general structure 3. A final re-
and has about the same contour length as the 3 -appendage ductive amination with 6 on the entire slide surface sealed
of the oligonucleotide probes of general structure 3, thus remaining aldehyde sites between DNA chains on the
avoiding blocking the base pairing of the 3 -terminal re- spots, as well as possibly providing additional tightening
gion of the oligonucleotides. To be suitable for immobili- of the passifying coat in the background area, resulting in
zation on the aldehyde modified slide surface, 5 had to be DNA arrays of general structure 12 (Schemes 1 and 3).
converted to amine 6.42,43 We decided to employ the syn-
thetic route shown in Scheme 2, which is one step shorter
(Figure 1) were prepared following the protocol shown in
DNA microarrays with sequences 13, 14, and 15
Synthesis 2002, No. 6, 816–824 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
Oligonucleotides Arrays on Aldehyde Bearing Glass Slides
819
Phthalimide,
O
PPh3, DIAD,
THF
O
N
O
OH
O
O
O
O
95%
O
7
5
1. N2H4, ∆
2. HCl conc., ∆
70% over 2 steps
O
NH2
O
O
6
Scheme 2
Scheme 3, with spotting every DNA probe strand in dupli- were observed (Figure 2). The mismatched probe 15 gave
cate. Oligonucleotide 13 contains a cholic acid moiety at less hybridization signal than fully matched 14, even
its 5 -terminus, a modification shown earlier to increase though the mismatch is very close to the terminus, and 5 -
target affinity and base pairing fidelity at the terminus of capped 13 gave the strongest signal.
a duplex.19 Sequence 14 is made up of unmodified DNA,
except for the 3 -linker to the surface, and so is sequence
15, but 15 contains a mismatched base pair at the penulti-
mate position of the duplex with Cy3 labeled target strand
16. When the slide presenting 13, 14, and 15 was incubat-
ed with 16, under a cover slip, i.e. with exposure of the
The DNA array also proved to be reusable. Removal of
bound target DNA with water at 90 °C gave back a slide
with no or virtually no fluorescence signal upon scanning.
When used three times in a row, less than 25% loss of hy-
bridization signal was observed between the first and the
last experiment. Together, these results demonstrated that
background surface to the fluorophore labeled DNA, se-
the slide with coated background operates successfully as
lective hybridizations at the sites of DNA immobilization
melted wax
9
8
aldehyde-derivatized slide
6, NaCNBH3, PBS
CH2Cl2
11
10
1. DNA-DP-Lys (3),
NaCNBH3, MES buffer
2. 6, NaCNBH3, PBS
12
Scheme 3
Synthesis 2002, No. 6, 816–824 ISSN 0039-7881 © Thieme Stuttgart · New York

820
K. L. Dombi et al.
SPECIAL TOPIC
OH
OH
OH
N
N
Cy3
O
PO2
5'
HO
5'
HO
HO
O
O
HN
PO2
T
T
A
T
T
O
A
A
G
G
O
O
G
T
G
T
G
T
C
G
C
A
G
T
T
T
T
G
A
C
G
A
C
G
A
C
T
T
T
C
A
A
C
C
A
G
C
G
A
T
G
C
G
A
T
G
C
G
A
T
O
T
O
O
PO2
DP
DP
DP
OH
3'
O
Lys
Lys
Lys
CO2
CO2
CO2
O
3'
3'
3'
NH
H2N
CO2
16
14
15
13
Figure 1 Oligonucleotides employed in hybridization experiments with DNA microarrays. Fluorophore-labeled 16 was employed in solution.
The three lysine bearing sequences 13, 14, and 15 were immobilized on the glass surface in separate regions. Every letter to the right of a bold
horizontal bar represents a nucleobase in a DNA sequence. The highlighted boxes show structural details of modified termini.
DNA microarray, and that the hybridization is selective, Several extensions of the current scheme can be imagined.
both in terms of sequence selectivity and in terms of stron- One would facilitate automation of the hybridization and
ger binding when an affinity-enhanced, modified probe is washing steps by introducing a fixed container around the
used.
