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Abstracts of Oak Ridge Posters |
(Ensemble Discovery, Cambridge, MA;
aaddress correspondence to this author at: Ensemble Discovery, 99 Erie Street, Cambridge, MA 02139; fax: 617 492 6689; e-mail lhaff{at}ensemblediscovery.com)
DNA-programmed chemistry (DPC) is a novel technology for synthesis of a wide variety of organic compounds at nanomolar concentrations under physiologic conditions (1). Annealing of small molecule-oligonucleotide precursors to DNA templates, each with an attached chemical moiety, can generate high amounts of effective molarities of these reactants. This phenomenon can enhance, by nearly 1-million-fold, the rate of a reaction, while increasing its specificity. We have developed a specific chemical process that generates a fluorescent product on annealing of 2 oligonucleotides to each other. Oligonucleotides containing a 3'-terminal azidocoumarin (AzC)1 and a 5'-terminal triphenylphosphine (TPP) will react in aqueous solution to form a fluorescent product, 7-aminocoumarin. The reaction rate is very slow at submicromolar concentrations of the single-stranded TPP and AzC groups in free solution. However, if these fluorophore precursor-containing oligonucleotides anneal to each other or to a common DNA target, as long as the 3' and 5' fluorophore precursors are annealed in close proximity, their localized high concentration supports their reaction to yield a fluorescent product. We have found that this principle can also be exploited for detection of proteins (and other high molecular weight analytes), with the advantages of a simple, homogeneous phase assay with potentially very low background.
We modified this architecture for detecting proteins and other non–nucleic-acid targets. The model target selected was platelet-derived growth factor (PDGF-BB), a protein that contains 2 identical B subunits, for which tight-binding aptamers have been previously identified.
We obtained human PDGF-BB and mouse monoclonal antihuman PDGF-BB from R&D systems. Except as indicated, all reaction and melting curve solutions contained, in a volume of 100 µL: 50 mmol/L Tris/HCl buffer at pH 8.0, 10 mmol/L magnesium chloride, 40 pmol of detection oligonucleotides, 40 pmol of PDGF-BB, and typically, 350 mL/L formamide by volume.
We determined melting curves by measuring fluorescence enhancement of SYBR Green I (Molecular Probes) dye binding to double-stranded DNA in a Bio-Rad iCycler under conditions similar to those described by Lipsky et al. (2). Melting curves were obtained in a 1000-fold dilution of SYBR Green dye, increasing temperature from 10 °C to 90 °C in 0.5 °C increments every 10 s. Melting temperature (Tm) was defined as the point of inflection of the first derivative of the melting curve, according to iCycler software. For fluorescence generation by TPP and AzC, the increase of fluorescence with time was monitored by incubation at 25 °C in a Wallac Victor 1420 luminometer, with excitation at 355 nm and emission at 460 nm.
We prepared TPP oligonucleotides from resin-bound 5'-amino oligonucleotides converted to their TPP derivatives (3) and azidocoumarin oligonucleotides by reaction of 3-amino oligonucleotides with the N-hydroxylsuccinimide ester of 7-azio-4-methylcoumarin-3-acetic acid (4). Oligonucleotides were purified by C18 chromatography. Within the oligonucleotide sequences, the anti-PDGF-B aptamer sequence (5) is underlined and reporter sequences are in italics. The spacer sequence between the aptamer and reporter sequences in all cases was CCCCCCCCCC. All sequences are indicated 5' to 3'.
Coumarin aptamer oligonucleotide: CAG GCT ACG GCA CGT AGA GCA TCA CCA TGA TCC TGC CCC CCC CCC ATA TTT AAG C-AzC;
TPP "matched" oligonucleotide: TPP-GCT TAA ATA TCC CCC CCC CCC AGG CTA CGG CAC GTA GAG CATCAC CAT GAT CCT G;
TPP "mismatched" oligonucleotide: TPP-TGG GAA TGG TGC CCC CCC CCC CAG GCT ACG GAC GTA GAGCAT CAC CAT-GAT CCT G.
We synthesized 3 oligonucleotides for this assay. The oligonucleotide containing the 3'-AzC group contained the 5' PDGF-BB aptamer sequence, a C10 spacer, and a 10-base reporter sequence with a 3' terminal AzC group. The TPP oligonucleotide contained the 3'-PDGF-BB aptamer sequence, a C10 spacer, and a 10-base 5' reporter sequence (complementary to the reporter sequence of the AzC oligonucleotide) containing the 5' TPP group. The 3rd oligonucleotide had the same architecture as the 2nd oligonucleotide, but the reporter sequence was mismatched to the AzC oligonucleotide.
