Reactivity-dependent PCR: Direct, solution-phase in vitro selection for bond formation

Article abstract of DOI:10.1021/ja903084a

(Figure Presented) In vitro selection is a key component of efforts to discover functional nucleic acids and small molecules from libraries of DNA, RNA, and DNA-encoded small molecules. Such selections have been widely used to evolve RNA and DNA catalysts

Full text of DOI:10.1021/ja903084a

Published on Web 06/12/2009
Reactivity-Dependent PCR: Direct, Solution-Phase in Vitro Selection for Bond
Formation
David J. Gorin, Adam S. Kamlet, and David R. Liu*
Howard Hughes Medical Institute and Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138
Received April 17, 2009; E-mail: drliu@fas.harvard.edu
In Vitro selection is a key component of efforts to discover
functional nucleic acids and small molecules from libraries of DNA,
RNA, and DNA-encoded small molecules.1 When the desired
activity is binding affinity, as is the case for aptamer evolution2 or
for the discovery of DNA-linked small molecules that bind to
protein targets,3 a direct selection is possible; the library is typically
incubated with immobilized target molecules, and bound library
members are washed and eluted before being subjected to PCR
amplification (Figure 1a).
intermolecular hybridization of two DNA strands of the same
sequence (2 and 3) is predicted to be far less favorable, with a Tm
of only 11 °C (Figure 2). We hypothesized that the significant
difference in intramolecular versus intermolecular duplex stability
could enable a new type of in Vitro selection, wherein bond
formation or bond cleavage is transduced into the formation of a
self-priming DNA hairpin. This hairpin enables the selective PCR
amplification of those DNA sequences that encode the reactive
species (Figure 2b).12
Figure 1. Traditional approaches to in Vitro selection.
Figure 2. Principles underlying reactivity-dependent PCR (RDPCR).
Conditions in (a): 10 nM DNA, 2 mM Mg2+, 100 mM NaCl.
In Vitro selections have also been applied to evolve RNA and
DNA catalysts4 and, more recently, to discover new reactions from
DNA-encoded libraries of potential substrates.5 For these applica-
tions, desired library members undergo bond formation or bond
cleavage. Selections for reactivity are significantly more complica-
ted than selections for binding affinity. Typically, libraries are
incubated with biotinylated substrates or potential reaction partners.
Bond formation results in the attachment of biotin to a library
member, which in turn enables its capture by immobilized avidin
(Figure 1b).6 For bond cleavage, an inverse approach is commonly
used in which immobilized, biotinylated library members liberate
themselves upon bond scission.7 While effective, such selections
for reactivity are indirect, require the synthesis of biotin-linked
substrates, and involve multiple solution-phase and solid-phase
manipulations.
We first assessed the ability of intramolecular self-priming to
result in preferential DNA amplification. We synthesized a series
of oligonucleotide pairs (4a+5), each predicted to hybridize
intermolecularly at their 3′ ends to form a short duplex region of
8 or 10 bp (Figure 3). The 5′ end of each DNA oligonucleotide in
the pair contained a sequence identical to either primer 6 or primer
7. PCR amplification cannot occur until after DNA hybridization
and 3′ extension take place to generate a single-stranded DNA
molecule containing both primer 6 and a sequence complementary
to primer 7. This 3′-extended species can then hybridize with primer
7 and initiate PCR amplification.
Motivated by our ongoing interest in applying in Vitro selection
to discovery problems that involve chemical reactivity,8 we sought
to develop a new method that more directly links bond formation
or bond cleavage with the amplification of desired sequences9 and
that obviates the need for solid-phase capture, washing, and elution
steps. Here we report the development and validation of such a
method, reactivity-dependent PCR (RDPCR).
RDPCR is based on the well-established observation that the
melting temperatures (Tm) of double-stranded nucleic acids are
substantially higher when hybridization occurs intramolecularly as
opposed to intermolecularly.10 For example, the DNA hairpin 1
with an 8 bp stem is predicted11 to exhibit a Tm of 48 °C, while the
Figure 3. Comparison of PCR efficiency of intramolecularly primed versus
intermolecularly primed DNA templates. PCR conditions for PAGE samples:
19 fmol of 8 or 19 fmol of 4a+5 in 30 µL, 25 cycles.
