I would like to request quotation for a thiol-modified oligonucleotides. Is it possible to cross link the thiol-modified oligos to gold surface with succinimidyl 4-[maleimidophenyl]butyrate (SMPB)? What is the differences between 3′ C3, 3′ C6 and 3′ C6 Disulfide linker? -Lim

Dear Lim,

Thank you for your interest in TriLink’s custom oligo synthesis. Thethree thiol linkers you mention differ by the position on the oligonucleotide (5′ or 3′) and the linker length (3 or 6 carbon chain). The structure of the reduced thiol linker can be found on each product page. Thiol modified oligonucleotides are shipped as the protected thiol (for example, C6-S-S-C6, aka disulfide) to avoid dimerization. Prior to use, the disulfide can be reduced using TCEP. Thiol modified oligos can be ordered through OligoBuilder®.

Thiol modified oligos can be direclty linked to a gold surface as described by Li Z, Jin R, Mirkin CA, Letsinger RL. .

Please let us know if you have any additional questions.

Best regards,

I would like to synthesize the following oligo: 5′-CAGGGGCCTACACGTTGTA-3′. The problem I understand is the 4 Gs (starting at base 3) in a row will potentially cause synthesis issue. Also this G-quadruplex will potentially form undesirable secondary structure for PCR applications in the presence of potassium. To mitigate, I learned that it’s possible to synthesize an oligo with a G analog, such as 7-Deaza-2′-deoxyguanosine to lessen the chance for the potential problems mentioned above. If this analog indeed is the best course of action, what’s the best position(s) to insert this G analog, and how many to insert? However, I also read inserting multiple 7-Deaza-2′-deoxyguanosine could be problematic because 7-deaza-dG is sensitive to the iodine-based oxidizer solution used in phosphoramidite-based DNA synthesis. So, a suggestion is to substitute 7-deaza-dG with 7-deaza-8-aza-dG, which I’m not sure you have available as an option. -Chun-Nan

Dear Chun-Nan,

Thank you for your interest in TriLink. Poly guanosine and deoxyguanosine stretches have the propensity to form tetraplexes (complexes consisting of four different strands all bound at the poly G region). These complexes are very tight and make oligonucleotide synthesis difficult.

The use of 7-deaza-dG during PCR reduces the secondary structure but, unfortunately, the 7-deaza-dG and 7-deaza-8-aza-dG amidites have the same innate properties as guanosine during oligonucleotide synthesis. Synthetic and processing capabilities would still be problematic. For multiple incorporations of 7-deaza-dG, CSO is recommended as the oxidizing solution to combat degradation.

TriLink has devised specialized protocols for multiple and/or consecutive insertions of guanosine or 7-deaza guanosine. Due to the complexity of manufacturing, there is typically reduced yield or purity compared to a sequence with better base distribution. The number of incorporations of guanosine, 7-deaza-guanosine and/or 7-deaza-8-aza- guanosine will determine the final effect.

I would recommend trying the unmodified compound before proceeding with the modified version as the cost of the modifications will lead to an overall higher price.

Best regards,
Katelyn Murphy
Product Manager

I would like to order a DNA oligo which contains 12bp random nucleotides (Ns) in the middle, if I wish to have all 4^12=16777216 possible oligos, what synthesis scale order I should place with you guys? Can you tell me how could assure that? -Lisa

Dear Lisa,

Thank you for interest in our randomer service. The expected yield of our various scales can be found on the Phosphodiester Product Information. An 0.2 μmole HPLC purified, DNA synthesis typically yields 5-15 OD260 units which is 25-75 nmoles of final product for an oligo with an extinction coefficient of 200 OD260/μmole. 25 nmoles of product is equivalent to 1.5 x 1016 sequences. You would expect on average 9.0 x 108 copes of each of your ~1.7 x 107 possible sequences within the 25 nmole yield.

TriLink has done extensive research to optimize the “n” wobble (A, C, G, T) to achieve as close to a 1:1:1:1 equal ratio as possible in the final oligo product. We also offer an enzymatic digest assay which measures the base distribution of the final product. You can order your oligo through OligoBuilder® and include your yield requirement and if you are interested in an enzymatic digest in the Notes.

Best regards,
Natasha Paul, PhD

I noticed you offer libraries with varying lengths of random regions, which one should I use?

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.

What is the effect of the fixed primer sequences flanking my random region?

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.