Figure 1.
DNA configuration of a proximity ligation assay (PLA) template for multiplexing.
One PLA template comprises two adaptor and connector DNA strands. The adaptor strands are conjugated to an antibody (Ab) and each exhibits two annealing sequences (ASs). The connector strands exhibit the complementary sequences of the two AS sites located on different adaptors (AS′). In cases where the two antibodies and, consequently, the adaptors are in close proximity, the connector strands with matching AS sites can hybridize and form a circular DNA ring. Upon ligation, the circular DNA can be amplified by rolling-circle amplification (RCA). Multiplexing of the PLA is enabled by encoding the target information of the antibody in the AS region of the adaptor for one connector. Arrowheads on all DNA strands mark the 3′ end of the oligonucleotides.
Figure 2.
In silico PLA template design procedure.
(A) Computational flow chart to obtain orthogonal proximity ligation assay (PLA) templates. The process is divided into two randomization steps. In the first step, the annealing sequence pairs (AS/AS′) are generated and subjected to GC content, nucleotide bias, secondary structure, and cross-hybridization filters. The second randomization step maximizes the differences in the hybridization energy between the largest observed value for a matched AS/AS′ pair and the lowest observed value for a matched AS/AS′ pair () in the composite. (B) Upper and lower bar charts show the DNA hybridization energies between all AS sites within the 24 PLA templates after the first and second selection and randomization steps, respectively. Grey and black bars denote hybridization events between unmatched and matched AS sites within the generated PLA template library.
Figure 3.
Microfluidic chip integration of a solid-phase rolling-circle amplification (RCA) and proximity ligation assay (PLA) system for testing PLA templates.
(A) Pairs of either adaptor or connector strands from a PLA template library are plotted on an epoxy-coated glass slide and aligned to a polydimethylsiloxane (PDMS)-based microfluidic chip. (B1) DNA-only PLA test system. Pairs of biotinylated adaptor strands from the library are spotted as microarray and aligned to the PDMS chip. The real image shows 1 out of 640 unit cells of the microfluidic chip. Each unit cell is divided into two chambers, i.e., the DNA storage chamber and the assay chamber. Microfluidic membrane valves separate the unit cells from each other (V1) and the DNA storage and assay chambers (V2). In the first step, a layered pull-down assay is developed using the button valve on the epoxy-coated glass surface within all assay chambers of the chip (see main text). NeutrAvidin forms the reactive top layer of the pull-down assay. Next, biotinylated adaptors are resolved from the microarray spot in the DNA storage chamber and allowed to diffuse to the pull-down area in the assay chamber. Thus, an array containing all binary adaptor combinations is created. A pair of connectors to complement the adaptors was introduced from the outside of the chip to obtain fully assembled PLA templates. (B2) For the spPLA test system, pairs of connector strands from the library are spotted on the microarray and aligned to the PDMS chip. Similar to B1, a pull-down assay is developed on a NeutrAvidin-reactive surface. In subsequent washing steps, an antibody “sandwich” was developed to detect vascular endothelial growth factor (VEGF). The two anti-VEGF antibodies from different hosts were labeled with the adaptor strands. Combinations of adaptor strands within the antibody sandwich were achieved through fluidic multiplexing on the chip (see main text). Next, the connector pairs were resolved and allowed to diffuse to the pull-down area and fully assemble PLA templates. (C) After connector hybridization, fluids for PLA template ligation, amplification, and product detection are perfused in automated washing steps.
Figure 4.
On-chip solid-phase rolling-circle amplification (spRCA) system for testing proximity ligation assay (PLA) templates.
(A) The image shows a merged fluorescence and bright-field micrograph of two circular pull-down areas within representative unit cells on the chip. In the lower and upper images, positive and negative RCA events were obtained with matched and mismatched shared connectors (SCs), respectively. The bar graph shows background corrected fluorescence signal corresponding to the pull-down area of the left images. Error bars are calculated from four repeats. The scale bar represents 75 µm. (B) Temperature dependence of a PLA template assembling in a spRCA reaction. The graphs show the local background corrected fluorescence signals of 160 spRCA reactions, including the screening of one CC/DC pair, of 100 binary adaptor combinations from the selected 10 PLA templates. Furthermore, 20 single adaptors, 20 non-specific DNA strands, and 20 tests without DNA were screened against the same connector pair as control.
Figure 5.
PLA template library screen with the solid-phase rolling-circle amplification (spRCA) system.
The matrix shows the Z-scores for the RCA reactions of 100 adaptor pairs screened against 10 coding connectors (CCs) from 10 in silico generated PLA templates. The detection connector sequence was kept constant in all RCA reactions. Diagonal elements of the matrix (white bars) are the 10 matched adaptor/connector pairs. Red bars in the matrix indicate false-positive events from mismatched adaptor/SC combinations. The lower histogram presents the absolute values of the Z-score for the complete screening.
Figure 6.
PLA template library screen with the solid phase proximity ligation assay (spPLA) system.
The matrix shows the Z-scores for the PLA reactions of 64 antibody pairs with the same adaptors as in Figure 4 screened against eight coding connectors (CC) from the in silico generated PLA templates. The detection connector sequence was kept constant in all rolling-circle amplification (RCA) reactions. Diagonal elements of the matrix (white bars) are the eight matched adaptor/connector pairs. The numbering of the adaptor and connector is the same as the numbering of the PLA templates from Figure 4B. The plates 4 and 8 were removed in this screening because of false-positive reaction results in the RCA screening. The lower histogram presents the absolute values of the Z-scores for the complete screening.