Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy

Krzywkowski, Tomasz; Nilsson, Mats

doi:10.1093/nar/gkx708

Cited by 40 publications

(48 citation statements)

References 31 publications

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“…We anticipate that improvements in probe design algorithms might directly yield improvements in detection efficiency. While PBCV1 based direct RNA detection offers a high detection specificity, the overall fidelity needs to be further improved for applications pertaining to single nucleotide polymorphism detection, without compromising sensitivity 31 . We are actively working on improving In-situ sequencing read depth, read-length and improved basecalling algorithms with error-correction codes.…”

Section: Limitationsmentioning

confidence: 99%

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

Iyer

Punthambaker

Liu

et al. 2018

Preprint

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We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallelIn-Situ analysis (BOLORAMIS), a reverse-transcription (RT)-free method for spatially-resolved, targeted, in-situ RNA identification of single or multiple targets. For this proof of concept, we have profiled 154 distinct coding and small non-coding transcripts ranging in sizes 18 nucleotides in length and upwards, from over 200, 000 individual human induced pluripotent stem cells (iPSC) and demonstrated compatibility with multiplexed detection, enabled by fluorescent in-situ sequencing. We use BOLORAMIS data to identify differences in spatial localization and cell-tocell expression heterogeneity. Our results demonstrate BOLORAMIS to be a generalizable toolset for targeted, in-situ detection of coding and small non-coding RNA for single or multiplexed applications. Manuscript:Single-cell transcriptomics is an exponentially evolving field, with recent developments in multiplexed in-situ technologies paving the way for spatial imaging of the genome and transcriptome at an unprecedented resolution 1 . We proposed Fluorescent In Situ Sequencing (FISSEQ) in 2003, and in 2014 demonstrated the generation and sequencing of highly multiplexed, spatially resolved in-situ RNA libraries in cells and tissues 2,3 . While FISSEQ's novelty lay in enabling unbiased discovery, it is primarily limited by poor detection sensitivity, and unsuitability for targeted in-situ transcriptomics (<0.005% compared to single-molecule (sm) FISH) 4,5 . Padlock probes have been demonstrated for in-situ sequencing for multiplexed transcriptomics, with singlebase resolution on a small number of transcripts 6 . However in both methods, detection efficiency is a function of RT efficiency, and subject to noise resulting from variable priming efficiency and 3 | P a g e random priming induced bias 7 . LNA modified primers have been demonstrated to increase RT efficiency with padlock probes (~30%), but require careful calibration and can be costprohibitively expensive for genome-wide applications (~2 order higher cost than unmodified primers) 5,6,8 . smFISH based multiplexed transcriptome imaging methods overcome the limitations of RT by directly hybridizing a plurality of oligo-paint like encoded DNA probes directly on target RNA, and subsequently reading out target locations using a two-stage hybridization scheme 9-14 . While smFISH based methods offer the highest in-situ RNA detection efficiency, it is best suited for large transcripts (>1500nt) and can yield lower signal than RCA based methods (~50-200 vs ~800-1000 fluorophores/transcript, respectively). As a result, a vast segment of biologically interesting RNA species, (including short-non coding RNA) remain vastly inaccessible to Multiplexed In situ (MIS) methods 1,15 . Consequently, there is a strong demand for robust MIS RNA detection methods that can overcome some of the limitations of each method (sensitivity, cost barrier and transcript-size limitation), while retaining the desirable features of these ...

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Section: Limitationsmentioning

confidence: 99%

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

Iyer

Punthambaker

Liu

et al. 2018

Preprint

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“…For example, DNA ligation assays perform much better on DNA substrates than on RNA substrates (Nilsson et al 2000). Most DNA ligases do not tolerate RNA templates, and the ones that do, for example, Chlorella virus DNA ligase (PBCV-1 DNA ligase), have poor end-joining fidelity (Krzywkowski and Nilsson 2017). We have recently improved the end-joining fidelity of PBCV-1 ligase on RNA by adopting the highly specific nucleic acid sensing property of Taq DNA polymerase.…”

Section: Introductionmentioning

confidence: 99%

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

Krzywkowski

Kühnemund

Nilsson

2018

RNA

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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.