slide, though the engineering effort for this and a circulat-
ing flow system with a pump may be substantial, without
offering major advantages for moderate throughput stud-
ies. Further, one could perform the immobilization step
with oligonucleotide duplexes, rather than single strands,
as reported for DNA immobilized on gold surfaces.44 The
probe strand, engaged in Watson–Crick duplex formation
with a complementary strand, would have any residual re-
activity of the exocyclic amino groups of the nucleobases
masked, and the presence of the complementary strand
would ensure that enough space is available for comple-
mentary strands to bind in later hybridization experi-
ments. However, the cost and effort for synthesizing the
complementary strands are not negligible, and complete
removal of the complementary strands after immobiliza-
tion is not trivial to establish either. Thirdly, it may be
worthwhile replacing Cy3 as the fluorophore of the target
strands. The phosphoramidite with which this cyanine dye
is introduced is currently the most costly chemical em-
ployed in our procedure. Labeling strands of general
structure 3 with fluorescein isothiocyanate or rhodamine
B isothiocyanate has already been performed successful-
ly, though the fluorescence intensity upon hybridization
of the labeled strands to the microarrays is currently lower
than that achieved with the corresponding Cy3 labeled
strands, possibly because the surface was not wetted suf-
ficiently prior to scanning.25
Figure 2 Results of a hybridization experiment with a model
microarray displaying oligonucleotides 15 (left lane), 13 (center la-
ne), and 14 (right lane), immobilized on a background coated glass
surface, prepared as shown in Scheme 3. Each oligonucleotide was
spotted in duplicate (upper and lower row) to test reproducibility.
Fluorescent oligonucleotide 16 (10 M) in buffer solution was incu-
bated with the surface of the array, as described in the Experimental
Part. (a) Fluorescence image, (b) fluorescence intensity profile for the
upper row of spots, generated with NIH Image/Scion Image.48
Synthesis 2002, No. 6, 816–824 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
Oligonucleotides Arrays on Aldehyde Bearing Glass Slides
N-(3,6,9-Trioxadecyl)phthalimide (7)
821
Even without additional extensions and improvements, it
is hoped that the method presented here will prove useful
for routine testing of modified oligonucleotides as hybrid-
ization probes in microarrays, and possibly for the prepa-
ration of arrays for biomedical applications themselves.
Since syntheses on lysine bearing solid support 1 produce
few side products, spotting without purification should be
possible, making the preparation of multispot arrays fea-
sible. These should enable the identification of hybridiza-
tion probes, whose mismatch discrimination is better than
that of unmodified DNA, particularly when the mismatch
is close to one of the termini of the duplexes, where
breathing and fraying prevent high fidelity recognition in
natural DNA and RNA.45
Phthalimide (3.53 g, 24 mmol) and PPh3 (6.3 g, 24 mmol) were
dried for 1 h at 0.1 Torr, placed under argon, and dissolved in THF
(100 mL). The solution was treated with triethylenglycol monome-
thylether 5 (3.2 mL, 20 mmol). After 15 min, DIAD (4.73 mL, 24
mmol) was added dropwise at r.t., resulting in a slight warming of
the solution. After 12 h at r.t., TLC showed that the reaction was
complete (petroleum ether (PE)–EtOAc, 1:1, educt Rf 0.1, product
Rf 0.45). The reaction was quenched by addition of EtOH (40 mL).
The solvent was evaporated in vacuo and the residue dried at 0.1
Torr for 1 h. The residue was treated with PE–EtOAc (1:1, 20 mL)
and stirred at 40 °C for 1 h. The white residue was filtered off and
washed with the same solvent mixture (10 mL). The filtrate was
evaporated to dryness, dried at 0.1 Torr, and chromatographed on
silica gel (PE–EtOAc , 4:1), yielding the title compound as colorless
oil (5.57 g, 19 mmol, 95%).
1H NMR (CDCl3, 250 MHz): = 3.28 (s, 1 H), 3.40 (m, 2 H), 3.51–
3.62 (m, 6 H), 3.67–3.71 (t, J = 5.5 Hz, 2 H), 3.83–3.84 (t, J = 5.5
Hz, 2 H), 7.65–7.69 (m, 2 H), 7.77–7.81 (m, 2 H).
13C NMR (CDCl3, 62.9 MHz): = 37.09, 58.84, 67.74, 69.93,
70.37, 70.39, 71.67, 123.05, 131.98, 133.76, 168.07.
