Single-stranded RNA Synthesis

Single-stranded RNA Synthesis

Create and order RNA molecules with a wide variety of chemical modifications or learn about our capabilities for long RNA oligos, dye labeling and custom amidites.

Single-stranded RNA Synthesis

NOTE: These tools are for ordering RNA and RNA-DNA chimeras only

Dharmacon RNA synthesis capabilities are well beyond the industry standard

  • Long RNA oligos
  • Custom RNA inhibitors for the study of microRNA biology
  • Custom phosphoramidites
  • Specialized chemical modifications or modified bases

Custom phosphoramidite production can be requested when the modified base you require is not commercially available or requires a chemical adaptation for our chemistry platform.

Please contact Technical Support for more information.

Dharmacon routinely synthesizes RNA oligonucleotides of 80 nt and longer and consistently obtains superior levels of purity.

The ability to synthesize longer oligos is highly sequence-dependent due to the potential for secondary structure formation and the inherent difficulty for chemical synthesis of certain sequences. Reduced overall yield is expected as oligo length increases.

The chemistry experts at Dharmacon can customize RNA phosphoramidites to accommodate your unique requests.

When you need a non-standard chemical modification or modified nucleobase that is not available online, we may still be able to help.

To learn more about our capabilities for custom amidite synthesis and to determine feasibility for your project, please contact Technical Support. You may also request a quote using our online submission form.

If contacting our scientists by email, please consider including a chemical structure or an attached publication where your request is described - this will help our chemists determine feasibility and will help to facilitate a more timely response.

How today's scientists use RNA oligos:

Single-stranded RNA may be used for:
Custom microRNA inhibitors
Aptamers
Ribozymes
tRNAs
FISH/ISH probes
FRET probes
RNA/DNA hybrids
Custom RNA duplexes
Dicer substrates
Antisense oligos
Enzyme substrates
Applications for RNA may involve:
in vitro or in vivo applications
Virology
miRNA functional studies
X-ray crystallography, NMR
RNAi
Transcription / translation
Assay development
Microarrays
RNA-mediated cellular mechanisms
RNA/protein interactions
RNA chemistry

Deprotection

Single-stranded RNA can be shipped in its 2' protected form to improve stability.

As a result of chemical RNA synthesis, all oligonucleotides will have chemical protecting groups at the 2' position of each nucleotide.

2'-ACE chemistry is unique among all others because no other chemistry platform offers the option of delivering the product in the protected form. Some of the benefits of working with 2'-protected RNA include:

  • Perform additional chemical manuipulations to the oligo prior to deprotection
  • Achieve an enhanced nuclease resistance conferred to the final product*
  • Maintain water-solubility and stability during long-term storage
  • Easily remove protecting groups under mildly acidic conditions

Protected RNA must be deprotected prior to use. You have two options for deprotecting your RNA oligos:

  1. Perform the deprotection reaction in your lab (default)
    This is a simple 30 minute procedure, and the deprotection buffer will be provided with your order at no charge. The protocol can be found here.
  2. Choose to have your RNA oligo deprotected prior to shipping
    For a small processing fee you will receive your oligo in the "ready-to-use" state following resuspension. If you wish to have your RNA deprotected by us, simply check the box "2'-Deprotect / Desalt" when placing your order.
Deprocated RNA

Dye Labels

Dharmacon offers a broad portfolio of dye modifications for fluorescent labeling of your custom siRNA and RNA oligonucleotides.

  • Include a fluorescent tag at the 3', 5' or internal positions of an RNA oligo
  • Potential for multiple labels within a single oligo not limited by position within the sequence
  • Synthesis of most dye-labeled oligos is scalable to gram quantities

Fluorescent dyes available online:

*Some 3' dye labels are only available upon request.
Fluorophore λ max abs (nm) λ max em (nm) Comparable to 5' or 3'
Fluorescein / 6-FAM 494 520 - both*
DY547 548 562 Cy3, Alexa 546, Alexa 555 both*
TAMRA 565 580 - both*
DY647 645 662 Cy5, Alexa 647 both*
DY677 684 698 CY5.5, Alexa 680 both*
Cy3 547 563 - both*
Cy5 646 662 - both*
Cy5.5 688 707 - both*

Some of the dye options listed in the above tables will produce higher oligo yields than others. For assistance in choosing the most appropriate dye label for your application, please contact Technical Support.You can also request a quote online if you already know that an alternative dye will be required for your experiments.

Alexa Fluor® is a registered trademark of Invitrogen Corporation. All other trademarks are the property of GE Healthcare Limited and its subsidiaries.

Chemical modifications available for custom RNA and siRNA

In addition to fluorescent dyes, we offer a broad portfolio of other chemical modifications that can be applied to both siRNA and single-stranded RNA.

Purification and Processing

We routinely achieve 80-85% purity for unmodified single-stranded RNA <35 nt. However, purification may be recommended when chemically synthesized RNA oligonucleotides are:

  • Especially long (>40-50 nt in length)
  • Chemically modified at the 3' end or internally
  • Dually labeled
  • Intended for use in sensitive assays or in vivo applications

Please contact Technical Support for information regarding recommendations for purification or purity estimates for unpurified material.