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“…The specificity of SCRINSHOT crucially relies on the specific targeting of the SplintR ligation activity to the correct sites of the interrogated mRNA. A recent study (33) reported that the fidelity of this ligase is poor as it tolerates mismatches at padlock probe junctions. In addition, SplintR shows 1% of its ligation activity on a 1-nucleotide gap of a nicked duplex DNA substrate and it is unable to join ends across a 2-nucleotide gap (35).…”

Section: Resultsmentioning

confidence: 99%

“…Not hybridized padlock probes were removed by washes with 10% Formamide in 2X SSC (Sigma-Aldrich, S6639). To minimize the effect of the previously documented SplintR ligase nucleotide preferences (26, 33), padlock probe ligation was performed overnight O/N at 25°C using the SplintR ligase (NEB, M0375) at a final concentration of 0.5 Units/μl, T4 RNA ligase buffer (NEB, B0216) and 10μM ATP, according to manufacturer recommendations (see New England Biolabs webpage).…”

Section: Methodsmentioning

confidence: 99%

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SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

Sountoulidis¹,

Liontos²,

Nguyen³

et al. 2020

Preprint

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16 17 ǂ Corresponding authors: Christos Samakovlis (christos.samakovlis@scilifelab.se), Alexandros 18 Sountoulidis (alexandros.sountoulidis@scilifelab.se) 19 20 Keywords: SCRINSHOT, padlock-probe, multiplex RNA FISH, spatial cell-type map, lung 21 22 2 23 Abstract 24 Changes in cell identities and positions underlie tissue development and disease progression. 25 Although, single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of 26 cell-states, spatially resolved single-cell mapping presents a challenging task. We developed 27 SCRINSHOT (Single Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex 28 RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by 29 circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock 30 probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles 31 thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on 32 murine and human organs. SCRINSHOT quantification of marker gene expression shows high 33 correlation with published scRNA-Seq data over a broad range of gene expression levels. We 34 demonstrate the utility of SCRISHOT by mapping the locations of abundant and rare cell types 35 along the murine airways. The amenability, multiplexity and quantitative qualities of SCRINSHOT 36 facilitate single cell mRNA profiling of cell-state alterations in tissues under a variety of native and 37 experimental conditions. 38 39 40 41 42 43 44 45 65addressed by the sequential fluorescence in situ hybridization (seqFISH) (14, 15) and the 66 multiplexed error-robust FISH (MERFISH) (4). These approaches utilize sequential rounds of 67 hybridization of FISH probes or barcode-based primary probes to detect multiple RNA species. 68The outstanding throughput of these methods makes them strong candidates for generation of 69 spatial transcriptome maps in tissues. However, since the principle of these techniques is similar 70 to smFISH, they require large number of gene-specific probes, confocal or super-resolution 4 71 microscopy to deconvolve the signals and complicated algorithms for both probe design and 72 analysis. Nevertheless, the low signal-to-noise ratio still remains a major technical challenge of 73 these methods, especially for tissue sections with strong auto-fluorescence from structural 74 extracellular matrix components like collagen and elastin (16). New strategies for signal 75 amplifications, such as branched-DNA amplification (RNAScope) (17) and hybridization chain 76 reaction (18, 19), have been recently combined with sophisticated probe design (AmpFISH) (20) 77 to increase sensitivity and specificity of smFISH. 78 Padlock probes have been successfully used to detect RNA species (21). They are linear DNA 79 molecules, with complementary arms to the target mRNA sequence and a common "backbone". 80 Upon hybridization with the target sequence, they can be ligated, creating circular single-stranded 81...

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Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy

Cited by 40 publications

References 31 publications

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

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

SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

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