Proximity extension of circular DNA aptamers with real-time protein detection

Giusto, Daniel A. Di; Wlassoff, Wjatschesslaw A.; Gooding, J. Justin; Messerle, Barbara A.; King, Garry C.

doi:10.1093/nar/gni063

Cited by 174 publications

(151 citation statements)

References 53 publications

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“…(39)(40)(41)(42) As mentioned earlier, the 50nt ssDNA aptamer construct employed in this study can be amplified via RCA. Although less sensitive than PCR, the RCA technique is convenient as it can be performed at a constant temperature, e.g.…”

Section: Rca Detectionmentioning

confidence: 93%

Protein detection via direct enzymatic amplification of short DNA aptamers

Fischer

Tarasow

Tok

2008

Analytical Biochemistry

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Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM).

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Section: Rca Detectionmentioning

confidence: 93%

Protein detection via direct enzymatic amplification of short DNA aptamers

Fischer

Tarasow

Tok

2008

Analytical Biochemistry

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“…46,[52][53][54][55] Consequently, the merging of aptamer and microarray technologies to create bioaffinity sensors for the simultaneous detection of multiple protein or biomarker targets from a biological sample is still at a very early stage.…”

Section: Iii1 Nucleic Acid Aptamer Microarraysmentioning

confidence: 99%

Microarray methods for protein biomarker detection

Lee

Wark

Corn

2008

Analyst

137

108

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The application of protein biomarkers as an aid for the detection and treatment of diseases has been subject to intensified interest in recent years. The quantitative assaying of protein biomarkers in easily obtainable biological fluids such as serum and urine offers the opportunity to improve patient care via earlier and more accurate diagnoses in a convenient, non-invasive manner as well as providing a potential route towards more individually targeted treatment. Essential to achieving progress in biomarker technology is the ability to screen large numbers of proteins simultaneously in a single experiment with high sensitivity and selectivity. In this article, we highlight recent progress in the use of microarrays for high-throughput biomarker profiling and discuss some of the challenges associated with these efforts. I IntroductionThe discovery of protein biomarkers whose change in expression level or state correlates with the progression of a disease is becoming increasingly important. Once validated, proposed biomarkers can be involved in achieving an earlier diagnosis, differentiating between disease types with greater accuracy, and assessing response to treatment. Prominent examples of biomarkers include proteins that are associated with a variety of cancers, 1-4 as well as heart, 5,6 renal, 7,8 andAlzheimer's 9,10 diseases.Most biomarker studies reported to date have focused on serum-, plasmaand urine-based samples. A number of other possibilities include saliva, tears, breath condensates, cerebrospinal fluid and tissue lysates extracted from biopsy samples. There are several excellent reviews detailing biomarker discovery and validation. [11][12][13][14][15] The potential benefits of biomarkers have greatly motivated both academic and industry researchers to apply new proteomic technologies for biomarker discovery and to develop quantitative analytical methodologies for rapid and sensitive biomarker detection. Many clinically relevant biomarkers reside in blood at picomolar concentrations and lower, which is five to seven orders of magnitude lower than the most abundant plasma proteins. Therefore, protein detection with high specificity and sensitivity is required; unfortunately, no universal ultrasensitive enzymatic amplification method (such as PCR for the case of nucleic acid detection) exists for proteins. Additionally, it is desirable to screen multiple proteins simultaneously in a single sample. Multiplexed measurements are attractive not only for economic reasons but also for identifying characteristic signal patterns associated with the relative changes in entire sets of proteins as this will provide much more insight and diagnostic accuracy than individual biomarker measurements.hyejinlee@knu.ac.kr. 14,19,[24][25][26][27][28][29][30] Although microarray methods are wellestablished for high-throughput nucleic acid studies, their application for protein detection has been limited by issues such as the surface immobilization of protein capture probes without loss in bioactivity as well a...

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“…When the anti-thrombin aptamer was adapted to the equivalent of an ELISA the limit of detection was <1nM [42]. The best results have been garnered by other real-time methods: a detection limit of 10 pM in the presence of serum with real-time PCR [11], a detection limit of 30pM by proximity rolling circle amplification [6], and down to several hundred of molecules by exonuclease protection coupled with real-time PCR [5].…”

Section: Thrombin-mediated Ligation Followed By Real-time Pcrmentioning

confidence: 99%

Real-time PCR detection of protein analytes with conformation-switching aptamers

Yang

Ellington

2008

Analytical Biochemistry

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We have developed a novel method that utilizes conformation-switching aptamers for real-time PCR analysis of protein analytes. The aptamers have been designed so that they assume one secondary structure in the absence of a protein analyte, and a different secondary structure in the presence of a protein such as thrombin or PDGF. The protein-bound structure in turn assembles a ligation junction for the addition of a real-time PCR primer. Protein concentrations could be specifically detected into the picomolar range, even in the presence of cell lysates. The method has advantages relative to both immunoPCR (since no signal is produced by background binding) and to the proximity ligation assay (PLA; since only one epitope on a protein surface must be bound, rather than two).

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Proximity extension of circular DNA aptamers with real-time protein detection

Cited by 174 publications

References 53 publications

Protein detection via direct enzymatic amplification of short DNA aptamers

Protein detection via direct enzymatic amplification of short DNA aptamers

Microarray methods for protein biomarker detection

Real-time PCR detection of protein analytes with conformation-switching aptamers

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