DNA

Part:BBa_K4130015

Designed by: Sarah Broas   Group: iGEM22_Rochester   (2022-09-28)

Sar11-5 Aptamer

ssDNA Aptamer for free sarcosine with a 3’ amine modification

This part selectively detects sarcosine with a dissociation constant of 134.8 ± 1.12nM and a literature detection range of 55nM-2uM [10]. Sarcosine (N-methylglycine) is an amino acid derivative that is a byproduct of creatine hydrolysis [1] as well as an intermediate of choline metabolism [2]. In maple trees, it is found to increase in concentration from 0.01uM to 0.12uM [8] as the tree matures and nears new-leaf production (aka “bud break”), containing methyl groups that contribute an off-taste to maple syrup when boiled down from sap [3] Besides maple trees, sarcosine is also a human metabolite, thought to be a marker for prostate cancer [1], and has been investigated for use in treatment against schizophrenia.

Usage and Biology

Aptamers are ssDNA or ssRNA molecules that have selective binding capabilities to target molecules such as proteins or small molecules. These oligonucleotides can be synthesized through PCR and selected in vitro through SELEX [4] Their antibody-like function can be utilized to detect target molecules, particularly in diagnostic biosensor applications known as “aptasensors” [4]. However, aptamers are often easier to synthesize and therefore cheaper than antibodies, in addition to being optimal for specific targeting of smaller molecules [5] such as sarcosine. In context, this aptamer’s function was utilized in an electrochemical aptasensor mechanism, in which the aptamer was pipetted, also known as dropcasting, onto an electrode, followed by an application of current.

Aptamer Specifications

Figure 1: The 5’ amination applied to Sar11-5. The 5’ end was chosen for modification instead of the 3’ end due to the literature usage of the aptamer with 5’ FAM labeling[0], suggesting the 5’ end is more ideal for modification.

The initial aptamer sequence and structure for Sar09-3 (Fig. 2) was selected based on a previous study that demonstrated effective binding of sarcosine to the picomolar levels [10] After selecting the sequence, in the interest of time and effort, the ssDNA aptamer was ordered to be synthesized by IDT. In addition, the 3’ end of the aptamer sequence was ordered to be modified with an amine group (Fig. 1). This was done to ensure that the aptamers could covalently bond to functional groups present on the surface-deposited nanomaterials applied to screen printed electrodes (SPEs) to improve their performance. Strong electrode-to-aptamer binding, known as immobilization[9], is critical for establishing the accuracy and sensitivity of the aptasensor, since aptamer-to-target binding events incur folding that impedes the current of the electrode, and therefore triggers a signal in the form of a resistance readout.

Figure 2: Predicted secondary structure of Sar11-5 as modeled by the Mfold web server[0]. Highlighted in blue is the consensus sequence identified by Clustalx 1.8.3, which is shown to be responsible for binding to the target molecule[6].

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 67
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 67
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 67
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 67
  • 1000
    COMPATIBLE WITH RFC[1000]


References

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