Nucleoside phosphoramidite monomers containing two protected hydroxyl functional groups utilized as building blocks allow the synthesis of branched oligonucleotides. During the biosynthesis of eukaryotic messenger RNA, branched oligoribonucleotides are naturally found as 'lariat structures' in splicing intermediates.
Branched DNA (bDNA) allows the design and synthesis of signal amplification assays to detect nucleic acid sequences in hybridization assays quantitatively. Examples are assays for the detection of early viral infection states in plasma. Sensitive assays are needed to measure small amounts of viral RNA as this is the case for Human Immunodeficiency Virus Type 1 (HIV-1) or SARS-CoV and SARS-CoV-2 (COVID-19) infections.
In addition, the incorporation of branched nucleotide monomers into DNA or RNA also allows the construction of complex DNA and RNA structures such as DNA origami or complex nanostructures based on nucleic acid sequences. Well-positioned branching monomers enable the synthesis of well-defined DNA or RNA structures such as oligonucleotide-based hydrogels or nanoparticles.
Today, bDNA, developed in 1989 by Horn and Urdea, is widely used in clinical and research laboratories for quantitative detection of specific nucleic acid sequences. Assays utilizing bDNA have a wide dynamic range and allow reliable detection of a few target molecules in various samples.
Unlike in PCR, in a bDNA assay, only the signal is amplified, lowering the risk for detecting contaminations. Also, a minor sequence variation in probe-binding regions of the targets does not compromise assay performance, resulting in a more robust quantitation across genotypes. At the lower end of the assay's dynamic range, reproducibility is usually superior to PCR assays.
The synthesis of branched DNA oligonucleotides is possible using protected phosphoramidites with two protected hydroxyl functional groups as the branching monomers. To synthesize branched oligonucleotides used as signal-amplifying multimers, Horn and Urdea utilized standard phosphoramidite chemistry to synthesize a primary oligonucleotide on controlled pore glass (CPG) supports. The scientists used two primary hydroxyl functional groups for the synthesis of branched oligonucleotides on a solid phase. The use of dimethoxytrityl protection or 0-levulinate protection enables the incorporation of the branching points into the oligonucleotide. The synthesis of protected phosphoramidites started from 4-(1,2,4-triazole)-1-(β,-D-5-O- dimethoxytrityl-3-O-t-butyldimethylsilyl-2-deoxyribofuranosyl)-5-methyl-2(1H)- pyrimidinone followed by triazole displacement with 6-aminohexanol.
The researchers used two strategies for the synthesis of bDNAs.
1. The first synthesis strategy utilizing dimethoxytrityl protection for both primary hydroxyl functions resulted in "fork" structures.
2. The second strategy is employing levulinate protection resulted in "comb" structures.
Figure 1: Synthesis strategies for branched DNA (bDNA): (left) Fork structures. (right) Comb structures.
The simultaneous deprotection of the two hydroxyl functions in the branching nucleotides allowed the simultaneous condensation of two additional branching monomers or of two identical nucleoside phosphoramidites to the first oligonucleotide sequence. Deprotection of the functional groups produced two reactive sites that allowed further synthesis. The incorporation of multiple branching monomers permitted the addition of several secondary sequences directly to the primary sequence. After successfully synthesizing the primary sequence, the researchers condensed the oligomer with nucleotide phosphoramidite derivatives that possessed two protected hydroxyl functions.
| Figure 2: Structures of the two phosphoramidites used for the branching monomers (BM1 and BM2). |
Fork structure synthesis
The synthesis of the fork structure required synthesis of the primary sequence followed by condensation of one branching nucleotide, two thymidines used as molecular spacers, and two more branching nucleotides, followed by the synthesis of the secondary sequence.
Comb structure synthesis
Using O-levulinate to protect the N4-(6-hydroxylhexyl) functional group allows the synthesis of comb structures. Selective removal of the levulinyl group created reactive groups on the branching nucleotides available for condensing the second sequence or additional branching nucleotides. Two thymidines function as spacers.
Deprotection of the functional groups produced two reactive sites needed for further synthesis. The incorporation of multiple branching monomers allowed the addition of several secondary sequences directly to the primary sequence. After successful synthesis, the researchers condensed the oligomer with nucleotide phosphoramidite derivatives that possessed two protected hydroxyl functions.
The first-generation signal-amplifying assay used large, branched single-stranded polydeoxyribonucleotides (bDNA) combined with a labeled short hybridization probe to amplify targeted nucleic acid sequences.
In subsequent years, Collins et al. developed quantitative hybridization assays based on branched DNA signal amplification further.
Figure 3: Illustration of the basic first generation bDNA assays showing assay component (Adapted from Collins et al. 1997).
Figure 4: Illustration of the second and third generation bDNA assays showing assay component. This assay approach utilizes preamplifier oligonucleotides (Adapted from Collins et al. 1997).
Reference
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