Journal: Expert review of proteomics

Article Title: The method developer's guide to oligonucleotide design.

doi: 10.1080/14789450.2024.2318565

Figure Lengend Snippet: Figure 1. (a) A schematic presentation of the oligonucleotide design for in situ PLA. (A)The sequences of the different oligonucleotides are presented in the table below, color coded to identify the different elements. Proximity probe oligonucleotides are presented as conjugated to antibodies. The sequence of proximity probe oligonucleotide 1 was originally designed with three mismatched 2´-O-methyl-RNA at its 3´end to ensure that RCA could only be performed from proximity probe oligonucleotide 2. This is however not needed, and we have since then removed them as it does not affect efficiency of in situ PLA. Addition of a three mismatched 2´-O-methyl-RNA at the 3´end of the detection oligonucleotides can be used if one wants to combine the RCA step with detection, to prevent Phi29 DNA polymerase to prime when the detection oligonucleotides hybridize to the RCA products. The 3´end of the oligonucleotides are indicated as arrowheads, the sequences are written 5´ to 3´. (b) A schematic presentation of the in situ PLA reaction. The proximity probes bind their targets. Proximity sensing is achieved by hybridization of circularization oligonucleotides and their ligation into a circular DNA molecule (reporter of proximity events). PLA signal amplification is obtained by rolling circle amplification of the DNA circle, templated by one of the proximity probes, and subsequent detection of the RCA product with fluorophore-labeled detection oligonucleotides.

Article Snippet: A beautiful oligonucleotide design to produce a long double stranded DNA molecule was developed by the Pierce lab almost two decades ago, called hybridization chain reaction (HCR) [18].

Techniques: In Situ, Sequencing, Hybridization, Ligation, Amplification, Labeling

Journal: Expert review of proteomics

Article Title: The method developer's guide to oligonucleotide design.

doi: 10.1080/14789450.2024.2318565

Figure Lengend Snippet: Figure 2. (a) A schematic presentation of the oligonucleotide design for variants of in situ PLA. (A) By just adding a compaction oligonucleotide, the RCA product of a regular in situ PLA will be condensed into a much smaller object. All other oligonucleotides are the same as in Figure 1. Addition of a three mismatched 2 ´-O-methyl-RNA at the 3´ends of the detection oligonucleotides and the compaction oligonucleotide is required to perform RCA combined with detection. (b) The circularization oligonucleotide 1 of the regular in situ PLA was replaced with three variants, having different detection elements (F, G or H), which can be targeted with the cognate detection oligonucleotide. All other oligonucleotides are the same as in Figure 1. The 3´end of the oligonucleotides are indicated as arrowheads, the sequences are written 5´ to 3´. (c) A tag sequence (F, G or H) was incorporated into proximity probe oligonucleotide 2 to allow multiplexing. The sequence of proximity probe oligonucleotide 1 and the circularization oligonucleotides are the same as the regular in situ PLA (Figure 1), with the addition of the tag oligonucleotides F´, G´ and H´. The DNA circles formed in the proximity sensing will contain the tag sequence corresponding to the specific proximity probe oligonucleotide 2. (d) To detect tertiary proximity events, we utilized the regular in situ PLA design, with addition of a third proximity probe oligonucleotide. Circularization oligonucleotide 2 was exchanged with two new ones, i.e. circularization oligonucleotide 3 and 4. All other oligonucleotides are the same as in Figure 1. The 3´end of the oligonucleotides are indicated as arrowheads, the sequences are written 5´ to 3´.

Article Snippet: A beautiful oligonucleotide design to produce a long double stranded DNA molecule was developed by the Pierce lab almost two decades ago, called hybridization chain reaction (HCR) [18].