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We also prepared an analogous series of DNA oligonucleotides
capable of hybridizing intramolecularly to form hairpin structures
with 10-, 8-, or 6-bp stems (8a-8c). As with the intermolecularly
hybridizing oligonucleotides, PCR amplification must be initiated
by primer extension of the 3′ end. Quantitative, real-time PCR13
(qPCR) was used to compare the ability of these oligonucleotides
to undergo PCR amplification.
Consistent with our initial hypothesis, under identical PCR
conditions and with equal starting concentrations of DNA, the
intramolecularly hybridizing templates were amplified much more
efficiently than their intermolecular counterparts (8a vs 4a+5a, 8b
vs 4a+5b). The intramolecularly hybridizing templates reached a
threshold level of amplified product 13 to 15 PCR cycles (CT) earlier
than the intermolecular templates, corresponding to a > 213-fold
(>8000-fold) difference in effective initial template abundance.
These qPCR results were corroborated by PAGE analysis; after 25
cycles of PCR, amplified product was only detected in reactions
containing hairpin DNA. Collectively, these findings demonstrate
that intramolecularly hybridizing templates can be amplified to
abundant levels under conditions that fail to appreciably amplify
the corresponding intermolecularly hybridizing templates. Subse-
quent experiments in this work were carried out with an 8-base
stem, which was found to optimally balance robust intramolecular
priming and poor intermolecular priming (see Supporting Informa-
tion).
To use RDPCR in a general selection for bond formation, the
covalently linked functional groups in the hairpin loop must not
interfere with the required hybridization and 3′-extension events.
To test the compatibility of non-natural linkers with the preferential
amplification of self-priming templates, we repeated the qPCR
experiment in Figure 3 with a series of non-natural linker structures,
including ether, disulfide, and amide hairpin linkers. In all cases
tested, DNA templates containing non-natural linkers (9a-9c) were
far more efficiently amplified than the analogous intermolecularly
hybridizing templates (4+10) (Figure 4).
Figure 5. Selectivity of RDPCR in a library-format mock selection. PCR
conditions: 19 fmol of 12 and 13 in 60 µL, 25-35 cycles.
10- to 1000-fold excess of 12 and 13) overwhelmingly provided
the desired product. These results demonstrate the ability of hairpin
templates to be preferentially amplified even in the presence of
large excesses of linear templates and indicate that RDPCR can be
applied to library-format selections.14
Following these foundational studies, RDPCR was applied to
two model in Vitro selections. First, RDPCR was validated as a
bond-formation selection for DNA-encoded reaction discovery.
Previously, DNA-encoded reaction discovery required the capture,
washing, and elution of active library members on avidin-linked
beads (Figure 1b).5 In a RDPCR version of reaction discovery, pairs
of functional groups are attached to encoding DNA strands (Figure
6). A disulfide linker temporarily joins each substrate pair (9b, 9d).
Exposure to a set of reaction conditions and subsequent cleavage
of the disulfide bond provide one of two possible outcomes. If a
new covalent bond has formed between the functional groups, the
hairpin-forming nucleotides remain tethered together through the
reaction product, leaving a self-priming DNA hairpin (14). If no
bond has formed, then only intermolecular hybridization is possible
(15+16), resulting in inefficient PCR amplification. In contrast with
previous reaction discovery selections, the RDPCR version requires
no solid-phase steps and minimal manipulation.
Figure 4. Non-natural hairpin linkers support self-priming PCR. R )
(CH2)6OH.
Figure 6. RDPCR-based DNA-encoded reaction discovery selection. PCR
conditions for PAGE samples: 1 fmol of 9 in 20 µL, 23 cycles.
Since RDPCR will ultimately be applied to mixtures of both
active (resulting in DNA hairpins) and inactive (resulting in separate
linear oligonucleotides) library members, we next tested the ability
of hairpin templates to undergo preferential amplification in the
presence of large excesses of corresponding linear molecules (Figure
5). For these library-format experiments, the hairpin (11) and linear
(12) template sequences vary only by a single base, such that DNA
amplified from the hairpin contains a cleavage site for the restriction
enzyme HindIII, while DNA arising from 12 does not.
