Custom Morpholinos, Controls and End Modifications

Morpholino Antisense Oligos function by binding to pre-messenger or messenger RNA, blocking access to the RNA. Morpholinos function solely by a steric block mechanism, so they do not require or use RNase H for their activity. When properly selected and targeted, Morpholinos generally afford equal or better efficacy and greatly superior specificity than any other antisense structural type. However, inappropriately selected or targeted Morpholinos will have little or no activity. Accordingly, Gene Tools provides an Oligo Design Service at no added cost. Gene Tools follows our Targeting Guidlines to select appropriate oligo sequences suitable for inhibiting the activity of the genes of your choice.

For quantity and price information on custom-sequence Morpholino antisense oligos go to Current Price List.

For selecting and ordering Morpholino oligos visit our Online Store.

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Control Oligos

Standard Control: A Morpholino Standard Control oligo is offered at a reduced price per nanomole for small orders (100 to 300 nanomoles) to minimize the expense of antisense experiments. This Standard Control oligo is a 25-mer having the sequence:

5'-CCT CTT ACC TCA GTT ACA ATT TAT A 3'

This oligo should have no target and very little biological activity - except in reticulocytes from thallasemic humans having a splice-generating mutation at position 705 in beta-globin pre-mRNA. In those cells this oligo will correct a splicing error and thereby generate a correctly-spliced mRNA which codes for normal beta-globin chains. However, slight changes from mock-injected embryos have been observed in zebrafish; any single sequence is likely to have some effect on gene expression. The Standard Control is a widely used and often reported sequence that is a pretty good, but not perfect, negative control.

For quantity and price information on this Standard Control oligo go to Current Price List.

Random Control: The Random Control 25-N is a mixture of many oligo sequences, made be adding a mixture of activated Morpholino subunits at each step of the synthesis cycle. There are in theory 4^25 different sequences in a large batch of the random control mixture. In a solution containing 1 mM of the Morpholino backbone, any one sequence is present in vanishingly small concentration.

Custom Negative Controls: Custom-sequence Morpholino negative control oligos can also be ordered. If the standard or random control oligos are unsuitable as negative controls for your experiments, we recommend using the invert of your antisense sequence as your negative control. This invert-antisense has the advantage of having the same length and base composition as your antisense oligo.

Here is an example based on an arbitrary antisense sequence:

antisense sequence: 5'-AAC GAA CGA ACG AAC GAA CGA ACG C

invert antisense: 5'-CGC AAG CAA GCA AGC AAG CAA GCA A

A less desirable negative control is the sense sequence. Sometimes odd expression modulation is observed with sense controls, such as upregulation of the gene of interest (hypothetically due to DNA interactions).

antisense sequence: 5'-AAC GAA CGA ACG AAC GAA CGA ACG C

sense sequence: 5'-GCG TTC GTT CGT TCG TTC GTT CGT T

Custom Specificity Controls: Custom-sequence Morpholino specificity control oligos include non-overlapping antisense control oligos and five-mispair control oligos.

For rigorous specificity studies we recommend using a second non-overlapping oligo targeting the same RNA as the first antisense oligo. If the outcome of a knockdown is recapitulated when using a second oligo targeting the same mRNA as the first, this supports the hypothesis that the outcome observed was due to interaction with the targeted RNA and not an unexpected RNA.

Another specificity control is the five-mispair oligo, an option used for antisense studies throughout the early history of antisense; we recommend that you do not use the five mispair oligo as a specificity control. Unexpected effects sometimes appear when using five-mispair oligos. To most effectively decrease interaction with the antisense oligo's target RNA, the mispairs should be distributed through the sequence of the five-mispair control oligo.

antisense sequence: 5'-AAC GAA CGA ACG AAC GAA CGA ACG C

5-mispair control: 5'-AAg GAA gGA ACc AAC GAA CcA AgG C

It is noteworthy that in early studies of sequence specificity utilizing such mispaired controls, Morpholino oligos were found to afford high-specificity target inhibition over a concentration range several hundred fold greater than that afforded by corresponding Phosphorothioate oligos (Summerton et al., 1997; Summerton & Weller, 1997a).

For quantity and price information on custom-sequence Morpholino control oligos go to Current Price List.

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End Modifications

Gene Tools can provide Morpholino oligos with modifications on the 3' and/or 5' ends of the oligos. Available structures are shown below.

Linked index of end modifications:

3'-Fluorescent tags and quencher

3'-Carboxyfluorescein (Now available as a 5' modification for Vivo Morpholinos; See here for description)
3'-Gene Tools Blue
3'-Lissamine
3'-Dabcyl
3'-Sulfo-Cyanine5
3'-AF488

3'-Affinity tag and functional groups for chemical linkage

3'-Biotin synthesized prior to 24 Jun 2016
3'-Biotin synthesized 24 Jun 2016 and after
3'-Primary Amine
3'-Disulfide Amide
3'-Pyridyl dithio
3'-Cholesterol

5'-end Modifications

5'-Primary Amine
5'-Dabcyl
5'-Triethylene Glycol
5'-Hydroxy
5'-Disulfideamide

Modifications for Click Chemistry

3'-Azide
3'-Alkyne
3'-Cyclooctyne v1, no longer available
3'-Cyclooctyne v2, no longer available
3'-Cyclooctyne v3, current version
5'-Azide (Now available as a 5' modification for Vivo Morpholinos; See here for description)
5'-Alkyne
5'-Cyclooctyne v1, no longer available
5'-Cyclooctyne v2, no longer available
5'-Cyclooctyne v3, current version

3'-Fluorescent tags and quencher

3'-Carboxyfluorescein
Green-emitting fluorescent tag
Adds 489 Daltons to oligo mass
Production number suffix:-F
Example: 12-04Apr05A-F

Excitation peak: 501.5 nm
Emission peak: 524.5 nm

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Custom Morpholinos, Controls and End Modifications