The following solvents were of the best commercial quality and
were used as received: DMF, THF and TFA (Fluka/Riedel deHaen,
Taufkirchen, Germany), CH3CN (Acros, Geel, Belgium), and
AcOH (Roth, Karlsruhe, Germany). Also used as received were the
following chemicals: Ac2O and DIAD (Fluka), NH4OH and PPh3
(Fluka/Riedel deHaen), sodium dodecylsulfate (SDS), sodium cit-
rate, hydrazine monohydrate, and NaCNBH3 (Acros), triethylengly-
col monomethylether (Aldrich, Taufkirchen, Germany), Boc-
Lys(TFA)-OH (NovaBiochem, Läufelfingen, Switzerland), 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexaflourophos-
phate (HBTU), and hydroxybenzotriazole monohydrate (HOBT)
(Advanced ChemTech, Louisville, KY). The phosphoramidite
building blocks for DNA synthesis were from Proligo (Hamburg,
Germany) for dABz, dCBz, and T; and from Glen Research (Sterling,
VA) for dGdmf. The controlled pore glass (CPG) for DNA synthesis
was long chain alkylamine (LCAA) CPG from Controlled Pore
Glass Inc. (Lincoln Park, NJ) and had the following specifications:
mean pore diameter 1088 °A, surface area 34.4 m2/g, particle size
80–120 mesh, loading 93.3 mol/g. The Cy3 phosphoramidite
building block for 5 -fluorophore labeling was from Glen Research
(catalog No. 10–5913–95). Oligonucleotides were purified by RP
HPLC C4 columns (Vydac, Hesperia, CA or Macherey–Nagel,
Düren, Germany), using a gradient of CH3CN (solvent B) in 0.1 M
triethylammonium acetate (TEAA) at pH 7. Yields of oligonucle-
otides are based on the intergration of HPLC traces of crudes. Par-
affin wax (purum, mp 50–52 °C) for protecting slide areas was from
Fluka. Aldehyde bearing slides were from Cell Associates, (Hous-
ton, TX) and were ordered via GenPak Limited (New Milton, UK),
who sells them as ‘silylated slides’. According to the supplier, there
is a three carbon linker between the silicon atom that is part of the
surface network and the aldehyde group. Spotting was performed
with a multichannel micropipette (Roth), using drill press Bo-
hrständer Basic*** (Emil Lux GmbH, Wermelskirchen, Germany)
for positioning. Saline sodium citrate buffer for hybridization ex-
periments (SSC, 150 mM NaCl, 15 mM sodium citrate) was pre-
pared as a 20–fold concentrated stock solution and diluted to the
desired concentration when needed. Phosphate buffered saline solu-
tion (PBS, pH 7) was prepared using a standard protocol.46 MAL-
DI–TOF mass spectra were acquired on a Bruker BIFLEX III mass
spectrometer in negative mode at 19 kV with a delayed extraction
voltage of 17.15 kV. A mixture of 2,2,6-trihydroxy acetophenone
(0.3 M in EtOH) and diammonium citrate (0.1 M in H2O) was used
as matrix and comatrix for the mass spectrometer. A Perkin Elmer
Lambda 10 spectrophotometer was used to acquire UV spectra.
NMR spectra were acquired on a Bruker AC 250 spectrometer. The
fluorescence images of DNA arrays with bound analyte strand were
obtained with a Molecular Imager Fx (BioRad, Hercules, CA) using
software Quantity One, version 4.2.
EI-MS (70 eV, 105 °C): m/z (%) = 293, 234, 218, 204, 190, 174,
59.
3,6,9-Trioxadecylamine (6)
The N-(3,6,9-Trioxadecyl)phthalimide precursor 7 (5.75 g, 20.08
mmol) was taken up in EtOH (55 mL) and treated with hydrazine
monohydrate (1.16 mL, 22.4 mmol). The resulting mixture was re-
fluxed at 100 °C for 5 h, whereupon a white precipitate formed.
TLC showed full conversion (EtOAc, educt Rf 0.6, product Rf 0).
The slurry was allowed to cool and then treated with concd HCl (4.8
mL), followed by refluxing again for 1 h. The slurry was allowed to
cool to r.t. and the white solid was filtered off. The filtrate was evap-
orated in vacuo and the residue taken up in H2O (30 mL), and the
solution brought to pH 11 with 1 N NaOH (20 mL). The aqueous
phase was saturated with NaCl and extracted with CH2Cl2 (8 30
mL). The combined organic phases were dried over Na2SO4, evap-
orated to dryness and briefly dried at 0.1 Torr, yielding a slightly
brownish liquid (2.77 g). Kugelrohr distillation (0.1 Torr, heating
chamber at 110 °C) gave the title compound as colorless liquid (2.3
g, 14 mmol, 70%).
Rf 0.55 (MeOH–NH4OH, 9:1).
1H NMR in agreement with the literature.13,43
13C NMR (CDCl3, 62.9 MHz): = 41.59, 58.88, 70.12, 70.39,
70.45, 71.80, 73.21.
