Dear Moran,

Thank you for your question. We see a modest amount of incorporation of 1-Thio-ATP (N-8005) using T7 polymerase (≤25%). However, preliminary studies have shown greater incorporation the other 1-Thio analogs (≥50%). For 2´F incorporation (N-1007) we recommend using T7 R&DNA™. Though we have not tested 2´ OMe analogs with wt T7 polymerase, this paper suggests that T7 polymerase with a Y639F/H784A double mutation is optimal for incorporation.

Please let me know if you have further questions.

Regards,
Brea

Dear Vivian,

Thank you for your question. Unfortunately, we cannot provide expiration dates on specific oligonucleotides, as this data could differ with each individual sequence. As long as an oligonucleotide is stored at -20°C or lower for long term storage and multiple freeze thaw cycles are avoided, we have seen that dye labeled oligonucleotides can be stable up to 6 months and unmodified oligonucleotides stable up to 1+ years.

Best regards,
Elizabeth

Dear Chris,

Thank you for your questions about our Aptamer Libraries. NeoVentures Biotechnology analyzed a TriLink library to show the randomness of the library by enzymatic digest and by sequencing.

The pSNAPf Vector from New England Biolabs contains both a NdeI site and an SpeI site. Alternatively, a PCR fragment amplified with Taq polymerase will have TA ends that can be cloned into aTOPO® TA vector from Life Technologies.

Best regards,
Elizabeth

Dear Jennifer,

You can order a synthesis of each of the oligos you need through our OligoBuilder® system. OligoBuilder® will provide a price based on the scale and purification required. If you unsure of the scale, you can include the amount of material needed in the comments sections.

You can reference our OligoBuilder® Help Section or contact us if you’d like a further explanation.

Best regards,
Elizabeth

Dear Samuel,

Thank you for your question. The inclusion of modified bases in a transcription can alter several parameters. If you are including ARCA in the reaction and reducing the GTP (e.g. 4:1 ARCA:GTP) in order to give good capping, this will reduce your expected transcription yields by about 3-4 fold. In our hands, full substitution of U and C with pseudo-U and 5-methyl-C do no significantly reduce transcription yields. For an ARCA cappedmRNA  fully substituted with pseudo-U and 5-methyl-C, we typically see crude yields of 1.5-2 mg/ml of transcription. We optimize transcription reactions by varying the MgCl₂ concentration. 4-6 mM over total NTPs usually works well.

With regards to the 260/280 ratio, the extinction coefficients and lambda maxes of pseudo-U and 5-methyl C differ from U and C. The lambda max for pseudo-U is 265 vs 262 and 5-Methyl-C is 279 vs 271. Thus, pseudo-U and 5-methyl-C substitution would be predicted to give you a higher 260/280 ratio than normal NTPs. If you are observing a lower 260/280 ratio, this might mean that you are actually doing a better job of removing protein in this prep. I would not be concerned by this.

With regard to mobility, we do sometimes see that some modifications change the mobility of the RNA on gels. Base modification can change the secondary structure of the mRNA and thus the mobility on non-denaturing gels. We recommend glyoxal treating the mRNA with NorthernMax®-Gly Loading Dye and then running it with NorthernMax® 10X Running Buffer (do not use TAE or TBE).

I hope this information is helpful. If you continue to have problems, you could considering order a custom mRNA.

Best regards,
Anton McCaffrey, PhD
Principal Scientist

Dear Andrew,

We typically not do recommend using a subtraction correction and recommend measuring the concentration of a T16 oligo at 260 nm, the wavelength at which thymidine absorbs. If you are trying to employ a correction for a specific buffer, the exact subtraction would depend upon the buffer you are using. Please contact us for additional information.

Best regards,
Elizabeth

Dear Molly,

Thank you for your questions. The 3′ Biotin TEG modification does include a phosphate group between the TEG linker and the first nucleotide in your sequence. We look forward to helping you with your project!

Best regards,
Elizabeth

Dear Aaron,

A paper by Clark et. al. which studied the affect of different analogs on DNA methyltransferase showed that while dG and 7-deaza-dG will inhibit methyltransferase to a similar extent, they did not review the scenario where dC is upstream from 7-deaza-dG. The findings show that dG and 7-deaza-dG have similar properties and therfore, dC followed by 7-deaza-dG may be tolerated by the DNA methyltransferase. The only sure answer can be found by experimental testing.

Best regards,
Elizabeth

The only analog that has the properties you described is our CleanAmp™ dNTPs. Although the protecting group will not be stable during the elevated temperatures of PCR, certain enzymes such as the Therminator series of enzymes can incorporate this type of analogs. However, incorporation would need to be performed at low temperatures, such as at room temp. Then, a heat activation step can be used to remove the protecting group.

