Appendix

Standard IDT Ordering Base Codes

• Standard Bases: A, C, G, T
• RNA expressed as 'r_' (for example rA, rU)
• 2' O-methyl RNA bases are entered as 'm_'
• Locked nucleic acid bases (LNA) are entered as '+_'
• Phosphorothioated DNA bases are entered as '_*'
• Phosphorothioated RNA bases are entered as 'r_*'
• Phosphorothioated 2' O-methyl bases are entered as 'm_*'
• Phosphorothioated LNA bases are entered as '+_*'

Please note that rT and mT are not valid, and must be expressed as rU and mU instead.

Mixed Bases Definitions

Mixed bases, which are also known as degenerate or wobble bases, can be introduced at any position in an oligomer sequence. For example, mixed base composition at a single position can include all 4 bases ("N"), C or T bases ("Y"), A or G bases ("R"), etc. Since there are 11 different possible combinations of 2, 3 or 4 bases, a universal nomenclature has been established that must be used when specifying nucleic acid content as a mixed base site.

Modification List

Melting Temperature

Melting temperature (T_M) is the temperature at which an oligonucleotide duplex is 50% in single-stranded form and 50% in double-stranded form. IDT's online Oligo Analyzer estimates T_M from the nearest-neighbor two-state model, which is applicable to short DNA duplexes,

where ΔH° (enthalpy) and ΔS° (entropy) are the melting parameters calculated from the sequence and the published nearest neighbor thermodynamic parameters, R is the ideal gas constant (1.987 cal· K^-1mole^-1), [oligo] is the molar concentration of an oligonucleotide, and the constant of -273.15 converts temperature from Kelvin to degrees of Celsius. The most accurate, nearest-neighbor parameters were obtained from the following publications for DNA/DNA base pairs (Allawi, H., SantaLucia, J.,Jr., Biochemistry, 36, 10581), RNA/DNA base pairs (Sugimoto, N. et al., Biochemistry, 34, 11211), RNA/RNA base pairs (Xia, T. et al., Biochemistry, 37, 14719), and LNA/DNA base pairs (McTigue, P.M. et al., Biochemistry, 43, 5388).

T_M calculations for oligonucleotides containing non-consecutive, isolated LNA nucleotides hybridized to a DNA template utilize LNA energetic parameters from McTigue, P.M. et al. All other LNA nucleotides (i.e., consecutive LNA bases on a DNA template or any LNA nucleotides on an RNA template) are approximated because nearest-neighbor parameters for these types of bases pairs have yet to be published. Applications requiring extremely accurate predictions of the T_M for LNA containing oligonucleotides should be reviewed with a technical support representative from Exiqon, Inc. (www.exiqon.com)

T_M depends on monovalent salt concentration ([Na⁺]) of the solvent. The linear T_M correction has been typically used in the past. Scientists at IDT performed a large set of UV melting experiments (~3000 measurements) on about 100 short DNA duplexes in a variety of sodium buffers and determined that this linear function is inaccurate. Oligo Analyzer employs the improved quadratic T_M salt correction function (Owczarzy,R. et al., Biochemistry, 43, 3537),

where f(GC) is the fraction of GC base pairs.

Modifications

Modified oligonucleotides also need special consideration to ensure accuracy when calculating molecular weight, extinction coefficient (ε₂₆₀), and melting temperature (T_M). Modifications can change oligo mass, and sometimes alter UV absorbance or T_M.

Examples:

Molecular Weight of an oligo containing an NHS Ester modification (such as 5' Texas Red® NHS Ester) is a sum of molecular weights of native oligo, the fluorophore group, and an amino modifier

Extinction Coefficients (ε₂₆₀) of modifications, such as fluorophores and base analogs, are usually added to the ε₂₆₀ of the native oligonucledotide. Calculations for base analogs (e.g., 5-bromo dC) and conjugated bases (e.g., fluorescein dT) are more complex. First, ε₂₆₀ of an oligo containing the unmodified base is calculated. An adjustment is made later for the contribution of the modification (fluorophore). Unfortunately, ε₂₆₀ values are not known for all modifications.

Melting Temperature (T_M) can be changed when nucleotides are modified or additional chemical groups are added. For example, introduction of phosphorothioated residues decreases T_M significantly. In contrast, LNA nucleotides increase T_M. Unfortunately, nearest neighbor thermodynamic parameters have not been determined for a majority of these modifications. Therefore, no accurate parameters and physical models exist that would allow us to calculate melting temperatures for many modified oligonucleotides. Internal base modifications (e.g., biotin-dT), could collide and interfere with the duplex structure. Because the quantitative effects of interference are unknon, they are neglected. If thermodynamic parameters are not available, Oligo Analyzer reports T_M values for the unmodified sequence. Melting temperature changes casued by modifications may be approximated from the published literature. If needed, a precise T_M can be measured experimentally.

Molar Extinction Coefficient

Optical absorbance at 260 nm is routinely used to measure the concentration of nucleic acids present in a solution. Approximate conversion factors estimate that duplex DNA is about 50 µg/OD₂₆₀, single-stranded RNA is approximately 40 µg/OD₂₆₀, and single-stranded DNA is approximately 33µg/OD₂₆₀. While this is true for randomized sequences, these conversion factors are less accurate for short oligonucleotides and repeating sequences. Since the absorbance of each base is different, base composition and sequence context influence the absorbance. For example, 1.0 OD₂₆₀ of d(CCCCCCCCCCCC) (homopolymeric deoxycytidine) has a mass of 39 µg while 1.0 OD₂₆₀ of d(AAAAAAAAAAAA) (homopolymeric deoxyadenosine) has a mass of 25 µg. The extinction coefficient (ε₂₆₀) describes the relationship between concentration and UV absorbance and can be calculated for any sequence. Greatest accuracy is therefore achieved when the exact value of ε₂₆₀ is calculated for each oligo. Further, it is possible to take into account the presence of modified groups, such as fluorescent dyes, which have significant absorbance at 260 nm.

