Wu et al. (2024) recently showed that biotinylated antisense oligonucleotides in combination with RT-PCR, enable the validation of KARR-sequencing data results of cellular higher-order RNA structures and RNA-RNA interactions. Based on their study results, the research group suggested that KARR-seq will contribute to our understanding of RNA biology soon. Understanding RNA biology is essential for health and disease, by providing a comprehensive view of RNA architecture and interactions.
Synthetic bio-ASOs
Synthetic biotinylated antisense oligonucleotides (bio-ASOs) are single-stranded DNA or RNA oligonucleotides conjugated to a biotin group or tag. The biotin tag enables specific binding to streptavidin or avidin-conjugated beads or particles, enabling various molecular and biochemical applications. Bio-ASOs allow targeted gene regulation by binding complementary RNA sequences, leading to mRNA degradation, translation inhibition, or splicing modulation.
The Biotin moiety facilitates target capture, purification, and detection assays using streptavidin-coated beads, plates, or surfaces. Typical applications of biotinylated ASOs are RNA pull-down assays allowing identification of RNA-binding proteins, Northern blotting & ELISA useful for detection and quantification of nucleic acids, affinity purification to isolate specific RNA-protein complexes, and drug delivery studies by tracking which ASO uptake and localization.
Oligonucleotide sequences of biotinylated ASOs used by Wu et al. for the validation of small nucleolar RNA (snoRNA)-18S ribosomal RNA interactions are listed in table 1. Table 1 contains a list of bio-ASOs allowing the enrichment of targeted RNA regions via hybridization. Bio-ASOs allowed the pull-down of RNA fragments using streptavidin-coated beads from crosslinked cells as the source, in combination with on-bead end repair and proximity ligation, followed by pair-end sequencing of amplified post-ligation RNA (see Wu et al. for details of methods used).
Table 1: Sequences of ASOs containing bridged nucleic acids and biotinylated oligonucleotides.
| Name | Sequences (5' to 3') | Application | Notes |
| ASO 5' ETS | mGmCmUmCmCAGGAGCACCmGmCmAmAmG | Blocking the interaction between U3 and pre-rRNA | "m" = 2'-OMe |
| ASO Control | mCmUmUmGmCGGTGCTCCTmGmGmAmGmC | Control ASO | "m" = 2'-OMe |
| LNA KANK 2 | T+C+CATCCATCCATCCAT+C+C+A | Blocking the interaction between RSV RNA and KARR2 mRNA | "+" = LNA |
| LNA ITGB1 | C+A+CAATGTCTACCAACACG+C+C+C | Blocking the interaction between RSV RNA and ITGB1 mRNA | "+" = LNA |
| LNA CD44 1 | C+T+GGGAGGTGTTGGATGTG+A+G+G | Blocking the interaction between RSV RNA and CD44 mRNA | "+" = LNA |
| LNA CD44 2 | C+C+TCCACAGCTCCATTG+C+C+A | Blocking the interaction between RSV RNA and CD44 mRNA | "+" = LNA |
| LNA Control | G+C+GACTATACGCGCAAT+A+T+G | Control LNA | "+" = LNA |
| 18S pull down 1 | Bio-GCCCGTCGGCATGTATTAGCTCT | Validating interactions between 18S and SNORD68 | Bio = 5'-biotin |
| 18S pull down 3 | Bio-ACACTCAGCTAAGAGCATCGAGGG | Validating interactions between 18S and SNORD62A | Bio = 5'-biotin |
| 18S pull down 4 | Bio-TCGCTCTGGTCCGTCTTGCG | Validating interactions between 18S and SNORD62A, SNORD12C, and SNORD65 | Bio = 5'-biotin |
| 18S pull down 5 | Bio-TGTCAATCCTGTCCGTGTCCGG | Validating interactions between 18S and SNORD100 | Bio = 5'-biotin |
ETS = 5′ external transcribed spacer.
KARR-seq
The advanced sequencing technique known as “Kethoxal-Assisted RNA-RNA” interaction sequencing or KARR-seq allows the mapping of higher-order RNA structures and RNA-RNA interactions within cells. This method utilizes N₃-kethoxal labeling and multifunctional chemical crosslinkers to covalently capture and identify RNA-RNA interactions and complex RNA conformations in vivo.
KARR-seq works independently of local protein binding. KARR-seq enables comprehensive RNA Interaction Mapping in cells.
- KARR-seq provides a detailed landscape of intramolecular and intermolecular RNA-RNA interactions across the transcriptome, offering insights into RNA structural organization and functional relationships.
- The technique allows the detection of a wide range of RNA interactions with high sensitivity and precision, facilitating the discovery of previously unrecognized RNA structures and interaction networks.
