Strategies for Covalent Labeling of Long RNAs

Abstract

The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site-specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field.

Keywords: RNA labeling; bioorthogonal labeling; click chemistry; lncRNA; ribozymes.

Figure 1

Enzymatic ligation strategies to prepare long modified RNA oligonucleotides beyond the size limit of chemical solid‐phase synthesis. Small, site‐specifically modified RNA strands prepared by RNA solid‐phase synthesis can be ligated using different enzymes to generate long, site‐specifically labeled RNA. The acceptor sequence is bearing a terminal 3′‐OH, while the donor RNA carries a 5′‐triphosphate. A) T4 RNA ligase I mediated ligation of two single stranded RNA oligonucleotides brought in close proximity due to secondary structure formation. B) T4 RNA ligase I mediated ligation of two single stranded RNAs brought in close proximity by a DNA splint. C) Templated T4 DNA ligase/T4 RNA ligase II mediated ligation of two RNA strands. A splint is used to form the necessary ternary pre‐ligation complex and to bring ends to be ligated in close proximity. D) Templated T4 DNA ligase/T4 RNA ligase II mediated ligation of two RNA strands. A splint is used to break up secondary structure formation enabling the enzyme to access the ligation site and to form the necessary ternary pre‐ligation complex.

Figure 2

9DB1* deoxyribozyme‐mediated ligation to prepare a 118 nt long, site‐specifically spin‐labeled SAM‐I riboswitch from two smaller RNA strands. The modification is inserted during chemical solid‐phase synthesis of RNA. The acceptor sequence is bearing a terminal 3′‐OH, while the donor RNA carries a 5′‐triphosphate.[ ⁵³ , ⁷¹ ] The ligation site is indicated with an arrow.

Figure 3

Cleavage/ligation cascade performed by an engineered twin ribozyme (black). A predefined patch of RNA (dark green) in the RNA substrate (light green) is exchanged with a small, synthetically prepared and modified RNA sequence (blue). At the cleavage site, a 2′,3′‐cyclic‐phosphate (2′,3′‐cP) and a 5′‐OH is formed which can in turn be ligated by the twin ribozyme in the same manner with a substrate bearing a 2′,3′‐cP and a 5′‐OH group. Cleavage and ligation sites are indicated with arrows.

Figure 4

Unnatural base pairs and their derivatives applied for site‐specific RNA labeling.

Figure 5

Site‐specific RNA labeling with an UBP via genetic alphabet expansion transcription and applications thereof. Upper panel: General scheme of genetic alphabet expansion transcription: a functionalized UB triphosphate is site‐specifically incorporated into RNA during T7 IVT enabling post‐transcriptional labeling via click chemistry. Left panel: For site‐specific labeling of lncRNA, a DNA template bearing the UBP at specific positions can be prepared by 2‐step assembly PCR and is then transcribed into RNA. This technique was applied to site‐specifically label the glmS ribozyme allowing the investigation of ribozyme self‐cleavage. Right panel: Via plasmid PCR, unnatural base pairs can also be incorporated into long functional RNA >400 nt by using modified primers. By this, nitroxyl spin labels were introduced into the A‐region of Xist RNA allowing EPR distance measurements (right panel).[ ⁵ , ⁸⁹ ] Reproduced with permission from Ref. [89], Copyright 2017 Methods.

Figure 6

Selective RNA labeling via transcriptional priming (left) and position selective labeling of RNA (PLOR, right panel). Left panel: In transcriptional priming, a functionalized starter nucleotide is selectively incorporated at the 5′‐end of RNA. Enzymatic ligation of 5′‐labeled donor RNA to an acceptor RNA results in an internal functionalized nucleobase that can be further modified via a compatible click or crosslinker chemistry.[ ¹¹³ , ¹¹⁵ ] Right panel: PLOR is divided into initiation, elongation and termination. Transcription proceeds on a DNA template immobilized to streptavidin agarose beads. Transcriptional pausing is caused by the lack of a canonical triphosphate. Elongation consists of many cycles each lacking one canonical NTP to cause transcriptional pausing. This allows for labeling with a modified NTP at a specific position. To avoid cross‐contamination between individual cycles, thorough washing after pausing is essential (SPE=solid‐phase extraction).[ ¹¹⁷ , ¹¹⁹ ]

