Compounds that enhance the tailing activity of Moloney murine leukemia virus reverse transcriptase

In a previous study, we showed that MMLV-RT has a strong terminal transferase activity, and that the C-, G-, and T-tailing activities are enhanced by dGMP, dCMP, and dAMP, respectively. In this study, to achieve faster reaction and higher tailing efficiency, we screened other compounds for the ability to enhance the tailing activities of MMLV-RT, and determined the corresponding optimal concentrations. The C-, G-, and T-tailing activities were enhanced by guanine, cytosine, and adenine, respectively, and by derivatives thereof, suggesting a transient Watson-Click base pairing between an enhancer molecule and the nucleotide to be incorporated. In the presence of some additives (GMP and GDP for C-tailing and CMP for G-tailing), the tail length increased continuously, resulting in tail lengths of 7 to 15 (GMP and GDP) or 13 to 22 (CMP) nucleotides. Among the compounds that do not induce continuous addition, adenosine, deoxycytidine, and deoxyguanosine mostly enhanced T-, G-, and C-tailings, respectively. The enhancing chemicals described here will improve the feasibility of N-tailing by MMLV-RT in various biotechnological applications.


Materials and Methods
DNA preparation. FAM70 DNA was prepared as described previously 6 . In brief, a PCR reaction was carried out using a FAM-labeled primer and the product was digested with PvuII. The resulting 70-bp fragment carrying FAM at one end was purified by polyacrylamide gel electrophoresis and ethanol precipitation. The nucleotide sequence of FAM70 is 5′-FAM-AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGC GTCAGATGTGTATAAGAGACAG-3′. The concentration of FAM-70 DNA was determined using an Infinite 200 Fluorescence Spectrophotometer (Tecan, Switzerland), and the FAM-labeled primer was used as a standard.
Tailing reactions. Wild-type MMLV-RT (200 U/µL) was purchased from Nippon Gene (Japan). MMLV-RT (200 U, approximately 1.8 µg of protein) was subjected to SDS-PAGE analysis to confirm its purity; a single band with a size consistent with the expected size (70 kDa) was observed. The reaction mixture contained, in a total volume of 10 µL, 100 fmols of substrate DNA, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 6 mM MgCl 2 , 2 mM DTT, 4 mM dATP, dCTP, dGTP, or dTTP, 4 mM MnCl 2 , and 50 U MMLV-RT. Reactions were carried out in PCR tubes using a thermal cycler (C1000; Bio-Rad, USA) at 30 °C. A and G-tailing reactions proceeded faster than C and T-tailing reactions, and to evaluate the effects of additives, A-and G-tailing reactions were conducted for 2 min and C-and T-tailing reactions for 5 min. An aliquot of 0.5 µL of the reaction mixture was analyzed by capillary sequencing (see below).
Reagents. Reagents were purchased from Takara (Japan), Sigma-Aldrich (USA), Tokyo Chemical Industry (Japan) or Nacalai Tesuque (Japan). Guanine was dissolved in 0.3 M NaOH, and the other chemicals were dissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Four reagents guanosine (rG), deoxyguanosine (dG), adenosine (rA), and deoxyadenosine (dA) were prepared as saturated solution in TE buffer. The saturated solutions were stored at 4 °C and were heated at 100 °C for 3 minutes and cooled to room temperature (25 °C) before use. The molar extinction coefficients of 24,000, 17,600, 13,300, and 17,200, which were obtained by measuring solutions of authentic rA, dA, rG, and dG, respectively, were used to spectrophotometrically calculate the saturated concentrations.

DNA length analysis.
Changes in size of the FAM70 DNA were analyzed by a capillary sequencing as described previously 6 . Briefly, samples were added to the HiDi formamide-containing GeneScan-500 LIZ Size Standard (Thermo Fisher Scientific, USA), which contains 16 fragments of known size, and size-separated in a 3130xl Genetic Analyzer (Thermo Fisher Scientific, USA). TraceViewer software 6 was used to analyze the data, in which it aligned different run data by using two peaks from the size standard.

