Templated 3ʹ terminal fluorescent labeling of RNA using Klenow DNA polymerase

A long-standing challenge in the study of RNA structure-function dynamics using fluorescence-based methods has been the precise attachment of fluorophores to structured RNA molecules. Despite significant advancements in the field, existing techniques have limitations, especially for 3ʹ end labeling of long, structured RNAs. In response to this challenge, we developed a chemo-enzymatic method that uses Klenow DNA polymerase to label RNAs. In this method:• Klenow DNA polymerase adds an amino-modified nucleotide to the 3ʹ end of the RNA, guided by the DNA oligonucleotide template.• An NHS-ester dye is then conjugated to the amino-modified RNA, forming a covalent amide bond.• For highly structured RNAs, DNA oligonucleotides complementary to the RNA disrupt pre-existing intramolecular RNA structures. This methodological advancement enables site-specific incorporation of a single modified nucleotide at the 3′ terminus of various RNA substrates, irrespective of their length or secondary structure. The user-friendly nature of the technique, with minimal modifications required for different RNA targets, makes it readily adaptable by a broad range of researchers. This approach has the potential to significantly improve the development of functionalized RNA for various applications.


Supplementary material and/or additional information
Additional notes: N+1 addition reaction optimizations 1. Klenow concentration was varied from 0.1 U/µL to 0.4 U/µL in a n+1 addition reaction that was incubated for 2 hours at 37 o C. At 0.1 U/µl we observed the least amount of AAdUTP incorporation, ~ 95%.Maximal incorporation (100%) was observed when the enzyme concentration was increased to 0.2U/ µL with no further efficiency achieved at higher concentrations of Klenow (0.3 and 0.4 U/ µL) (Fig. S3B).2. AAdUTP concentrations ranging from 0.2 mM to 1 mM were evaluated in an n+1 addition reaction time course experiment (2-, 4-, 6-, 8-, and 16-hour incubation) (Fig. S3C).a.Following a 2-hour incubation period, we show that maximal addition (~95%) is observed at 1 mM AAdUTP.At lower concentrations of AAdUTP 0.2, 0.4, and 0.6 mM, the incorporation rate was approximately 20%, 50%, and 70% respectively.b.The optimal conditions maximizing the addition of AAdUTP to the RNA were seen when the AAdUTP concentration was 1 mM with incubation at 4+ hours (100%).The reaction efficiency did not increase when higher incubation times were evaluated.A similar efficiency was also seen when 0.6 mM AAdUTP and a 16-hour incubation were used.3. DNA template concentrations ranging from 1-fold to 10-fold of the RNA concentration were evaluated in native gel shift assays.For the shorter RNAs M1 A , M1 A_Shortened , M1 A_extended ,and M1 B , a 2-fold excess of the DNA template was sufficient to maximize the hybridization of the template with the RNA of interest (Fig. S4).However, for the longer HIV RRE RNA, increasing the DNA template concentration up to 10-fold was not successful in achieving maximal hybridization (Fig. 5).4. We varied the length of the DNA template for M1 B and HIV RRE RNA as described in the earlier section on nucleic acid design and synthesis.Our analysis of native gels in which the different length templates were hybridized to the RNAs revealed that shorter templates were inefficient at hybridizing to the RNA (12 and 17 nucleotides for M1 B RNA) (Figure S4).While we do not suggest a specific length of DNA template to bind to the RNA of interest, we recommend optimizations to identify the optimal length based on the RNA of interest.

Fig. S1 :
Fig. S1: Predicted secondary structures of RNAs used in this study.M1 A_shortened (16 nucleotides) RNA does not have a predicted structure.M1 A (22 nucleotides), M1 A_extended (26 nucleotides), and M1 B (54 nucleotides) have simple, hairpin-like structures.The 233-nucleotide HIV RRE RNA has a more complex structure of 6 stems around a central junction.There is a long stem towards the terminal end of the HIV RRE RNA.The nucleotides complementary to the DNA template are color-coded in blue, and the DNA capture oligonucleotides are color-coded in red, for the HIV RNA.All structure predictions were done using the RNAstructure software.

Fig. S2 :
Fig. S2: Preliminary data showing the templated addition of modified nucleotides 2-thiouridine, pseudouridine, 5methylcytidine and N 6 -methyladenosine (A-D respectively) by Klenow DNA polymerase to the 3ʹ end of RNA.The n+1 addition reaction contained 1 mM of the respective nucleotide and was incubated for 2-16 hours.It is important to note that the preliminary data presented in this figure suggests degradation of the RNA under the tested conditions (A, B, and D) and aberrant gel running conditions (B and D).However, despite these limitations, the figure suggests promising results for the Klenow n+1 addition reaction with modified nucleotides.Further optimization is expected to improve the incorporation efficiency of these and other modified nucleotides.E.Table summarizing the addition of different modified nucleotides in an n+1 addition reaction employing Klenow with a 2-hour incubation.

Fig. S3 :
Fig. S3: Summary of the optimizations for the 3ʹ addition of AAdUTP to RNA.All experiments in this series were carried out using the model RNA M1A (22 nt) at 2.67 µM in a final volume of 25 µL with 1-hour of incubation at 37 o C unless otherwise stated.The n+1 reactions analyzed on the 20% denaturing analytical gels.A. Comparison of the n+1 addition reaction efficiency when the 5ʹ DNA template overhang is either 1-nucleotide or 2-nucleotides.B. The n+1 addition reaction at increasing concentrations of enzyme.C. Comparing the efficiency of the n+1 addition reaction with increasing incubation time and AAdUTP concentration.The 200µM and 400µM samples appears slightly twisted, which may be due to a minor issue during gel electrophoresis.However, the interpretation of the trends in the migration is not impacted because there is a significant shift in the bands upon addition of the AAdUTP and increased incubation time.

Fig. S4 :
Fig. S4: Summary of optimizations of DNA template concentration and length in M1 B RNA.A.A native gel shift analysis was used to test the efficient hybridization of the 37 nucleotide DNA template with the M1 B RNA.The DNA template concentration was increased from a 1-fold to a 10-fold excess of the RNA.Upon increasing the concentration of the DNA template, an RNA-DNA complex was formed.B. A native gel shift assay to test how the length of the DNA template at a 2-fold concentration affected the annealing of the template to the M1 B RNA at room temperature incubation.For the shorter DNA templates (12 and 17 nucleotides), the RNA band was not shifted, suggesting inefficient hybridization under our tested incubation temperature.However, for the longer DNA templates (24 and 37 nucleotides), the RNA band was completely shifted, indicating hybridization of the DNA template with the RNA.