The quantity of 11 spiked into an equimolar mixture of 12 and
13 was varied to determine the selectivity of RDPCR in a library
format. As little as 1 attomol of 11 (600 000 molecules) could be
selectively amplified in the presence of a 10 000-fold excess of 12
and 13. The use of larger quantities of hairpin (corresponding to a
To test the ability of RDPCR to support reaction discovery, we
synthesized a disulfide-linked substrate (9d) with pendant alkene
and aryl iodide groups, which should undergo a Pd-mediated Heck-
type reaction (Figure 6).15 An unreactive control substrate (9b)
containing an azide and an aldehyde was similarly generated. Each
substrate was treated with 1 mM Na2PdCl4 (which is reduced to
Pd(0) in situ) in aqueous pH 7.5 buffer for 30 min at 65 °C, followed
by DTT to cleave the disulfide bond. The resulting material was
subjected to PCR. The DNA attached to the alkene-aryl iodide pair
(9d) amplified efficiently (Figure 6). Omission of Na2PdCl4 resulted
in much less efficient amplification. Likewise, the unreactive
substrate pair 9b did not undergo PCR amplification after identical
treatment. Omission of DTT, however, enabled the disulfide-linked
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starting substrate to amplify efficiently. Collectively, these results
indicate that RDPCR can selectively and efficiently amplify DNA
templates that have undergone bond formation and that amplification
is dependent on the intramolecularity of the resulting template-
primer species.16 These experiments were corroborated using an
azide/alkyne substrate that undergoes a Cu(I)-catalyzed cycload-
dition reaction (see Supporting Information).
In principle, RDPCR can also enable efficient selection for bond
cleavage, which has yet to be studied in a DNA-encoded context.
To explore this possibility, we evaluated the ability of DNA-linked
peptides to undergo cleavage mediated by a protease (Figure 7).
We anticipated that protease-mediated cleavage of a DNA-peptide
conjugate would expose a primary amine group, which would then
undergo DNA-templated amide bond formation to generate a hairpin
template for efficient PCR.17 In contrast, the absence of proteolysis
should result in no amide formation and thus inefficient PCR
amplification.
that catalyze bond-forming or bond-cleaving reactions including
those that do not generate nucleic acid-like products.
Acknowledgment. This work was supported by the Howard
Hughes Medical Institute and the NIH/NIGMS (R01GM065865).
D.J.G. gratefully acknowledges a joint American Cancer Society-
Canary Foundation postdoctoral fellowship and research support
from the Kavli Foundation. We thank Dr. Yinghua Shen for
assistance with mass spectrometry.
Supporting Information Available: Experimental procedures and
compound characterization data. This material is available free of
charges at http://pubs.acs.orgThis material is available free of charge
via the Internet at http://pubs.acs.org.
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Figure 7. RDPCR-based protease-mediated peptide cleavage selection. PCR
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small scale.
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A DNA-N-acetyl-pentapeptide conjugate (17), synthesized by
solid phase cosynthesis, was exposed to subtilisin A. The peptide
sequence (Ac-N-AFGPA) was designed to include cleavage sites
for subtilisin A.18 The enzyme-treated DNA was combined with a
carboxylic acid-linked DNA primer (10c) under conditions (DMT-
MM or sNHS+EDC) that support DNA-templated amide bond
formation.19
Addition of the protease-digested and carboxylate-ligated
DNA-peptide conjugate (18) to a PCR reaction resulted in efficient
PCR amplification. In contrast, no PCR product was detected by
PAGE when unfunctionalized DNA (4a) was used in place of the
pentapeptide or when subtilisin A was omitted. Likewise, omission
of the amide formation reagents also resulted in inefficient PCR
amplification, consistent with the necessity of intramolecular primer
hybridization for rapid amplification. These findings together
demonstrate the ability of RDPCR to rapidly detect DNA-linked
peptide substrates of protease enzymes.
In conclusion, we have developed and validated RDPCR as a
new, entirely solution-phase method for the selective amplification
of DNA sequences encoding molecules that undergo bond formation
or bond cleavage.9 By obviating the need to perform solid-phase
capture, washing, and elution steps, RDPCR can greatly streamline
the selection process for applications such as DNA-encoded reaction
discovery and protease activity profiling. Compared with the
performance characteristics of previous in Vitro selection methods,3c,5a
the data above suggest that RDPCR may also offer superior
enrichment factors (signal:background ratios). In addition, RDPCR
may be applicable to the evolution of ribozymes and DNAzymes
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