Spacer Structure
RNA structure freedom scoring — one of nine assay score components (weight 0.10).
Overview
The spacer_structure component quantifies how freely the spacer RNA avoids self-folding, which directly affects its ability to hybridize with the target. It carries a default weight of 0.10 in the assay score. Spacers that fold into stable secondary structures compete with target binding, reducing effective on-target activity.
Seed Accessibility
SPACER evaluates structure using 10 nt of flanking context around each target site. This seed region is critical because the initial nucleation of the crRNA-target duplex occurs at the seed — if this region is occluded by self-folding, the entire binding event is impaired.
Self-Folding Energy
The minimum free energy (MFE) of the spacer RNA, expressed in kcal/mol, is the primary metric. More negative values indicate more stable self-folding (worse for guide activity):
| MFE (kcal/mol) | Score | Interpretation |
|---|---|---|
| Near 0 | ~1.0 | Minimal self-folding — ideal for target binding |
| -5 to -10 | 0.5–0.8 | Moderate structure — acceptable for most applications |
| -10 to -20 | 0.2–0.5 | Significant self-folding — may reduce activity |
| < -20 | 0.0–0.2 | Strong self-folding — poor candidate |
Centroid Structure
In addition to MFE, SPACER can predict the centroid structure of the spacer assembled with its direct repeat sequence. This models how the full crRNA folds after transcription, capturing interactions between the spacer and the scaffold that are not visible from the spacer sequence alone.
SelfFold Quality Flag
When the computed self-folding free energy drops below -2 kcal/mol, the SELF_FOLD quality flag is raised at Warning severity. This threshold flags spacers with non-trivial self-complementarity that could interfere with proper crRNA folding and target recognition.