Support for synthesis of DNA to be immobilized (1)
The preparation of the controlled pore glass on which the DNA to
be immobilized was synthesized followed largely a methodology
reported earlier.37 Briefly, coupling of 12-trityloxylauric acid (100
mol, 45.6 mg) with HBTU (90 mol, 34.6 mg), HOBT (100 mol,
15.5 mg), and N,N-diisopropylethylamine (DIEA) (233.6 mol, 40
L) in DMF (600 L) to LCAA CPG (45 mg) for 1 h was repeated
once, followed by a capping step, which was performed using Ac2O
(1 mL) for 3 min. Detritylation with CH2Cl2–TFA (1:1, 6 mL) was
followed by coupling of Boc-Lys(TFA)-OH (35.3 mg, 100 mol)
with the same activation mixture as for trityloxylauric acid for 20
min. After another capping step and Boc-removal with CH2Cl2–
TFA (1:1, 2 mL), 2,2-dimethyl-3-trityloxypropionic acid (36.1 mg,
100 mol) was coupled for 1 h under the same activation conditions
as for the other two acids. Capping with Ac2O (2 mL, 3 min) and
detritylation, followed by the usual rinsing with CH3CN (12 mL)
and drying at 0.1 Torr gave the support ready for DNA synthesis.
Synthesis 2002, No. 6, 816–824 ISSN 0039-7881 © Thieme Stuttgart · New York

822
K. L. Dombi et al.
SPECIAL TOPIC
DNA Synthesis
Rt 32.5 min.
Oligodeoxyribonucleotides were synthesized on an ABI 380B DNA
synthesizer on a 1 mol scale, following the manufacturer’s recom-
mendations. Extended coupling times of 15 min were used for all
non standard phosphoramidites. After completion of the DNA syn-
thesis, using the trityl off mode, the DNA bearing CPG was placed
in Eppendorf-type polypropylene vessels in portions of 10 mg/ves-
sel, and concentrated aqueous ammonia (‘NH4OH’, 1.5 mL) was
added, followed by vortexing. The vessels were tightly sealed. After
16 h at r.t., excess ammonia was blown off with a gentle stream of
air directed on the surface of the solutions. The supernatants were
removed, the CPG washed with H2O (0.5 mL), and the combined
solutions lyophilized to dryness. The residues were purified by
HPLC.
MALDI–TOF MS: m/z [M – H]– calcd for C164H207N56O83P14,
4722.5; found: 4725.3.
Coating the slide background.
Melted paraffin wax (7 L per spot, at approx. 60 °C) was applied
to selected areas, using a multi channel pipetman attached to a drill
press. For this the pipet tips were heated for a few seconds using a
heat gun before being placed into the hot wax. The wax was taken
up, and quickly applied to the aldehyde bearing slide surface in rows
of 6 spots. The wax spots were allowed to solidify to create protect-
ed areas that would be unreactive in the subsequent step. The slide
was then photocopied on a standard paper copier to record the
placement of the wax spots. To coat the free surface, a mixture of
triethyleneglycol amine 6 (50 mM in PBS buffer, 5 mL) and Na
BH3CN (31 mM in PBS, 150 L) was applied to the aldehyde slide
and spread, using a glass pipet. The reductive amination was al-
lowed to proceed for 2.5 h at r.t. The slide was then rinsed with SSC/
0.2% SDS by dipping it into a 25 mL Erlenmeyer flask and then ro-
tating it therein for 2 min. H2O was rinsed over the slide to remove
excess salt, using a squirt bottle. The slide was then spun dry in cu-
vette dryer (Zentrax 280, Heildolph, Kelheim, Germany) adapted to
hold microscope slides. To remove the wax, the slide was placed in
a beaker with CH2Cl2 until the wax was entirely dissolved. The slide
was then rinsed with fresh CH2Cl2 and dried under a stream of ar-
gon.
5 -TGGTTGACTGCGAT-DP-Lys (14)
Yield 58%.
HPLC gradient of 0% B for 5 min, to 30% B in 35 min, to 100% B
in 10 min.
Rt 20.4 min.
MALDI–TOF MS: m/z [M – H]– calcd for C149H193N53O91P14,
4615.8; found: 4617.5.
5 -TAGTTGACTGCGAT-DP-Lys (15)
Yield 62%.
HPLC gradient of 0% B for 5 min, to 30% B in 40 min, to 100% B
in 10 min.
Spotting DNA.