Techniques: In Situ, Sequencing, Multiplexing

Journal: Expert review of proteomics

Article Title: The method developer's guide to oligonucleotide design.

doi: 10.1080/14789450.2024.2318565

Figure Lengend Snippet: Figure 3. (a) A schematic presentation of the oligonucleotide design to increase the efficiency of in situ PLA. (A) A schematic presentation of possible linear ligation products in the proximity sensing step of in situ PLA. (b) a schematic presentation of the unfold oligonucleotide design, where the circularization oligonucleotides are incorporated into proximity probe oligonucleotide 2. dU represent deoxyUridine (dU) in the sequence. Proximity probe oligonucleotide 1 was designed as a hairpin to block interactions to proximity probe oligonucleotide 2. Upper panel shows proximity probe binding and lower panel shows proximity sensing after dU residues have been removed by uracil-DNA glycosylase and endonuclease IV treatment. The 3´end of the oligonucleotides are indicated as arrowheads, the sequences are written 5´ to 3´. The detection oligonucleotide is the same as the regular in situ PLA (Figure 1).

Article Snippet: A beautiful oligonucleotide design to produce a long double stranded DNA molecule was developed by the Pierce lab almost two decades ago, called hybridization chain reaction (HCR) [18].

Techniques: In Situ, Ligation, Sequencing, Blocking Assay, Binding Assay

Journal: Expert review of proteomics

Article Title: The method developer's guide to oligonucleotide design.

doi: 10.1080/14789450.2024.2318565

Figure Lengend Snippet: Figure 4. A schematic presentation of the oligonucleotide design for MolBoolean. The sequences of the different oligonucleotides are presented in the table below, color coded to identify the different elements. Proximity probe oligonucleotides are presented as conjugated to antibodies. The 3´end of the oligonucleotides are indicated as arrowheads, the sequences are written 5´ to 3´. The DNA circle is ordered in two pieces that contains complementary elements (the black hairpins of the DNA circle), so that spontaneously hybridize to each other and facilitates ligation into a complete circle. The recognition sequence for the nickase Nt.BsmAI is shown in the table and where it cuts is indicated by (*) in the DNA circle sequence. Once the DNA circle is cut by Nt.BsmAI (position indicated by black arrows) the tag oligonucleotides can hybridize to the proximity probe oligonucleotides. This allows the oligonucleotides to be re-ligated into a DNA circle, that would be the template for RCA.

Article Snippet: A beautiful oligonucleotide design to produce a long double stranded DNA molecule was developed by the Pierce lab almost two decades ago, called hybridization chain reaction (HCR) [18].

Techniques: Ligation, Sequencing

Journal: Expert review of proteomics

Article Title: The method developer's guide to oligonucleotide design.

doi: 10.1080/14789450.2024.2318565

Figure Lengend Snippet: Figure 5. (a) A schematic presentation of the oligonucleotide design for proxHCR. (A) A schematic presentation of HCR. An initiator oligonucleotide (green and blue) will bind and invade an HCR hairpin. As it opens up it will reveal the initiator sequence (red and blue) to bind and invade an HCR hairpin of the other polarity. As it opens up it will reveal the first initiator sequence (green and blue). The HCR reaction will continue and generate a long nicked double-stranded DNA molecule. (b) A schematic presentation of the oligonucleotide design for proxHCR, where the activator oligonucleotide targets the loop region. The sequences of the different oligonucleotides for proxHCR are presented in the table below, color coded to identify the different elements. Proximity probe oligonucleotides are presented as conjugated to antibodies. The 3 ´end of the oligonucleotides are indicated as arrowheads, the sequences are written 5´ to 3´. (c) A schematic presentation of the proxHCR reaction, using the oligonucleotide above. The different elements are not presented in the figure, instead focusing on how the different oligonucleotides bind to each other. (d) A schematic presentation of the oligonucleotide design for proxHCR, where the activator oligonucleotide targets an external toehold. The sequences of the different oligonucleotides for proxHCR are presented in the table below, color coded to identify the different elements. Proximity probe oligonucleotides are presented as conjugated to antibodies. The 3´end of the oligonucleotides are indicated as arrowheads, the sequences are written 5´ to 3´. (e) A schematic presentation of the proxHCR reaction, using the oligonucleotide above. The different elements are not presented in the figure, instead focusing on how the different oligonucleotides bind to each other.

Article Snippet: A beautiful oligonucleotide design to produce a long double stranded DNA molecule was developed by the Pierce lab almost two decades ago, called hybridization chain reaction (HCR) [18].

Techniques: Sequencing