DNA-based nanosensors for next-generation clinical diagnostics via detection of enzyme activity

Abstract

Specific and sensitive detection of DNA-modifying enzymes represents a cornerstone in modern medical diagnostics. Many of the currently prevalent methods are not preferred in the clinics because they rely heavily on pre-amplification or post-separation steps. This editorial highlights the potential of adopting DNA-based nanosensors for the assessment of the activities of DNA-modifying enzymes, with emphasis on the topoisomerase and tyrosyl-DNA phosphodiesterase families. By underlining the existing challenges, we expect that the DNA-nanosensors may soon be promoted to clinical diagnostics via enzyme detection.

Keywords::

• biosensors
• cancer diagnostics
• single-cell analysis
• topoisomerase I
• tyrosyl-DNA phosphodiesterase 1

Why DNA-modifying enzymes?

The activity of DNA-modifying enzymes has been exploited extensively as targets in anticancer therapy. Generally, the drugs convert the enzymatic activity into DNA damage ultimately causing apoptosis. An example of such an enzyme is the human topoisomerase I (hTopI). hTopI is the sole cellular target of drugs from the camptothecin (CPT) family currently used in systemic treatment of colon, ovarian and small-cell lung cancers [1]. hTopI is an essential cellular enzyme: it regulates the topology of the DNA by introducing transient single-stranded breaks in the DNA followed by controlled rotation of the cleaved DNA strand around the non-cleaved strand. During cleavage of the DNA, hTopI becomes covalently attached to the 3′-end of the DNA and is only released upon religation of the nick. CPT exhibits its toxicity by selectively inhibiting the religation through specific binding of the TopI-DNA cleavage intermediate, resulting in hTopI being trapped on the DNA [2]. The trapping of hTopI may also occur naturally as a result of endogenous DNA lesions, such as abasic sites, nicks or mismatches [3]. Under these circumstances, however, the trapping is quickly repaired mainly by tyrosyl DNA-phosphodiesterase 1 (TDP1) in combination with other repair factors. Repair of the TopI-DNA complexes is crucial since collision of transcription or replication forks with the complexes may result in DNA breaks and ultimately cell death or hyper-recombinogenic phenotypes. Cancer cells often have increased TopI activity and divide more rapidly than benign cells, thus the use of CPT results in more TopI-DNA complexes and a higher demand of repair, which explains why cancer cells often are more sensitive to CPT than benign cells. Increased resistance toward CPT has been seen both as the result of mutations in the TOP1 gene, downregulation of TopI activity and through overexpression of TDP1 [4,5]. Thus, collectively measuring the activities of TopI and TDP1 could be useful for predicting CPT treatment response in individual patients.

Detecting the functionality of DNA-modifying enzymes

Duty of DNA-modifying enzymes in fundamental biological processes has rendered their activities indispensable indications in current clinical evaluation [6]. Commonly adopted measurements based on ELISA or immunohistochemical staining suffer from low detection sensitivity or labor intensive procedures such as amplification and separation, which limits their clinical applicability particularly in the so-called point-of-care settings [7,8]. Measurements based on quantitative reverse transcription-PCR assessing the mRNA, although improving the sensitivity considerably, presents an obvious disadvantage of quantifying the enzyme indirectly [9]. Additionally, cellular regulations between the mRNA and the enzyme may scramble the correlations between mRNA and enzyme levels [10]. Perhaps more importantly, the currently available methods measure the amount rather than activities or functionalities of enzymes. However, it is the activities, not the quantities per se of enzymes that dictate their biological functions. Furthermore, enzyme activities and functionalities may be modulated by post-translational modifications without affecting the amount of enzymes [11]. Collectively, these findings suggest that the enzyme quantities, and the mRNA amount, may not directly reflect the level of enzyme activity [12]. One of the biggest challenges in clinical diagnostics is to determine which biomarkers to monitor across the wide spectrum of disease processes. In cancer, for instance, both DNA mutations and changes in RNA levels are used as biomarkers. This may be sufficient in some cases but may also lead to wrong conclusions, since post-translational modifications of enzymes may change both the stability [13] and activity [14] of enzymes without changing the status of DNA or RNA. Thus, since enzymes may vary among cell types, change over time and show different post-translational modifications, they are preferable indicators for treatment outcome and changes of disease states.

Inspiration from DNA-based nanosensors?