Under experimental conditions of pH 8, 25 °C, 10 mmol/L magnesium chloride, and 350 mL formamide, the complementary reporter sequences of the oligonucleotides (measured Tm = 20 °C in 35% formamide) did not form a stable duplex (determined by SYBR Green melting curve analysis), and in the DPC-based assay, the fluorophore precursors on the oligonucleotide probes reacted very slowly in the absence of a target (Fig. 1
). When we added the target for the aptamers, PDGF-BB, the fluorescent product 7-aminocoumarin was generated, producing a fluorescent signal by the 2 aptamer-containing AzC and TPP oligonucleotide reporter molecules. Substitution of an aptamer-TPP oligonucleotide, in which the reporter sequences were mismatched to the AzC oligonucleotide, abolished the reaction (Fig. 1
), demonstrating the requirement that the reporter regions of the oligonucleotides be complementary to generate fluorescence. Omission of either of the oligonucleotides in the pair or the target prevented the reaction. Titration of the 2 matched oligonucleotides with each other revealed that the optimal ratio of AzC- to TPP-containing aptamer oligonucleotides was 1:1, and the optimal ratio of total oligonucleotides to PDGF-BB was 2:1, consistent with a mechanism in which the 2 different aptamer-containing oligonucleotides bind to a single molecule of PDGF-BB with 2 binding sites. (However, only half of the ternary complexes, on average, will contain the productive mixture of the 2 complementary pairs of TPP and AzC oligonucleotides, half will contain nonproductive mixtures of 2 TPP oligonucleotides or 2 AzC oligonucleotides.) Titration curves also confirmed that the reaction rate was linearly proportional to PDGF-BB concentration, up to the approximate point of PDGF-BB equivalence. In the presence of a greater than molar equivalence of PDGF-BB, the reaction rate actually decreased. This decrease was expected, because in an excess of target, fewer molecules will be in a complex bound to 2 aptamer oligonucleotides.
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Thermal melting studies revealed that the PDGF-BB binding event increased the Tm of the reporter regions of the complex by >30 °C.
The experimental system has also been tested in the presence of crude insect and mammalian cell lysates. These lysates failed to inhibit the reaction or to produce false positive rates of reaction, except, as expected, lysates prepared in the presence of strong reductants such as dithiothreitol produced false positive rates of reaction.
This homogeneous phase detection assay is based on the simultaneous binding of 2 aptamer-containing oligonucleotides, each linked to a complementary DNA reporter sequence containing 2 reactive fluorophore precursor molecules. These fluorophore precursors reacted to generate fluorescence only in the event of simultaneous binding of both oligonucleotides to a common target. The precursors reacted as a complex because of their localized high concentration and proximity to each other at the same end of a double-stranded reporter region. Because localized high concentration of the DNA leads to the shift of Tm, this region is double-stranded only when it binds to the target. This assay requires ambient assay temperatures slightly above the Tm of the duplex in the absence of target, as achieved by the use of relatively short reporter sequences, typically 8–12 bases long, and relatively low ionic strength buffer solutions, and the presence of formamide and/or increased temperatures. In our experience, the typical increase in Tm of this duplex upon target binding was 25–40 °C, which is consistent with the observed melting behavior of DNA oligonucleotides, in which each 10-fold increase in local concentration is expected to give a Tm increase of 7 °C (6).
Because signal generation depends on the simultaneous interaction of 2 binders with a common target in a ternary complex, we expected and observed increased avidity of binding. In a competitive assay with a free monomeric aptamer sequence, not linked to a reporter, a 25-molar excess of aptamer sequence was required to decrease the assay response 2-fold. This property of enhanced avidity is likely to be useful for low level detection of analytes for which strong binders are not available. We are currently using this technology with antibody-oligonucleotide DPC detection reagents against targets with homo- and heterodimeric binding sites. In the case of protein targets with more than 1 epitope, bispecific aptamer or antibody-based DPC detection reagents are expected to exhibit not only increased avidity but also greatly enhanced specificity for their targets. The response of the experimental system to targets added to crude mammalian and insect cell lysates appears unaffected, although high concentrations of reducing agents such as dithiothreitol produce positive interferences. In addition, we are exploring the use of this technology to synthesize novel fluorophores in addition to 7-amino coumarin. We are also investigating the synthesis of novel fluorophores with desired properties, such as lower background fluorescence of the fluorophore precursors and near-infrared dyes.
References
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, sample contains both probes and 0.4 µmol/L PDGF-BB. 
, sample contains only both probes. •, sample contains AzC and mismatched TPP probe and PDGF-BB.