DNA analysis by dynamic chemistry

Article abstract of DOI:10.1002/anie.200905699

Finding flaws: An enzyme-free method of DNA analysis raises the possibility of analyzing slngle-nucleotlde polymorphism, indel, and abaslc sites using mass spectrometry as a readout tool. The methodology Is suitable for the dual analysis of heterozygous samples. (Figure Presented)

Full text of DOI:10.1002/anie.200905699

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Chemie
DOI: 10.1002/anie.200905699
DNA Analysis
DNA Analysis by Dynamic Chemistry**
Frank R. Bowler, Juan J. Diaz-Mochon,* Michael D. Swift, and Mark Bradley*
A single-nucleotide polymorphism (SNP) is a
genetic variation for which two or more alter-
native alleles are present at appreciable frequency
in the human population.[1] Methods for SNP
analysis[2] are multifarious, but typically rely on an
enzymatic primer extension[2b] with fluorescence
and mass spectrometric (MS) detection. Several
nonenzymatic methods of DNA analysis have
been reported. One approach is based on the
differential melting temperatures of allele-spe-
cific probes[2c] while another has been to use DNA
mimics such as peptide nucleic acids (PNAs)[3] in a
number of ligation-based chemical approaches,
most notably in the elegant work of Seitz et al.[4]
Nonenzymatic ligation has also been achieved in a
DNA–DNA sense by Kool et al.,[5] who ligated
DNA strands containing a 3’-phosphorothioate
with a 5’-iodothymidine, and Richert et al.,[6] who Scheme 1. Dynamic chemistry applied to SNP analysis.
reacted nucleotides possessing an activated phos-
phate with a DNA strand containing a free 3’-
amino group. DNA-templated dynamic chemistry has
attracted interest for the preparation of stimuli-responsive
polymers and for gaining insight into the chemistry of
primordial self-replicating systems.[7] Most recently, Liu and
Heemstra have reported PNA-templated base-filling reac-
tions on PNA strands.[7h]
complementary iminium nucleobase. Subsequent reduction
and MALDI-TOF mass spectrometry would allow rapid
determination of base incorporation.
The first question that arises relates to the degree of
selection achievable through this dynamic approach. This was
addressed by the synthesis of the 15-mer PNA 1 with a single
“blank” position (Table 1 and Figure 1), complementary to
Herein we report the application of dynamic chemistry[8]
to DNA analysis, offering the prospect of nonenzymatic
genotyping of genomic DNA amplified by polymerase chain
reaction (PCR). This was achieved by the synthesis of a PNA
strand that contained a “blank” position opposite the
nucleobase under analysis in a complementary DNA tem-
plate. A reversible reaction, between this PNA/DNA duplex
(specifically the secondary amine of the “PNA blank”) and
four aldehyde-modifed nucleobases (Scheme 1), means that
the templating power of Watson–Crick base pairing and base
stacking[9] would be expected to drive the selection of the fully
Table 1: PNA sequences used for DNA analysis.
PNA oligomer
Sequence (N–C)[a,b]
1
Ac-TAC TAC ATC _CT TCC
2[c]
3[c]
4[c]
5
phosphonium-PEG-GTG GAG _TC AAC GA
phosphonium-PEG-GTG GAG _ _C AAC GA
phosphonium-PEG-GTG GAG _ _ _ AAC GA
phosphonium-CT TTC CT _ CAC TGT
phosphonium-TC GTT GA _ CTC CAC
6
[a] _ Represents a blank site (see Figure 1). [b] All PNA oligomers were
synthesized by solid-phase synthesis and had a C-terminal primary
amide. [c] See the Supporting Information for structures of the
phosphonium-polyethylene glycol (-PEG) units.
[*] F. R. Bowler, Dr. J. J. Diaz-Mochon, Dr. M. D. Swift, Prof. M. Bradley
School of Chemistry, University of Edinburgh
EH9 3JJ, Edinburgh (UK)
Fax: (+44)131-650-4820
E-mail: jj.diaz@ed.ac.uk
mark.bradley@ed.ac.uk
Homepage: http://www.combichem.co.uk
four 21-mer DNA templates I–IV (see Table 2). Treatment of
PNA 1 with one of the complementary DNA oligomers and
equimolar amounts of the four nucleobase aldehydes T, C, A,
and G (Figure 1), followed by reduction, addition of Q Se-
pharose[10] and MALDI-TOF MS analysis (see Figure 2 for
representative spectra), demonstrated highly selective incor-
poration of the nucleobase complementary to the SNP
position on the DNA template (see Table 3). As anticipated
for iminium ion formation, conversions were optimal at
mildly acidic pH (i.e. 5 ꢀ pH ꢀ 7; see the Supporting Infor-
[**] This concept was first described in a UK patent entitled “Nucleo-
base Characterisation” GB 0718255.3 filed on September 19, 2007
by Juan J. Diaz-Mochon and Mark Bradley (University of Edinburgh)
and published on March 26, 2008 (WO/2009/037473). This project
is funded by Scottish Enterprise. We are grateful to Dr. K. Finlayson
and Dr. Ann-Marie Stannard for their continuing support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200905699.