Figure 7

Site‐selective RNA labeling via RNA acylation at induced loop (RAIL) structures. A complementary helper DNA strand is hybridized exposing a loop at a predefined site within the endogenous RNA of interest to enable site‐selective 2′‐OH acylation with an acylimidazole reagent (here: nicotinyl acylimidazole azide). Remaining RNA segments and their 2′‐OH groups are shielded by the helper DNA‐RNA duplex. After acylation, the helper DNA strand is degraded by DNase treatment. Site‐selectively acylated RNA can be further modified by CuAAC reactions with functionalized alkine‐conjugates.

Figure 8

Refined deoxyribozyme‐catalyzed labeling (DECAL) of pre‐existing RNA with the 10‐DM24 DNAzyme applying assisting heavy metal ions and the phosphorothioate‐modified GTP derivative Sp‐GTP^S.[ ¹³⁰ , ¹³¹ ] The further engineered 10‐DM24 deoxyribozyme catalyzes the attachment of modified guanine mononucleotides to a 2′‐OH group within the RNA strand of interest. Addition of the oligonucleotide cofactor R_Δ compensates for a formerly longer oligonucleotide substrate and is necessary for sufficient conversion. The deoxyribozyme's binding arms hybridize to the target RNA and can be adapted for individual target sites to enable site‐specific RNA modification. Phosphorothioate‐modified Sp‐GTP^S is used as ligation substrate to prevent subsequent phosphodiesterase hydrolysis of the 10‐DM24 catalyzed 2′,5′‐branch modification due to the particular steric configuration of the newly formed linkage in 2′‐Rp‐P^S‐labeled RNA. Tb³⁺ acts as an accelerating cofactor that enables efficient conversions at pH 7.5 and reduces the required concentration of modified GTP triphosphate. Thiophilic Cd²⁺ ions are used to prevent phosphorothioate interfering effects on the metal ion coordination by the 10‐DM24/RNA/GTP^S ternary complex.

Figure 9

Site‐specific orthogonal double labeling of an endogenous RNA of interest using nucleotidyl and Tenofovir transferase ribozymes. The in vitro selected 2′‐5′ adenylyl transferase ribozyme FH14 can be utilized for internal labeling of 2′‐OH groups with N ⁶‐modified ATP analogs yielding 2′‐5′‐phosphodiester bond branched RNA. Additionally, the in vitro selected Tenofovir transferase ribozyme FJ1 can be applied for internal 2′‐OH group labeling with N ⁶‐modified acyclic nucleoside phosphonate Tenofovir derivatives yielding 2′‐5′‐phosphonate ester bond branched RNA.

Figure 10

Tag‐free internal RNA labeling based on mRNA methyltransferases METTL3‐14 and METTL‐16. The approach of methyltransferase‐directed transfer of activated groups (mTAG) can be used for covalent and sequence‐specific modification of endogenous RNA. Methyltransferases are applied to transfer various functional groups from S‐adenosyl‐L‐methionine (SAM or AdoMet) analogs to the N ⁶‐position of target adenosines within a RNA of interest. Therefore, a distinct recognition sequence defining the modification site for the methyltransferase needs to be included in the RNA sequence. Additionally, METTL3‐14 is able to transfer photoactive benzylic AdoMet analogs like ortho‐nitrobenzyl (ONB) and 6‐nitropiperonyl (NP) groups. Upon irradiation with light, the previously attached photoactive modification is removed from the RNA of interest. Both methyltransferases, METTL3‐14 and METTL‐16 can be used in a combined approach for orthogonal labeling of different sites within the same RNA strand. After sequential modification with a propargyl group and photoactive ONB group, the latter can be removed reversibly by irradiation with light.