Software

Overview

Padlock probes are single-stranded DNA (ssDNA) sequences that can detect short RNA biomarkers such as microRNAs (miRNAs). The “arms” or ends of the padlock probe are the reverse complement to the target miRNA sequence, and the middle sequence contains the chosen reporter mechanism. When coupled with a rolling circle approach, a rolling circle product (RCP) can then be used to determine the initial biomarker concentration (see RCA). Due to the lack of straightforward directions, padlock probes can be complicated to create for first-time users. The most recent software for generating padlock probes was ProbeMaker, which was released in 2008 with an outdated interface, complicated setup process, and Java runtime requirement (Stenberg et al., 2005). To address this lack of software, Lambert iGEM developed Probebuilder: a novel, intuitive, and user-friendly program for designing padlock probes.

Probebuilder's source code is availabel on it's Gitlab repository.

Design

Probebuilder provides an easy-to-use graphical user interface (GUI) that makes the design of padlock probes straightforward and simple: users insert both the target microRNA (miRNA) sequence and desired reporter sequence into the software, which generates a padlock probe (see Fig. 1a).

The GUI of Probebuilder is a web app that is built using Vue.js, HTML, CSS, and JavaScript. Probebuilder’s GUI provides a step-by-step layout, with two options for inserting a reporter sequence: manually inputting a reporter sequence or choosing from a wide range of reporters in Probebuilder’s Reporter Library (see Fig. 1b). After the padlock probe is generated, a text box is displayed on the screen with a copy button that allows the user to easily copy the sequence and order it from companies that offer DNA synthesizing services (see Fig. 1c).

Figure 1. Probebuilder’s target miRNA sequence input interface (a), reporter sequence input interface (b), and results interface (c).

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Algorithm

The algorithm that generates the padlock probe is written in Python and uses the BioPython and DNA_tools python libraries (Cock et al., 2009; Borry, n.d.). Probebuilder designs the arms of the padlock probes by first taking the reverse complement of the target microRNA (miRNA) sequence, and then determines a way to split the reverse complementary DNA (cDNA). This produces the two most efficient arms based on the differences in annealing temperatures, which determines the ability for the padlock probe to bind to the target miRNA, calculated using the formula shown below (Rychlik et al., 1990).

To find the best split for producing the single-stranded cDNA arms, the algorithm loops through every possible index of the reverse cDNA and compares the annealing temperatures between the segments created by the split (see Fig. 2). After the optimal arms are determined, the algorithm inserts the reporter sequence in between the arms to complete the padlock probe.

Figure 2. Probebuilder’s padlock arm creation algorithm.

Results

We compared the padlock probes generated by Probebuilder to those created manually (see Fig. 3). Padlock probes manually created by our team members with SnapGene require comparing multiple iterations of various splits in the reverse cDNA, which tends to be a lengthy and confusing process. We validated the padlock probes generated by Probebuilder with the padlock probes made by hand for the three microRNAs (miRNAs) we are working with and observed a 100% accuracy of the sequences. We also ordered a padlock probe for miRNA-451a that was designed using Probebuilder with linear probes, an RCA reporter mechanism. We tested the padlock probe and observed a fluorescent band of DNA very close to the well, which indicates that a long strand of DNA, greater than 1 kB, was produced due to the RCA reactions (see Fig. 4) (see RCA).

Figure 3. Comparison of padlock probes generated by SnapGene and Probebuilder.

Figure 4. Gel of RCA reaction with miRNA-451. Well 1 contained the DNA ladder. Wells 2-4 contained the padlock and miRNA control. Wells 5-9 contained the RCA reaction.

To further validate the effectiveness of the miR-451a padlock probe developed via ProbeBuilder, the perceived RCP seen in Figure 4 was introduced to linear DNA probes (see RCA). Figure 5 displays a significant decrease in the fluorescence intensity of a triplicate with FAM probes, BHQ probes, and the RCP produced compared to a triplicate of just FAM tagged probes. This finding experimentally validates the use of Probebuilder as a means of producing effective padlock probes.

Figure 5. Fluorescent reading of RCP produced by miR-451a padlock probe (generated via Probebuilder). BHQ-1 and FAM probes compared to solely FAM probes.

References