Serinol nucleic acid (SNA) is an acyclic phosphodiester backbone based on serinol (2-amino-1,3-propanediol). It shares the same three carbon skeleton as ribose and has nucleobases that are one atom further away from the skeleton than standard nucleic acids (Figure 1). SNA was first described almost 30 years ago, and in the years since, many investigations have been published on their properties and usage.
Early studies focused on the properties of oligonucleotides containing a small number of SNA substitutions. In these studies, it was found that SNA was destabilizing in terms of duplex stability. One single substitution reduced melting temperatures by 0.5-12 °C depending on the sequence and substitution location.1, 2 Later studies also looked at oligonucleotides composed entirely of SNA. SNA oligonucleotides will hybridize with SNA, RNA as well as DNA in an antiparallel fashion to form right-handed helices, and the resulting structures are relatively stable.3 In fact, SNA binds RNA more strongly than DNA does. As one would expect, SNA is very stable to nucleases. Even in the presence of snake venom phosphodiesterase, a standard exonuclease for base composition analysis of oligonucleotides, SNA is relatively stable.4
One property of SNA that sets it apart from most other modified nucleic acid backbones is its chirality. At the monomer level, serinol building blocks are achiral. It is only when the monomers are assembled in a linear chain that there can be chirality, and even then, it is sequence dependent. To differentiate one terminus from the other, it is convenient to label them as the S and R termini based on the stereochemistry of the terminal monomer (Figure 1). Instead of sequences expressed as 5′ to 3′, they are written as S to R instead. Palindromic sequences will be achiral while all others will be chiral. For example, ATT is chiral while TAT is not. Along these same lines, the enantiomer of an SNA oligonucleotide is the same sequence in reverse. For ATT, that would be TTA. Furthermore, an SNA oligonucleotide can hybridize with both DNA and L-DNA (Figure 2). Using the ATT example again, SNA ATT can hybridize with both DNA AAT (right hand helix) and L-DNA TAA (left hand helix).
In recent years, SNA has been used in several applications. SNA has been used in RNA interference.5 When SNA was substituted into the 5′ and 3′ termini of the passenger strand and the 3′ terminus of the guide strand, nuclease resistance, guide strand selectivity and RNA interference activity were all improved. SNA has also been used in molecular beacons to detect RNA in cells.6 Sequences constructed of entirely SNA backbones, including the fluorophores and quenchers, successfully detected target RNA with signal to noise ratios as high as 930. Furthermore, an all SNA sequence was evaluated for its ability to facilitate splice switching.4 In an in vitro cellular model of Duchene muscular dystrophy, 41-52% exon skipping was observed. Finally, the use of SNA as the foundation of a helical amplification system that is triggered by hybridization was demonstrated.7
To give researchers access to more tools for therapeutic oligonucleotides as well as other unique applications, Glen Research is introducing the SNA phosphoramidites of A, C, G and T (Figure 3). These reagents are chiral and stereopure. They should be used in the same way as 3′ phosphoramidites to give oligonucleotides with an S terminus that can be treated as the 5′ and an R terminus that can be treated as the 3′. To synthesize the mirror image of an SNA sequence, one only needs to reverse the order of addition of the phosphoramidites. These SNA phosphoramidites are somewhat different from our Serinol line of modifiers.8, 9 While both share the same achiral serinol backbone, the modifiers are not stereopure (Figure 4).
In our hands, the SNA phosphoramidites can be attached with standard coupling times. Deprotection is carried out as dictated by the nucleobase protecting groups and will typically be for 4-8 h at 55 °C with ammonium hydroxide (more on that below). The use of methylamine-containing solutions should be avoided to prevent any N4-methylation of C. SNA can be processed and purified using the same techniques as standard oligonucleotides.
|
Nucleobase |
Nucleobase loss (%) |
|
A |
2.0 |
|
C |
0.7 |
|
G |
1.3 |
|
T |
0.3 |
During our initial literature review of SNA, we found one report that showed SNA with diaminopurine was susceptible to loss of the nucleobase via cleavage of the amide bond to give a pseudo abasic site.10
To evaluate how susceptible this was in the standard bases, oligonucleotides were synthesized with multiple insertions of either SNA A, C, G or T. The oligonucleotides were deprotected at 55 °C and aliquots were removed after about 4, 7 and 22 h. In each case, we were able to find loss of the nucleobase-acetic acid by ESI-MS, and the relative amounts are summarized (Table 1). In the case of A and G, the cleavage was high enough that a nucleobase-acetic acid was identified by RP-HPLC. After 22 h of deprotection, the average loss was just over 1%. For a 4 h deprotection, that would be about 0.2%, which is relatively manageable. Deprotections at 55 °C should not exceed 8 h, and oligonucleotides with many purine insertions will likely be more challenging to purify.
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