Purification and processing options for RNA

* PAGE and HPLC purification options can be requested with or without Desalt / Deprotect
Unprocessed Desalt/Deprotect PAGE HPLC In vivo In vivo HPLC
Deprotected Deprotected Desalt Deprotect RNA PAGE Deprotected RNA* HPLC Deptrotected RNA* In vivo Deprotected RNA In vivo HPLC Deprotected RNA
Desalted Desalt Deprotect Desalted RNA PAGE Desalted RNA* HPLC Desalted RNA* In vivo Desalted RNA In vivo HPLC Desalted RNA
Endotoxin tested In vivo Endotoxin tested RNA In vivo HPLC Endotoxin tested RNA
Sodium counter-ion exchange In vivo Sodium counter-ion exchange RNA In vivo HPLC Sodium counter-ion exchange RNA
Recommended for 3' or dually labeled RNA PAGE Recommended for 3 or dually labeled RNA HPLC Recommended for 3 or dually labeled RNA In vivo HPLC Recommended for 3 or dually labeled RNA
Recommended for in vivo use In vivo Recommended for in vivo use RNA In vivo HPLC Recommended for in vivo use RNA

Description of RNA Processing Terms

  • Unprocessed: The RNA oligo has not undergone any post-synthesis processing; it is not desalted, deprotected, or purified.
  • Desalted: The RNA oligo has been desalted by either ethanol precipitation or C18 column desalting; column desalting is typically employed for PAGE-purified RNA and RNA <10 nt in length.
  • Deprotected: The 2'-ACE protecting groups of the RNA bases have been removed (deprotected).
  • PAGE: The RNA oligo has been purified by polyacrylamide gel electrophoresis.
  • HPLC: The RNA oligo has undergone ion exchange high performance liquid chromatography for purification.
  • In vivo: The RNA oligo has been processed by counter-ion (Na+) exchange, desalting, sterile filtration, and endotoxin testing.
  • In vivo HPLC: The RNA oligo has undergone both in vivo processing as well as HPLC purification.

What is an NHS Ester?


An NHS ester is a reactive group found on the dye that provides the functionality for labeling amino groups found on the oligonucleotide.

Some DyLight dyes are available as phosphoramidites (our standard dyes), and others as NHS esters (our non-standard Alexa alternatives). If applicable, we recommend DyLight dyes as a first choice since this form is most compatible with our 2'-ACE RNA synthesis chemistry and will therefore be more likely to produce higher yields.

5 Floirescein

5'-Fluorescein

Description: Fluorescein is often used in fluorescence experiments to demonstrate the kinetics of folding or substrate binding. Fluorescein is also used as a donor to track optimal changes related to folding or substrate binding to intermolecular interactions.

  • Mol. Wt.: 537.46
  • Special Note: Light sensitive. Fluorescein is very sensitive to photo bleaching.
  • Absorbance/Fluorescence: 494/520 nm

Reference: Science 266: 785-789 (1994), EMBO J. 17: 2378-2391 (1998)

5 DY547 Cy3 alternate

5'-DY547 (Cy3™ alternate)

For more information please contact Technical Support:

ts.dharmacon@ge.com
1-800-235-9880 (US)
00800-222 00 888
+44 (0) 845 3 63 04 25 (UK / EMEA)

  • Mol. Wt.: 491.25 g/mol
5 TAMRA-hexyl linker

5'-TAMRA-hexyl linker

Description: TAMRA is a strongly absorbing dye with a wide variety of applications. This modification is coupled from the 5'- or 3'-end of an oligonucleotide to either the 5th or 6th position of the dye.

  • Special Note: Light sensitive
  • Absorbance/Fluorescence: 565/580 nm

References: Nucl. Acids. Res. 24: 4535-4542 (1996), Biochem. 39: 14487-14484 (2000)

5 DY647 Cy5 alternate

5'-DY647 (Cy5™ alternate)

For more information please contact Technical Support:

ts.dharmacon@ge.com
1-800-235-9880 (US)
00800-222 00 888
+44 (0) 845 3 63 04 25 (UK / EMEA)

  • Mol Wt.: 517.26 g/mol
5 DY677 Cy5.5 alternate

5'-DY677 (Cy5.5™ alternate)

For more information please contact Technical Support:

ts.dharmacon@ge.com
1-800-235-9880 (US)
00800-222 00 888
+44 (0) 845 3 63 04 25 (UK / EMEA)

  • Mol Wt.: 617.29 g/mol

Modification Name: 5’-Cy3

Modification Code: Cy3

Unit Molecular Weight: 507.59 g/mol

Cy3 Extinction Coefficient: 136,000

Excitation/Emission Max: 547 nm/563 nm

Unit Structure:

Unit Structure: 5’-Cy3

Modification Name: 3’-Cy3

Modification Code: Cy3-3’

Unit Molecular Weight: 800.85 g/mol

Cy3 Extinction Coefficient: 136,000

Excitation/Emission Max: 547 nm/563 nm

Unit Structure:

Unit Structure: 3’-Cy3

Modification Name: 5’-Cy5

Modification Code: Cy5

Unit Molecular Weight: 533.63 g/mol

Cy5 Extinction Coefficient: 250,000

Excitation/Emission Max: 646 nm/662 nm

Unit Structure:

Unit Structure: 5’-Cy5

Modification Name: 3’-Cy5

Modification Code: Cy5-3’

Unit Molecular Weight: 826.88 g/mol

Cy5 Extinction Coefficient: 250,000

Excitation/Emission Max: 646 nm/662 nm

Unit Structure:

Unit Structure: 3’-Cy5

Modification Name: 5’-Cy5.5

Modification Code: Cy5^5

Unit Molecular Weight: 633.75 g/mol

Cy5.5 Extinction Coefficient: 209,000

Excitation/Emission Max: 688 nm/707 nm

Unit Structure:

Unit Structure: 5’-Cy5.5

Modification Name: 3’-Cy5.5

Modification Code: Cy5^5-3’

Unit Molecular Weight: 927.00 g/mol

Cy5.5 Extinction Coefficient: 209,000

Excitation/Emission Max: 688 nm/707 nm

Unit Structure:

Unit Structure: 3’-Cy5.5