The pSNAPf plasmid from New England Biolabs (cat no. N9183) has an NdeI site (nt 484) and SpeI site (nt 249) that can be used for cloning. Alternatively, a PCR fragment amplified with Taq polymerase will have TA ends that can be cloned into a TA cloning vector such as TOPO® TA vector from Life Technologies.
Yes, due to secondary structure formation chemical synthesis becomes challenging around 70 bases. However, under TriLink’s RNA transcription service much longer aptamers can be made. Learn More>
Libraries with longer random regions have more unique sequence motifs than do libraries with shorter random regions. However, not all possible unique sequences can be represented in each selection. For example, a library with a 30 nucleotide random region has the potential for 1.1 x 1018 unique sequences and a library with a 20 nucleotide random region has 1.1 x 1012 unique sequences. Typical selections start with 1015 copies of library. For a library with 20mer random region, approximately 1,000 copies of each unique sequence are present, while for the corresponding library with a 30mer random region, only one out of every 1,000 unique sequences is represented.
In contrast, libraries with shorter random regions will give you a better representation of all possible sequences but are inherently less complex than a library with a longer randomer region. However, once an aptamer is selected shorter aptamers are easier and less expensive to synthesize.
We offer libraries with different random region lengths, allowing the right balance of library complexity and library representation to be experimentally determined for your selection.
TriLink’s random sites are optimized to produce as close to a 1:1:1:1, A:C:G:T base ratio in the resulting wobble site as possible. TriLink has done extensive research in the area of randomer oligonucleotides. In aptamers any bias can cause misrepresentation of the sequence space, which can limit the search for aptamers that bind the desired target with high specificity. The randomness of TriLink’s library manufacturing process was confirmed by NeoVentures and featured in the whitepaper Validation of Random Library for Aptamer Selection.
Although one would assume the flanking fixed primer regions would play a major role in the resulting aptamer structure, it has been demonstrated, in a bioinformatics study performed in Dr. Andrew Ellington’s lab that these constant regions are only minimally involved in the structures of selected aptamers.
Despite these findings, a number of selections are performed using fixed regions which are shorter than traditional primer binding sites. We have designed our libraries to contain an Ndel site (CA/TATG) upstream of the random region and a Spel site (A/CTAGT) just downstream. This allows the use of restriction endonucleases and ligases in a workflow to minimize the role of the fixed sequence in the selection.
An RNA Library can be made from the DNA Library for DNA Aptamer Selection with the optional T7-Promoter Forward Primer. The T7-Promoter Forward Primer is essentially the same as the forward selection primer, with the exception that it has the T7 promoter sequence on the 5′ end. Use the T7-Promoter Forward Primer in combination with the reverse selection primer in PCR to generate the corresponding DNA template which contains the T7 promoter sequence. This DNA template can be used to generate the RNA library by in vitro transcription using T7 RNA polymerase.