Efficient RNA 5′-adenylation by T4 DNA ligase to facilitate practical applications

Abstract

We describe a simple procedure for RNA 59-adenylation using T4 DNA ligase. The 59-monophosphorylated terminus of an RNA substrate is annealed to a complementary DNA strand that has a 39-overhang of 10 nucleotides. Then, T4 DNA ligase and ATP are used to synthesize 59-adenylated RNA (59-AppRNA), which should find use in a variety of practical applications. In the absence of an acceptor nucleic acid strand, the two-step T4 DNA ligase mechanism is successfully interrupted after the adenylation step, providing 40%-80% yield of 59-AppRNA after PAGE purification with few side products (the yield varies with RNA sequence). Optimized reaction conditions are described for 59-adenylating RNA substrates of essentially any length including long and structured RNAs, without need for sequestration of the RNA 39-terminus to avoid circularization. The new procedure is applicable on the preparative nanomole scale. This 59-adenylation strategy using T4 DNA ligase is a substantial improvement over our recently reported adenylation method that uses T4 RNA ligase, which often leads to substantial amounts of side products and requires careful optimization for each RNA substrate. Efficient synthetic access to 59-adenylated RNA will facilitate a range of applications by providing substrates for in vitro selection; by establishing a new protocol for RNA 59-capping; and by providing an alternative approach for labeling RNA with 32 P or biophysical probes at the 59-terminus.

Use of Deoxyribozymes in RNA Research

“…Although a method for appending a 5′-triphosphate to a synthetic RNA oligonucleotide via solid-phase synthesis has been reported (Paul et al , 2006), in our hands this approach is technically demanding and difficult to reproduce. Alternatively, an activated phosphorus at the RNA 5′-terminus can be provided by a 5′-adenylate, which can be installed onto readily obtained 5′-phosphorylated RNA using T4 RNA ligase (Silverman, 2004) or T4 DNA ligase (Wang and Silverman, 2006b). RNA-ligating deoxyribozymes that readily accept a 5′-adenylated R substrate still await development.…”

“…A branch-site guanosine is tolerated, albeit with about 50-fold lower ligation rate constant than with branch-site adenosine. The 5′-triphosphorylated RNA nucleotide that reacts with the 2′-OH can be either A or G. 5′-Adenylated C (Wang and Silverman, 2006b) is also accepted with lower rate and yield; 5′-adenylated U shows little reactivity. Other than these two nucleotides directly at the ligation junction, the two RNA substrates may have nearly any sequence while still allowing substantial ligation rate and yield.…”

New Deoxyribozymes for the Native Ligation of RNA

“…5 -AppRNA is the active intermediate during RNA ligation by protein enzymes (such as T4 RNA ligase), which activate the 5 -phosphate of the RNA donor by adenylation with ATP, and upon reaction with the 3 -OH of the acceptor RNA, AMP is released as a leaving group [23]. A number of convenient routes for the synthesis of 5 -adenylated oligonucleotides have been reported [24][25][26], and these were previously used as donor substrates for in vitro selections of catalytic DNA. Several deoxyribozymes are known to form 2 ,5 -branched nucleic acid architectures with 5 -adenylated RNA and DNA [27,28].…”

Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I

“…Early efforts to halt the ligase reactions after the second step, allowing the adenylylated intermediate to accumulate as the product, mainly used T4 DNA ligase. To stop the reaction after the second step, the fragment to be adenylylated (i.e., the phosphate donor oligonucleotide) was hybridized to a template strand but the acceptor oligonucleotide was either omitted (Chiuman and Li 2002;Vigneault et al 2008) or included with optimized mismatches to the template near the ligation site (Wang and Silverman 2006;Patel et al 2008). Although useful, these early attempts did not work well for all sequences and required the synthesis and subsequent removal of the template and mismatched acceptor DNA.…”