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or thymine (attributed to the greater number of
templating hydrogen bonds). Furthermore,
purine bases were incorporated with greater
selectivity than pyrimidines (A > T, G > C by
approximately twofold). The selectivity of the
incorporation could be further improved by
altering the starting ratio of the bases (see
Figure S15 and Table S1 in the Supporting Infor-
mation). The reversibility of the nucleobase
incorporation (prior to reduction) was demon-
strated by analyzing the reaction of PNA/DNA
(1/IV). In the absence of G small levels of
misprimed incorporation were detected after
reduction; however, when G was added to the
reaction mixture (immediately before reduction)
the removal of virtually all misprimed binding
Figure 1. Top: The four aldehyde-modified nucleobases (T, C, A, and G) and
1-pyreneacetaldehyde (P). Bottom: General structure of a modified “blank” PNA
resulted, showing the reversibility of the selection
strand.
process (see Figures S22 and S23 in the Support-
Table 2: DNA oligomers subjected to analysis.
ing Information).
Sequence (5’–3’)[a,b]
To serve as a technique for SNP analysis, any
DNA oligomer
approach must permit the genotyping of heterozygous
individuals who possess two different alleles of a particular
gene. Heterozygotes present a greater challenge than homo-
zygotes, as the signals associated with each allele should
ideally be detected with approximately equal intensity to
facilitate confident genotyping. To allow “heterozygous” SNP
analysis, the relative concentrations of the four aldehyde
monomers were altered to normalize the selection ratio
between the bases (see Figure S24 in the Supporting Infor-
mation for a representative spectrum).
SNPs are important in determining the severity of cystic
fibrosis (CF), a life-threatening inherited disease. DNA
oligomers representing CF-linked SNPs (W1282X and
G551D; see Table 2) were analyzed[11] using PNAs 5 and 6,
respectively, which employed a triphenylphosphonium tag[10]
that improved the detection limit of MALDI-TOF MS by an
order of magnitude (compared to PNA 1; see the Supporting
Information). The resulting mass spectra permitted confident
“calling” of the homo- and heterozygous models for both
SNPs (see Figures S25–S30 in the Supporting Information).
Simultaneous analysis was also performed by combining all
four DNA strands (VI–IX) and profiling with PNAs 5 and 6,
thereby modeling the situation for an individual heterozygous
at both SNP locations. The resulting mass spectra showed the
highly selective incorporation of the expected nucleobases
(Figure 3).
I
II
III
IV
TTT TTT GGA AGG GAT GTA GTA
TTT TTT GGA AGA GAT GTA GTA
TTT TTT GGA AGT GAT GTA GTA
TTT TTT GGA AGC GAT GTA GTA
TCG TTG ACC TCC AC
V
VI wt codon 551
VII (G551D mutant)
VIII wt codon 1282
IX (W1282X mutant)
X abasic
GTG GAG GTC AAC GA
GTG GAG ATC AAC GA
ACA GTG GAG GAA AG
ACA GTG AAG GAA AG
GTG GAG ZTC AAC GA
[a] DNA/PNA hybridize with the 3’-end of the DNA matched to the
N terminus of the PNA. [b] Nucleobase subjected to analysis is in bold.
Z=abasic site.
The dynamic incorporation of nucleobases into multiple
consecutive “blanks” was explored using PNAs 2–4 (Table 1)
with DNA V (Table 2). The resulting mass spectra showed
selective incorporation of the correct bases in all cases (see
Figures S32–S34 in the Supporting Information). These
results offer the possibility of indel (insertion and deletion
of one or more nucleobases within a DNA strand) analysis.[12]
Abasic sugars are found naturally in the genome as a
result of spontaneous lesions, or chemical or physical
damage.[13] Kool and Matray observed that pyrene nucleoside
triphosphate (dPTP) could be enzymatically incorporated
opposite a templating abasic site.[14] A DNA template X
containing an abasic site (see Table 2 and Scheme 2) was
Figure 2. Mass spectra recorded after DNA-templated reductive amina-
tions using an unoptimized equimolar ratio of the four aldehydes.
a) DNA template I directs incorporation of C, b) DNA template II
directs incorporation of T, c) DNA template III directs incorporation of
A, and d) DNA template IV directs incorporation of G. I=percentage
intensity.
mation for details). Under these conditions guanine and
cytosine were found to be incorporated more efficiently and
(approximately fivefold) more selectively than either adenine
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Table 3: MALDI signal ratios for nucleobase incorporation (all ratios
reported to the nearest integer).
the selective incorporation of the specific nucleobase alde-
hyde. Analysis with the addition of P (Scheme 2) gave clean
incorporation of only P (see Figure S36 in the Supporting
Information).