The first step is to identify the aptamer for the analyte of choice using one of the many selection protocols available. We recommend that you use a random library with no more than 40 random bases. Longer ones will yield aptamers that will be more difficult and expensive to prepare synthetically, especially if the aptamer is heavily modified. You can review our stocked libraries to see if one may be used during your selection.

Once you have the sequence, it is fairly easy for us to modify your aptamer with a reactive moiety for conjugation to a metal. For instance, we can put sulfur on the aptamer for direct conjugation to a gold particle. Once you have identified the metal(gold is well established in the literature) we can help with the decision regarding chemistry.

Please contact us for more details.

Thank you for your interest in our RNA transcription service. We believe our capabilities are only limited by customers imaginations. Please submit a quote request with the details of your project to initiate discussions with our Product Management Team.

We understand you are looking to perform an aptamer selection for a metabolite of interest. Once selected, you will put this into one of the intergenic regions. In the absence of metabolite, this region will be linear, while in the presence of metabolite, this region will fold into a complex structure. The presence of this structure will then block translation. Therefore, we believe you are asking if you can design your aptamer library to have a 5’- and 3’- end that is fixed to match the intergenic sequences, with a random number of nucleotides (N20, N30) in between. And the answer to this is yes! We would be happy to make a custom library for you. This construct can be priced and ordered through OligoBuilder.

A 5′ amine modified oligonucleotide will be delivered ready to conjugate to a succinimidyl ester.

Please keep in mind that TriLink can perform the conjugation and purify the final product for you. We can conjugate most commercial succinimidyl esters or one you supply.

We run mass spec and PAGE analysis on every oligo ordered as part of our quality system. The PAGE analysis allows qualitative assessment and mass spec confirms identity. An HPLC or gel densitometry is required for quantification of purity. HPLC and other analytics can be requested at the time of order. If you are not sure which analytical would be best for your requirement, just ask!

In regards to the DNP-TEG labeled oligonucleotides, we recommend requesting an AX-HPLC analysis.

In this whitepaper, we quantified the amount of amplicon formed as a function of the amount of input biotinylated nucleotide by gel densitometry. In particular, the percent amplicon yield for a given percentage of biotinylated nucleotide was normalized to an reaction which contained only natural nucleotides.

While this approach can be used to identify optimal reaction conditions that will allow for biotinylated nucleotide incorporation in PCR without sacrificing amplicon yield, this does not measure the amount of biotin in the PCR product itself. Should direct quantification of the biotin content be of interest, enzymatic digestion of the isolated product is one way to do this. Eadie, et. al.and Andrus, et. al.describe this technique in their publications.

A. Photobleaching of different fluorophores has been reviewed for use in a number of applications including quantitative microscopy and fluorescence photobleaching recovery (FPR). Dailey, et. al. reviewed studies of photobleaching in different systems, as well as different methods employed to reduce photobleaching.

While 6-FAM is less susceptible to photobleaching than FITC, newer dyes have been developed that are more stable. For example, Panchuk-Voloshina, et al. demonstrated the stability of Alexa Dyes over FAM and FITC.

A. We have documented Biotin-AA-dCTP (N-5002) incorporating at a very high percent substitution for dCTP, up to 97%, in our white paper: PCR incorporation of modified dNTPs: the substrate properties of biotinylated dNTPs. We do not have analagous PCR data on the efficiency of the N4-Biotin-OBEA-dCTP (N-5004), but since the modification is to the N4 nitrogen, it may interfere with Watson-Crick base pairing. Therefore, while this dCTP analog can be incorporated in PCR, the percent substitution for dCTP may not be as high as for Biotin-AA-dCTP.
As you eluded to, different analogs will incorporate in PCR with varying efficiency and some optimization may be needed. We recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions for modified nucleotide incorporation with robust amplicon yield.

1-Thio-2′-Deoxyadenosine-5′-Triphosphate (N-8001) and 1-Thio-2′-Deoxycytidine-5′-Triphosphate (N-8002) can be incorporated by Taq in PCR. However, not all analogs will incorporate with the same efficiency in PCR; some optimization may be needed. We recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions for modified nucleotide incorporation with robust amplicon yield.

We recommend resuspending oligonucleotides in aqueous, neutral solution. It would be best to resuspend your trimer oligonucleotide in DDI water. If you have any further questions, please do not hesitate to contact us.

Have a question about specialty oligonucleotides? Post a comment below.

Thank you for your question about how to best conjugate 2′-F-RNA aptamers onto nanoparticles. Depending on the type of nanoparticle you use (e.g., gold, quantum dot, magnetic or carbohydrate nanoparticles), there are different approaches for how to perform the conjugation… Read More

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