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 I.
Table I. PBS length and preferred tRNA types of well-studied retroviruses
Name of retrovirus |
Length of PBS |
Preferred tRNA type |
Reference |
Moloney murine leukemia virus (MMLV) |
18 |
tRNAPro |
Kosloff et al. |
Human immunodeficiency virus type 1(HIV-1) |
18 |
tRNALys |
Kosloff et al. |
Rous sarcoma virus (RSV) |
17 |
tRNATrp |
Kulpa et al. |
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.