The molar extinction coefficient is a physical constant that is unique for each sequence and describes the amount of absorbance at 260 nm (A₂₆₀) of 1 mole/L DNA solution measured in 1 cm path-length cuvette. This definition is derived from the Beer-Lambert law,

A = log(I_O/I) = ε * c * p

where A is the absorbance, I_O and I are, respectively, the intensities of incident and transmitted light, c is the molar concentration of an oligonucleotide(mole/L), p is the length of the light path through the sample (cm), and ε is the molecule molar extinction coefficient (L/(mole ·cm)). The ε₂₆₀ value of an oligonucleotide is calculated from the following equation (Cantor,C.R. et al., Biopolymers, 9, 1059-1077., Cavaluzzi,M.J. and Borer,P.N. Nucleic Acids Res., 32, e13),

where ε_{Nearest Neighbor} is the nearest neighbor coefficient for a pair of bases, ε_Individual is the coefficient for an individual base, and N is the length of the oligonucleotide.

Molecular Weight (Anhydrous)

Molecular weight (MW) is the sum of the atomic masses of the constituent atoms for 1 mole of oligonucleotide. The anhydrous molecular weight represents the pure oligo free of any of the counter ions or water molecules that are normally weakly bound to an oligo after synthesis. This calculation gives with the molecular weight measured by mass spectroscopy.

Molecular weight of an oligomer is a sum of the weights of individual bases and chemical modifications. Oligos are typically synthesized without a 5'-phosphate group, which must be subtracted.

Anhydrous MW = Σ_{Individual Base MW} + Σ_{Individual Mod MW} - PO₂H + H₂

where PO₂H + H₂ = 63.980 and H₂ = 2.016

Molecular weights of DNA bases:

RNA bases: The molecular weight of an RNA nucleotide is the weight of a DNA nucleotide + 15.999, accounting for the additional oxygen atom present (Example: rA is dA (313.209) + 15.999 = 329.208). When determining the weight of uracil (rU), start with dU and not thymine (dT).

2'-O-Methyl bases: The molecular weight of an 2'-O-Methyl RNA nucleotide is the weight of a DNA nucleotide + 30.026, accounting for the the addditional methoxy group (-OCH₃) present (For example: mA is dA (313.209( + 30.026 = 343.235). When determining the weight of uracil (mU), start with dU and not thymine (dT).

LNA bases: The molecular weight of an LNA nucleotide is the weight of a DNA nucleotide + 28.011, accounting for the bridging oxygen and carbon from the 2' carbon to the 4' carbon (-OCH₂-) present (For example: +A is dA (313.209) + 28.011 = 341.220). The exception is LNA C, which contains an additional methyl group off of the 5-carbon (14.026)

Phosphorothioated bases: The molecular weight of a phosphorothioate nucleotide is the weight of a nucleotide (DNA, RNA, 2'-O-Methyl, LNA) + 16.061, accounting for the substitution of one sulfur atom for a non-bridging oxygen atom in the phosphodiester backbone (For example: A* is A (313.209) + 16.061 = 329.270). Phosphorothioate modification refers to substitutions affecting the internucleoside linkages and does not involve the free 3'- or 5'- ends. Thus a 20-mer phosphorothioate oligonucleotide has 19 phosphorothioate linkages.

nmole/OD₂₆₀

The amount of oligonucleotide in naomoles that, when dissolved in 1 mL volume, results in 1 unit of absorbance at 260 nm with a standard 1 cm path-length cuvette. nmmole/OD₂₆₀ is calculated from an oligonucleotide's molar extinction coefficient. OD₂₆₀ is calculated from the following equation,

OD₂₆₀ = (A₂₆₀ * V) / p

where A₂₆₀ is absorbance at 260 nm, V is the solution volume in mL, and p is the length of the light path through the sample (cm). Thus, OD₂₆₀ has the units mL/cm. Starting from the oligo molar extinction coefficient, ε₂₆₀,

ε₂₆₀ = L/(mol · cm) = 10³ mL/(mol · cm)

Since OD₂₆₀ has the units of mL/cm, the equation can be written as,

10³ OD₂₆₀/mol = 10³ OD₂₆₀/ 10⁹ nmol = OD₂₆₀/ 10^-6 nmol

Combination of both equations yields,

ε₂₆₀ = OD₂₆₀ / 10^-6 nmol

and this can be rearranged as,

nmole/OD₂₆₀ = 10⁶ / ε₂₆₀

µg/OD₂₆₀

The amount of oligonucleotide in micrograms that, when dissolved in mL volume, results in 1 unit of absorbance at 260 nm with a standard 1 cm path-length cuvette. µg/OD₂₆₀ is derived using molecular weight and nmole/OD₂₆₀ values:

µ/OD₂₆₀ = nmole/OD₂₆₀ * molecular weight (g/mol) * 10^-3

The definition of OD₂₆₀ can be found within the nmole/OD₂₆₀ definition above.