Development of aptamers specific for potential diagnostic targets in Burkholderia pseudomallei

Gnanam, Annie J.; Hall, Bradley; Shen, Xiaofan; Piasecki, Shawn K.; Vernados, Anna; Galyov, Edouard E.; Smither, Sophie J.; Kitto, G. Barrie; Titball, Richard W.; Ellington, Andrew D.; Brown, Katherine A.

doi:10.1016/s0035-9203(08)70015-4

Cited by 13 publications

(10 citation statements)

References 4 publications

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“…Hence, the need of an improved diagnostic reagent is vital for early detection. For this purpose, high affinity aptamers were selected against recombinantly expressed B. pseudomallei specific proteins: a putative oxidoreductase BPSL2748 and secretion pathway proteins (BipD and BopE type III), which are considered to be potential targets for applications in diagnostic assays for B. pseudomalle (Gnanam et al 2008).…”

Section: Bacteriamentioning

confidence: 99%

Application of aptamers in diagnostics, drug-delivery and imaging

et al. 2016

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Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their target with high specificity and affinity. Although aptamers are analogous to antibodies for a wide range of target recognition and variety of applications, they have significant advantages over antibodies. Since aptamers have recently emerged as a class of biomolecules with an application in a wide array of fields, we need to summarize the latest developments herein. In this review we will discuss about the latest developments in using aptamers in diagnostics, drug delivery and imaging. We begin with diagnostics, discussing the application of aptamers for the detection of infective agents itself, antigens/ toxins (bacteria), biomarkers (cancer), or a combination. The ease of conjugation and labelling of aptamers makes them a potential tool for diagnostics. Also, due to the reduced off-target effects of aptamers, their use as a potential drug delivery tool is emerging rapidly. Hence, we discuss their use in targeted delivery in conjugation with siRNAs, nanoparticles, liposomes, drugs and antibodies. Finally, we discuss about the conjugation strategies applicable for RNA and DNA aptamers for imaging. Their stability and self-assembly after heating makes them superior over protein-based binding molecules in terms of labelling and conjugation strategies.

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Section: Bacteriamentioning

confidence: 99%

Application of aptamers in diagnostics, drug-delivery and imaging

et al. 2016

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“…DNA and RNA aptamers [1] , [2] , [3] , [4] have affinities for their targets similar to antibodies [5] and are useful in biosensors [6] , [7] , [8] , [9] , diagnostics [5] , [10] , [11] and therapeutics [12] , [13] , [14] . Unlike antibodies, aptamers are not limited to binding immunogenic epitopes but have been reported to specifically bind an array of small organic molecules, macromolecules, and cells [3] , [4] , [9] , [15] .…”

Section: Introductionmentioning

confidence: 99%

Acyclic Identification of Aptamers for Human alpha-Thrombin Using Over-Represented Libraries and Deep Sequencing

et al. 2011

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BackgroundAptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution.Methodology/Principal FindingsWe show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2–6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts), and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (Kd = 12 nM for Thb1). The signal-to-noise ratio for this experiment was roughly 10,000∶1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts) being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (Kd = 0.5 µM for α-methyl-mannoside); ConA was used to immobilize α-thrombin.Conclusions/SignificanceOver-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets.

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“…15,17 Protein concentrations were determined using the BCA kit (Pierce) using bovine serum albumin as a standard, and sample purity assessed by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

“…9 Therefore we have adopted a strategy which involves the development of high affinity reagents, aptamers and antibodies, to detect surface-associated or secreted macromolecules from Burkholderia species. 15,16 Such reagents would have the flexibility to be used in both rapid readout assays to address clinical needs in endemic areas (e.g., rural Southeast Asia), as well as in more sophisticated high throughput multiplexed platforms for security-based applications. In this paper we describe our current progress in diagnostic target selection and production, including the generation of an engineered Fab directed against a B. mallei protein, BimA.…”

Section: Introductionmentioning

confidence: 99%

Development of reagents and assays for the detection of pathogenic Burkholderia species

et al. 2011

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Rapid detection of the category B biothreat agents Burkholderia pseudomallei and Burkholderia mallei in acute infections is critical to ensure that appropriate treatment is administered quickly to reduce an otherwise high probability of mortality (ca. 40% for B. pseudomallei). We are developing assays that can be used in clinical laboratories or security applications for the direct detection of surface-localized and secreted macromolecules produced by these organisms. We present our current medium-throughout approach for target selection and production of Burkholderia macromolecules and describe the generation of a Fab molecule targeted to the B. mallei BimA protein. We also present development of prototype assays for detecting Burkholderia species using anti-lipopolysaccharide antibodies.

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Development of aptamers specific for potential diagnostic targets in Burkholderia pseudomallei

Cited by 13 publications

References 4 publications

Application of aptamers in diagnostics, drug-delivery and imaging

Application of aptamers in diagnostics, drug-delivery and imaging

Acyclic Identification of Aptamers for Human alpha-Thrombin Using Over-Represented Libraries and Deep Sequencing

Development of reagents and assays for the detection of pathogenic Burkholderia species

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