- KARR-seq allows studying RNA structural dynamics under various physiological and stress conditions. The resulting data show how processes like translation influence mRNA compaction and how viral RNAs interact with host transcripts during infection.
| Figure 1: Kethoxal (3-ethoxy-1,1-dihydroxy-2-butanone) Kethoxal C6H12O4, Mw: 148.158 g·mol−1  | Figure 2: N3-Kethoxal (3-(2-azidoethoxy)-1,1-dihydroxybutan-2-one) C6H11N3O4, Mw: 189.17 g·mol−1  |
| Kethoxal reacts with nucleic acids, particularly guanine, in DNA and RNA. This reaction allows the study of RNA structure, transcription, and DNA sequencing. | Adding N3-kethoxal to single stranded oligonucleotide enables click reaction type modification reactions for bioconjugation. |
For researchers interested in implementing KARR-seq, detailed protocols, and software tools are available to facilitate data analysis and interpretation at github, KARR-seq.
N3-Kethoxal Reaction
Kethoxal reacts with guanidine bases in single-stranded DNA and RNA. As Weng et al. have found, the presence of Watson-Crick base-pairing in double-stranded DNA blocks the labeling reaction.
The kethoxal reaction can be reversed at higher temperature.
However, the N3-kethoxal modification in RNA can be fixed in the presence of borate buffers.
Kethoxal reacts with guanine in single-stranded DNA and RNA. Kethoxal has a high specificity for guanine over other ribonucleotides and reacts preferentially with guanine residues that are not involved in hydrogen bonding. The reaction is reversible, allowing the removal of the kethoxal moiety and recovery of the original RNA molecule. Weng et al. (2020) found that N3-kethoxal only reacted with guanine in ssRNA and did not react with other nucleic bases. N3-kethoxal specifically modified the N1 and N2 positions of guanine. N3-kethoxal labeled all guanines in synthetic guanine-containing oligonucleotides, while the oligonucleotides without guanine showed no reaction.
Typical applications of kethoxal reactions are:
- Kethoxal-assisted single-stranded DNA sequencing allowing the study of how transcription changes in response to stimuli like UVB exposure.
- Mapping of RNA structure and RNA-RNA interactions.
- Probing of RNA interactions with secondary structures in other RNA molecules.
- Introducing of azido groups into RNA within a cellular environment.
- Inactivation of 30S ribosomal subunits and enabling the characterization of native and denatured forms of tRNA Trp.
References
Huang J, Zhao R, Mo J, Wang F, Weng X, Zhou X. N3 -Kethoxal-Based Bioorthogonal Intracellular RNA Labeling. Chembiochem. 2021 May 4;22(9):1559-1562. [Wiley]
Marinov GK, Kim SH, Bagdatli ST, Higashino SI, Trevino AE, Tycko J, Wu T, Bintu L, Bassik MC, He C, Kundaje A, Greenleaf WJ. CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping. Genome Biol. 2023 Apr 21;24(1):85. [PMC]
Oligo-biotin-labeling
Sabanayagam CR, Smith CL, Cantor CR. Oligonucleotide immobilization on micropatterned streptavidin surfaces. Nucleic Acids Res. 2000 Apr 15;28(8):E33. [PMC]
Shapiro R & Hachmann J The Reaction of Guanine Derivatives with 1,2-Dicarbonyl Compounds*. Biochemistry 5, 2799–2807 (1966). [PubMed]
Site-specific-labeling-of-long-rna-with-biotin-for-diagnostic-or-other-purpose
Weng X, Gong J, Chen Y, Wu T, Wang F, Yang S, Yuan Y, Luo G, Chen K, Hu L, Ma H, Wang P, Zhang QC, Zhou X, He C. Keth-seq for transcriptome-wide RNA structure mapping. Nat Chem Biol. 2020 May;16(5):489-492. [PMC]
Wu T, Cheng AY, Zhang Y, Xu J, Wu J, Wen L, Li X, Liu B, Dou X, Wang P, Zhang L, Fei J, Li J, Ouyang Z, He C. KARR-seq reveals cellular higher-order RNA structures and RNA-RNA interactions. Nat Biotechnol. 2024 Dec;42(12):1909-1920. [PMC, nature]
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Bio-Synthesis provides a full spectrum of high quality custom peptides and oligonucleotides including chemical modification services such as 5'-triphosphate and back-bone modifications, conjugation to fatty acids, biotinylation of peptides and oligonucleotides by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain synthetic modified peptides or artificially modified oligonucleotides, such as BNA (LNA) antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides capped and biotinylated mRNA as well as long circular oligonucleotides.
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