Our software simplifies the primer design process for target-specific mutagenesis via reverse transcriptase (RT). We called it tRNA primer designer. Studies have shown that tRNA functions as the primer for in vivo reverse transcription initiation: the 5' end of the tRNA interacts with RT, and the 3' end matches with the mRNA encoding the target. The software consists of 4 parts: reverse transcriptase selection, target sequence input, designed-tRNA visualization, and primer output. Although we only test MMLV-RT experimentally, the software can adjust its designing method based on the properties of well-studied RT from 3 species, MMLV, HIV-1 and RSV. Users could design their tRNA primers even for eukaryotic experiments. In addition, we calculate and output the tRNA acceptor stem annealing temperature, as this might be used as an indicator for likelihood to success.

Motivation

Previous studies have shown that a tRNA primer is required for the initiation of reverse transcription (Dahlberg et al.). In our system, we express the tRNA primer in E. coli by cloning it onto the plasmid that is used for generating the tools for mutation, i.e. P_mutant. However, designing the primer sequence according to different target sequences is time-consuming and needs many adjustments to find the perfect match. This motivates us to build a software for tRNA primer designing.

Theoretical basis

Previous studies have reported that the interactions between tRNA primer and mRNA template as well as the reverse transcriptase are crucial in directing subsequent cDNA synthesis (James E. Dahlberg et al.). Specifically, according to the model for reverse transcription proposed by Kulpa et al., reverse transcription includes 5 steps (Figure 1), in which the annealing of tRNA primer to the primer binding site (PBS) region on mRNA template is crucial for the synthesis of minus strand strong stop DNA (–ssDNA) and the following cDNA synthesis process.

Many researchers have studied the reverse transcription process in viruses, from which we find two critical properties in the annealing process of tRNA primer and PBS that should be taken into consideration when building the tRNA primer designer.

The first property is that the 3'-terminal of the tRNA primer should be complementary to the PBS on mRNA template (Kosloff et al.). The second one is that different viruses prefer specific type of tRNA primer for reverse transcription (Kulpa et al., Kosloff et al.). What should also be noted is that for different viruses, the lengths of PBS as well as the types of tRNA primer are different. The PBS lengths and the preferred tRNA types of 3 most well-studied retroviruses are listed in Table 1.

These discoveries serve as the theoretical basis for our tRNA primer designer. So basically, the function of our tRNA primer designer is to change the tRNA template in order to suit the basic properties of the reverse transcriptase (MMLV RT / HIV-1 RT / RSV RT) selected by the user as well as to replace several nucleotides (17 or 18) on 3'-terminal of the tRNA templates to match with nucleotides at the 5'-terminal of the GOI which users input. Also, to make sure that the RNA sequence is a tRNA sequence, the secondary structure should be revealed. We achieve this goal by using the similar tRNA secondary structure prediction scheme as the one implemented in the opensource software tRNAfinder (Kurokawa et al.).

Studies have shown that the primary factor guiding the selection of tRNA primer for MMLV-RT is the PBS sequence instead of the inherent nature of reverse transcriptase (A. H. Lund et al., S. P. Goff et al.). So, by making mutations on both the PBS and tRNA sequence, the researchers have found that reverse transcription could still successfully take place while the virus’ titer is not greatly affected. Also, after several cycles of replication, the mutated sequence is not changed back to its original version (Pedersen et al., 1997). Even though it is found that the primer is not stringent for MMLV, studies have revealed that the tRNA-like structure is necessary. A study that the inclusion of one single non-Watson-Crick base pair between PBS and tRNA primer would improve the replication efficiency (F. S. Pedersen et al., 1993), but we didn’t adopt this construct as the one base pair mismatch would often be changed to the full-complementary version after the first cycle of replication (Pedersen et al., 1997), making this addition unnecessary.

User guidelines

Our tRNA primer designer is a web tool for potential users of our mutagenesis system to design their own tRNA primers according to their experimental setups. Here we provide a step-by-step guide to using this software.

Step 1. Select the type of reverse transcriptase (RT) that you want to use based on your experimental design, as shown in the figure below. Note that this software only allows you to choose from MMLV RT / HIV-1 RT / RSV RT.

Step 2. Input a DNA sequence that you want to mutate, as shown in the figure below (upper panel). You can find a demo for it if you click on the icon on the right side of the web page, as shown in the figure below (lower panel). The last 17/18 nucleotides (nt) of the sequence are selected to be PBS, depending on the type of RT that you have chosen in Step 1. The length of PBS is 18nt if MMLV RT / HIV-1 RT is selected, and is 17nt if RSV RT is selected.

Note that the target sequence should be longer than 17 or 18 nucleotides based on your selected RT. Besides, it shouldn't contain any characters other than A/T/C/G. If any occurs, there will be an error message.

Step 3. Click on the "submit" button and see the result, as shown in the figure below (upper panel). The result is composed of two parts. The first part shows you the secondary structure of the template tRNA that you will be using as well as the designed tRNA primer. The fragment that can be annealed to PBS of the input DNA sequence is shown in red. The second part will give you the DNA sequence encoding the tRNA primer that satisfies your need. You can just copy it and use it elsewhere. These two parts are demonstrated in the figure below (lower panel).

References

1. [1]. Peters G , Dahlberg J E . RNA-directed DNA synthesis in Moloney murine leukemia virus: interaction between the primer tRNA and the genome RNA.[J]. Journal of Virology, 1979, 31(2):398-407.
2. [2]. Kulpa, D. Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors[J]. EMBO (European Molecular Biology Organization) Journal, 1997, 16(4):856-865.
3. [3]. Palmer M T , Kirkman R , Kosloff B R , et al. tRNA Isoacceptor Preference prior to Retrovirus Gag-Pol Junction Links Primer Selection and Viral Translation[J]. Journal of Virology, 2007, 81(9):4397-4404.
4. [4]. Kinouchi M , Kurokawa K . [Special Issue: Fact Databases and Freewares] tRNAfinder: A Software System To Find All tRNA Genes in the DNA Sequence Based on the Cloverleaf Secondary Structure[J]. Journal of Computer Aided Chemistry, 2006, 7:116-124.
5. [5]. Lund, Anders H. et al. “Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication.” Journal of virology, 67 12 (1993): 7125-30.

tRNA primer

A demonstration of our software is embeded below. And, we have a beautified version running on Amazon EC2.