DNA-based nanosensors have led to previous successes in genomic and proteomics studies, however, their potential for analyzing DNA-modifying enzymes has been overlooked. Detection of enzyme activities via DNA nanosensors usually takes advantages of the functionality of DNA-modifying enzyme, such as DNA cleavage, ligation, synthesis and repair [15]. For example, specific and sensitive measurements of the cleavage-religation activities from the DNA topoisomerase and the tyrosine recombinase families have previously been demonstrated, by combining DNA nanosensors with rolling circle amplification [16,17]. Even though this assay enables a single molecular sensitivity, the highly delicate fluorescence microscope and the time-consuming procedures have limited its applicability for point-of-care settings. Adopting a concept similar to molecular beacons, we have recently presented a variety of DNA nanosensors for real-time measurement of TDP1 [18] and hTopI [19]. However, besides problems associated with organic fluorophores, the detection sensitivity is not comparable with the microscopy-based approaches. Although perfect solutions have yet to be seen, the initial development has recognized the urgent need of a clinically assessable quantification of DNA-modifying enzymes’ activities.

How far are we: moving from test tubes to real clinics

We believe the existing development in DNA-based nanosensors, particularly molecular configurations, readout formats and substrate designs, may serve as a stepping stone toward accurate and simple detection schemes of enzymatic activities for the prediction of the treatment outcome of enzyme-targeting therapies as well as an easy accessible tool for large-scale drug screening. However, challenges are anticipated when clinical diagnostics is considered: most enzymatic sensors are validated only using purified enzymes in controlled in vitro settings where there is minimum interference to sensor specificity. In contradiction to this, the clinical evaluation of complex biological samples, such as crude extract from clinical specimens contains various enzymes that might cause false positives. Therefore, it is essential to verify the specificity thoroughly, also in complex biological systems. Additionally, concentrated cellular extract has been constantly prescribed for enzyme-based in vitro detection, which may be difficult using clinical samples and it is thus inevitable to aim for highly sensitive assays. Specificity and sensitivity in complex biological fluids are of particular consideration for the point-of-care settings, where the crude samples are usually evaluated. Further challenge remains on the complex cellular heterogeneity, for example, to resolve heterogeneous cancer tissue which might contain various percentages of tumor cells, it may be necessary to develop fully quantitative analysis with sensitivity down to single cell level. Stringent validation is of particular demand when investigating tissue samples, because they, due to the presence of connective tissue and various different cell types, are disparate to cell lines simulations and fluidic format of samples, such as blood and saliva. Furthermore, normalization in the form of per mg or per mm³ between different clinical tissue samples may be imperfect since they may vary not only in cell types but also cell density. Thus, normalization using tissue weight or geometric sample size may result in a varying amount of cells, which would bias the verdict of measured enzymatic activities.

Moving forward, measuring enzymatic activities directly through DNA-based nanosensors demonstrates several crucial dissimilarities over picking up the genetic variances: true functionality: measuring activities indicate the exact functionality from the enzymes, rather than the upstream genetic instructions from the DNA or mRNA; environmental effect: enzymatic activities may be changed by disease progression or chemotherapeutic treatment while the concentration of genetic materials remains unaltered; intrinsic amplification: each enzyme may generate numerous products provided with excess ‘substrates’ [20]. Such an inherent amplification may potentially allow diagnosis of diseases at an early stage, with subtle disordered enzyme activities. Therefore, we expect the achievements from the field of DNA-based nanosensors may cause: a further shift away from preimplantation diagnosis and more toward routine clinical diagnostics, particularly in situations where only small amounts of tissues are available for analysis; a single cell-based analysis for the interrelationship among different enzymatic activities, such as TopI and TDP1. With the vivid development of DNA-based nanosensors and their validation in complex biological samples, nanosensors may start to move into the clinics and pave the way for more accurate predictions of treatment response or disease progression.

Acknowledgements

The authors would like to acknowledge the support from the Danish Research Council (116325/FTP), Lundbeck Foundation (R95-A10275), Karen Elise Jensen Foundation, Carlsbergfondet, the Aase og Ejnar Danielsens Foundation, the Købmand Svend Hansen og hustru Ina Hansens Foundation, the Familien Hede Nielsens Foundation, the Marie & MB. Richters Foundation, the Fabrikant Einar Willumsens Mindelegat, the Kong Christian Den Tiendes Fond, the Lykfeldts legat, the Fhv. Dir. Leo Nielsen og Hustru Karen Margrethe Nielsens Legat for Lægevidenskabelig Grundforskning, the Simon Fougner Hartmans Familiefond, Fuhrmann-Fonden and the Familien Erichsens mindefond.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

No writing assistance was utilized in the production of this manuscript.