Results
Compounds that enhance MMLV-RT tailing reactions. Because C-, G-, and T-tailings are specifically enhanced by dGMP, dCMP, and dAMP, respectively, and transient Watson-Crick base pairing between an enhancer molecule and a deoxyribonucleoside triphosphate molecule about to be incorporated has been proposed 6, 10 , we questioned whether any compound with a base moiety that can base-pair with the base moiety of a nucleoside triphosphate to be incorporated can enhance the tailing reaction. We expected that some such compounds may exhibit enhancing activities superior to those of dGMP, dCMP, and dAMP.
The tailing assay was conducted as described previously 6 , using a 70-bp DNA molecule having one 5′ end labeled with FAM (designated FAM70) and the 3′ end of the FAM-labeled strand being the target for tailing reactions. The FAM-labeled strand was subjected to capillary sequencing, and TraceViewer software was used to analyze the results.
Optimal concentrations for tailing reaction enhancement. We investigated the optimal concentrations of each compound for enhancing the tailing reaction (Fig. 2a), and Fig. 3 shows the results of tailing experiments conducted using compounds at optimal concentrations. We excluded bases, as the enhancing effects were limited and small. We also excluded NTPs because MMLV-RT incorporates NTPs to the ends of double-stranded DNA, although to a limited extent (see Fig. 4). The effects of each compound were evaluated by calculating the tailing index values, which are the average number of nucleotides that a single DNA end has acquired (Fig. 2b). For example, in the absence of additives the G-tailing index was 1.97. In the presence of 10 mM deoxycytidine (dC) the value increased to 2.15, and a concentration of 200 mM dC led to the highest observed G-tailing value of 3.85.

Continuous tail extension induced by GMP, GDP, and CMP.
We investigated whether the tail is extended continuously or the extension stops after reaching a certain length. Figure 5 shows the tail length profile after 2 h, 5 h, and 10 h of incubation. For C-tailing, GMP and GDP induced continuous addition that led to extensions of 7 to 15 Cs. For G-tailing, CMP induced the continuous addition of Gs (13 to 22). Specificities of the tailing reaction enhancements. We investigated the enhancement specificity of compounds that considerably enhanced the tailing reaction, including those that induced continuous extension ( Table 1). Most of these compounds enhanced specific tailing reactions, whereas adenosine, which mostly enhanced T-tailing, also enhanced C-tailing. Likewise, CDP enhanced G-tailing and also C-tailing. Some compounds suppressed tailing reactions; dC suppressed A-tailing, GDP suppressed G-tailing, and GMP suppressed G-tailing.

Discussion
In this study, the strong tailing activity of MMLV-RT 6 was further characterized by identifying additional enhancing compounds. The molecular mechanism involved in the tailing enhancement could be the transient Watson-Click base pairing formed between an enhancer molecule and the deoxyribonucleotide triphosphate molecule to be incorporated. We propose that an enhancer molecule binds to the active center of MMLV-RT in an orientation similar to that taken by a single ribonucleotide moiety in an RNA molecule in a revers transcription reaction. Of note, bases are not efficient enhancers, suggesting that the base-moiety and the ribose or deoxyribose moiety of the compounds are involved in the binding. Although we have tested different compounds, other nucleotide analogues, such as cyclic AMP, NADH, and cyclic diGMP, or the wealth of compounds developed for anti-cancer therapy, might have even stronger effects, and the 3D structure of MMLV-RT 11 and molecular docking approaches may help the search for better tailing enhancers.
Although compounds that are supposed to form Watson-Crick base pairing with dATP were tested, none of them enhanced A-tailing. The base-stacking interaction of dATP with the deoxyribose or base of the DNA ends 7 might be so strong that the two hydrogen bonds provided by the enhancer molecule do not promote the tailing reaction. In fact, in the absence of enhancer, dATP is incorporated at the fastest rate among dNTPs, and three to five As are added within 10 minutes.
The continuous nucleotide addition by MMLV-RT in the presence of GMP, GDP, and CMP, similar to that of terminal nucleotidyl transferase 12 , might be useful in technological applications, such as labeling of DNA ends, TUNEL assays 13 , RACE 14 , and homopolymer tail-mediated ligation PCR 15 . The different extent of effects of enhancers on the different tailing reactions described in this study will form a basis for utilization of the template-independent tailing activity of MMLV-RT. Although its practical use needs further improvements for each specific application, the activity may be utilized for efficient ligation of adaptor molecules to DNA ends in next-generation sequencing [16][17][18]traditional TA 19 or CG cloning 20 , and cosmid library preparations.