DNA oligomer
Templating base
MALDI signal ratios[a]
C/T/A/G
Dynamic chemistry has thus been developed as an
effective method of DNA analysis, demonstrating high base
selectivity and the potential for enzyme-free SNP genotyping
of PCR-amplified DNA. MALD-TOF MS enabled the dual
analysis of “heterozygous” samples. Variations in base
selectivity were attributed primarily to the number of hydro-
gen bonds templating the incorporation reaction; G and C
were incorporated approximately fivefold more selectively
than A and T. Within these subsets, purine bases were
incorporated around two times more selectively than the
pyrimidines (i.e. A > T, G > C); this is attributed to differ-
ences in p-stacking interactions. The approach also raises the
possibility of analyzing other sources of genetic variation and
mutation, such as indels and abasic sites, in a manner simply
not possible with current approaches. Moreover, it offers an
approach to identify nucleobase mimics to expand the genetic
alphabet.[15] The approach has also demonstrated the tem-
plating role of DNA in promoting selective nucleobase
incorporation.
I
II
III
IV
G
A
T
19:1:1:1
1:4:1:1
1:1:8:1
1:1:1:39
C
[a] Based upon relative intensities of most common isotope. The
nucleobase complementary to the position under interrogation on the
DNA template is in bold.
Experimental Section
Figure 3. Profiling of CF-relevant sequences with oligonucleotides
VI–IX and PNAs 5 and 6, illustrating the potential for dual analysis of
SNPs by dynamic chemistry. In this case the ratio of PNAs 5 and 6
was 10:6 to allow approximately equal product peak intensities (see
Figure S31 in the Supporting Information). I=percentage intensity.
Typical protocol for DNA-templated reductive aminations: A PNA
blank (2.5 mL, 40 mm aq.), DNA template (1 mL, 100 mm aq.),
aldehydes A, G, C, and T (1.6 mL of each, 1.7 mm aq.), and pH 6
phosphate buffer (8.1 mL, 10 mm aq.) were combined in a 1.5 mL
Eppendorf tube and placed in an Eppendorf Thermomixer comfort at
808C and 1200 rpm for 5 min. The reaction mixture was then cooled
to 408C (at 38CminÀ1) before NaBH3CN (2 mL, 1m aq.) was added
and shaking continued for 1 h. Pre-equilibrated Q Sepharose Fast
Flow (5 mL, see the Supporting Information) was added before the
reaction mixture was agitated at room temperature for 20 min. The
reaction tube was centrifuged and the supernatant removed, then the
Q Sepharose was washed centrifugally with 3% MeCN in water (3 ꢀ
200 mL). Sinapinic acid matrix (10 mL) was added to the resin, and this
mixture was spotted (1 mL in duplicate) onto a stainless steel MALDI
plate. Reactions were performed in duplicate, and five MALDI
spectra acquired for each. Spectra are presented
therefore analyzed by dynamic incorporation with PNA 6 and
aldehydes T, C, G, and A with and without the pyrene base
analogue 1-pyreneacetaldehyde P (Figure 1). In the absence
of P this yielded minimal base-incorporation products (see
Figure S35 in the Supporting Information). The major signal
corresponded to starting material, demonstrating the role of
the complementary base of the DNA template in promoting
unprocessed. Relative peak intensities were
determined for the most common isotopes of
the PNA-incorporation products. Product signal
ratios were determined by averaging over the ten
spectra. A control reaction was performed with-
out DNA (see Figure S37 in the Supporting
Information).
Received: October 9, 2009
Revised: December 3, 2009
Published online: February 12, 2010
Keywords: DNA analysis ·
.
dynamic chemistry · genotyping ·
peptide nucleic acids ·
single-nucleotide polymorphisms
Scheme 2. Analysis of abasic DNA without (top) and with (bottom) 1-pyreneacetaldehyde,
P. a) Hybridization, b) dynamic reversible incorporation, c) reduction, and d) MS detection.
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