Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection

Abstract

Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1 DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3 ′ ′ ′ ′ ′-OH RNA/5 ′ ′ ′ ′ ′-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5 ′ ′ ′ ′ ′ flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide polymorphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.

SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution

“…In summary, we confirmed the previously reported tolerance of SplintR ligase for single-nucleotide mismatches at the ligation site [ 26 , 30 , 37 ]. Our experimental data show that the specificity of SCRINSHOT largely depends on the stringent hybridization of the 20-nucleotide-long padlock arms (40 nucleotides in total) and less on the specificity of the used ligase.…”

Squaramides and Ureas: A Flexible Approach to Polymerase‐Compatible Nucleic Acid Assembly

“…Since the DNA oligonucleotides will be directly used for qPCR, the introduction of “tails” (sequences) that are not found naturally in the genome enables the design of primers that are highly specific to the DNA template. Similar in concept to Padlock probes, the key differentiator is that squaramide chemical ligation enables efficient ligation irrespective of nucleic acid substrate, allowing RNA detection that is otherwise challenging with ligase enzymes …”

“…[33] Since the DNAo ligonucleotides will be directly used for qPCR, the introduction of "tails" (sequences) that are not found naturally in the genome enables the design of primers that are highly specific to the DNAtemplate.Similar in concept to Padlock probes, [34,35] the key differentiator is that squaramide chemical ligation enables efficient ligation irrespective of nucleic acid substrate,a llowing RNAd etection that is otherwise challenging with ligase enzymes. [36] In designing our pair of RNA-detecting DNAo ligonucleotides ( Figure 5A), we split each oligonucleotide into a20mer region that hybridises to the RNAtarget and a10-mer 5'tail for PCR primer binding.T he two 20-mer regions were designed to minimise self-complementarity,w hich would otherwise lead to template-independent ligation, and to be highly specific to the target as determined by BLAST [37] alignment against ar eference database.T he 10-mer sequences were designed to be non-complementary to the target, and provide ar elatively unstructured tail on which PCR primers can efficiently bind to for amplification, the latter being determined by mfold. [38] As ap roof of concept, we initially designed DNA oligonucleotides that target a1 00-mer RNA( Figure 5A and Supporting Information, Table S9).…”

Direct RNA targeted transcriptomic profiling in tissue using Hybridization-based RNA In Situ Sequencing (HybRISS)

“…Currently, alternative protocols that involve the direct probing of RNA with PLPs and RCA, such as SCRINSHOT (16) and targeted ExSeq (17) have also exhibited improved transcript detection efficiency. However, PBCV-1 DNA ligase used in these protocols have shown high tolerance for mismatches in ligation and extensive optimization would have to be undertaken for different tissue types to prevent off-target detection (13,16) . The increased efficiency of dRNA has allowed for improved detection of lower expressed transcripts, which would otherwise be challenging to detect with the cDNA approach.…”

“…Direct hybridization and probing of mRNA in situ for improved efficiency has been attempted with different commercially available ligases, including Chlorella virus DNA ligase (PBCV-1 DNA ligase), but showed high tolerances of mismatches for ligation (12) , and consequently worse specificity compared to cDNA template DNA ligation using thermophilic DNA ligases. Recently, it was reported that T4-RNA Ligase 2 showed good ligation efficiency with 3'-RNA/5'-DNA PLPs (Chimeric PLPs) on RNA templates, and exhibits higher ligation fidelity on single nucleotide variations, compared to PBCV-DNA ligase (13) . Here we evaluate such a direct RNA (dRNA) chemistry in situ with HybISS (HybRISS: Hybridization-based RNA in situ sequencing) that targets RNA with chimeric PLPs while still retaining the fundamental benefits of ISS technology.…”