A solution of the 3 -lysine bearing DNA (3) (2 L, 2 M) in 2-(N-
morpholino)ethanesulfonic acid (MES) buffer (0.1 M, pH 6.8) was
applied to the aldehyde slide at a site previously protected by wax,
following the pattern recorded on the photocopy, which was placed
underneath. A solution of NaBH3CN (0.5 L, 31 mM in PBS) was
added to the droplet on the slide and mixed by pipetting up and
down, without touching the glass surface. The remaining spots were
generated in the same manner. The slide was placed in a polypropy-
lene storage/transport chamber for individual microscope slides
(Merck Eurolab, Darmstadt, Germany), with H2O drops placed on
the ends of the slide to ensure humidity and avoid evaporation loss-
es. The reaction was allowed to proceed for 16 h at r.t. in the closed
chamber. Then, spots were removed with a paper towel, and the sur-
face washed in SSC/0.2% SDS for 2 min. After a brief H2O rinse,
the slide was spun dry as described above. Unreacted aldehyde
groups were then reacted with 6 in a reductive amination, performed
as described above, except that the entire surface of the slide was al-
lowed to react for 1 h. After removal of the reaction solution, soak-
ing in SSC/0.2% SDS for 2 min, and washing with filtered H2O (0.2
m pore size filter) to eliminate dust, the slide was spun dry.
Rt 34.3 min.
MALDI–TOF MS: m/z [M – H]– calcd for C149H193N53O90P14,
4599.8; found: 4597.6.
5 -chl-T*GGTTGACTGCGAT-DP-Lys (13)
Following standard DNA synthesis on support 1, except that the last
coupling step was performed with the phosphoramidite building
block of 5 -amino-5 -deoxythymidine,47 a mixture of cholic acid
(40.9 mg, 100 mol), HBTU (35.1 mg, 90 mol), and HOBT (15.5
mg, 100 mol) in DMF (600 L) was treated with DIEA (40 L,
233.6 mol), the solution was vortexed and pushed into a synthesis
cartridge containing the loaded CPG (15 mg) using two syringes.
After 1 h, the support was rinsed with DMF (6 mL) and CH3CN (6
mL), and dried at 0.1 Torr for 1 h. Deprotection and purification
were the same as described above.
Yield 36%.
HPLC gradient of 0% B for 5 min, to 30% B in 40 min, to 100% B
in 10 min.
Rt 44.7 min.
Hybridization.
MALDI–TOF MS: m/z [M – H]– calcd for C173H230N53O95P14,
A solution of fluorophore labeled DNA 16 (10 L, 10 M) in ten
fold concentrated SSC/0.2% SDS buffer was applied to the alde-
hyde slide and spread with a glass coverslip in the fashion typical
for microscope slides. Drops of H2O were applied on the edges of
the slide to prevent evaporation losses. The slide was transferred to
a plastic slide chamber and incubated for 16 h at r.t. (22 °C). After-
wards, the coverslip was removed while dipping the slide in a wash
solution of SSC/0.2% SDS. The slide, while in the wash solution (in
a 25 mL Erlenmeyer flask), was sonicated in a bath sonicator (USR
18 Qualilab, Merck Eurolab, Bruchsal, Germany) for 10 second per
washing step. The washing solutions were SSC/0.2% SDS, ten fold
diluted SSC/0.2% SDS, and ten fold diluted SSC without SDS. Af-
ter the final washing step, the slide was rinsed with filtered H2O and
spun dry. To determine the extent of hybridization, the slide was
scanned in the imager, using the filter setting for Cy3 provided with
the instrument at a resolution of 100 m. The intensity profiles were
5007.4; found: 5008.3.
5 -Cy3-ATCGCAGTCAACCA-3 (16)
The synthesis of this fluorophore labeled target sequence was per-
formed on standard CPG (Proligo) loaded with the N6-benzoyl-2 -
deoxyadenosine building block for the 3 -terminal residue using the
DNA synthesis conditions given above. For the labeling step, the
phosphoramidite of the dye Cy3 (Glen Research) was coupled with
an extended coupling time of 10 min. The trityl group protecting the
dye moiety was removed on the DNA synthesizer. Cleavage from
the support and purification were performed as given for the other
oligonucleotides.
Yield 38%.
HPLC gradient of 0% B for 5 min, to 30% B in 30 min, to 100% B
in 10 min.
Synthesis 2002, No. 6, 816–824 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIAL TOPIC
Oligonucleotides Arrays on Aldehyde Bearing Glass Slides
823
generated from scans using the public domain NIH Image program
developed at the U.S. National Institutes of Health.
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Supporting Information Available: NMR spectra of compounds
6 and 7, and MALDI–TOF mass spectra of compounds 13–16 (7
pages total).
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Acknowledgments
The authors wish to thank Siegfried Herzberger for providing syn-
thetic intermediates. Financial support for this work is provided by
Deutsche Forschungsgemeinschaft (grants No. RI 1063/1, RI 1063/
2, and FOR 434/1), and Fonds der Chemischen Industrie, which in
turn, is supported by BMBF (40%).
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Synthesis 2002, No. 6, 816–824 ISSN 0039-7881 © Thieme Stuttgart · New York