Scoring Overview

siRNAforge uses research-backed thermodynamic metrics to rank siRNA candidates. Higher composite scores indicate better predicted efficacy.

Quick Reference

Metric	Optimal Range	What It Means
composite_score	7-10	Overall quality (higher = better)
asymmetry_score	≥0.65	Guide strand selection preference
gc_content	40-55%	Stability vs. accessibility balance
melting_temp	55-65°C	Duplex stability
mfe	-4 to -7 kcal/mol	Secondary structure stability

Composite Score

The composite score combines multiple factors with research-validated weights:

• Thermodynamic asymmetry (25%) - Guide strand preferentially enters RISC

• GC content (20%) - Balance between stability and accessibility

• Target accessibility (25%) - mRNA region accessibility

• Off-target potential (20%) - Specificity prediction

• Empirical rules (10%) - Position-specific sequence features

Interpreting scores:

• 8-10: Excellent candidates for experiments

• 6-8: Good candidates, may need validation

• <6: Consider alternatives

Asymmetry Score

The most important single predictor of siRNA efficacy.

Score	Interpretation
0.8-1.0	Excellent - strong guide strand bias
0.65-0.8	Good - likely correct strand selection
0.5-0.65	Moderate - mixed strand loading possible
<0.5	Poor - passenger strand may dominate

Research basis: Khvorova et al. (2003), Schwarz et al. (2003)

GC Content

Affects duplex stability and target accessibility.

Range	Effect
<35%	Unstable duplex, poor RISC loading
35-40%	Acceptable, monitor stability
40-55%	Optimal range
55-60%	Acceptable, may reduce accessibility
>60%	Overly stable, poor target release

Melting Temperature

Temperature at which 50% of duplexes dissociate.

• <55°C: Unstable, may dissociate prematurely

• 55-65°C: Optimal for mammalian cells

• 65-75°C: Acceptable, verify experimentally

• >75°C: May resist RISC processing

Minimum Free Energy (MFE)

Predicts secondary structure stability of the guide strand.

• >0 kcal/mol: Unstable, poor structure

• -2 to -4 kcal/mol: Minimal structure (good)

• -4 to -8 kcal/mol: Moderate structure (optimal)

• <-10 kcal/mol: Strong self-structure (may reduce activity)

Filtering Recommendations

Standard (most applications)

sirnaforge workflow GENE --gc-min 35 --gc-max 60

Stringent (publication-quality)

sirnaforge workflow GENE --gc-min 40 --gc-max 55 --top-n 30

Relaxed (difficult targets)

sirnaforge workflow GENE --gc-min 30 --gc-max 65

Output Columns

The *_pass.csv and *_all.csv files include:

Column	Description
sirna_id	Unique identifier
guide_sequence	21nt guide strand (5’→3’)
passenger_sequence	Passenger/sense strand
position	Start position in transcript
composite_score	Overall quality score
asymmetry_score	Thermodynamic asymmetry
gc_content	GC percentage
melting_temp_c	Melting temperature (°C)
mfe	Minimum free energy (kcal/mol)
quality_flags	Any warnings or notes

References

1. Khvorova A et al. (2003) - Thermodynamic asymmetry and RISC loading

2. Schwarz DS et al. (2003) - Asymmetry rule for siRNA strand selection

3. Reynolds A et al. (2004) - Rational siRNA design guidelines

4. Ui-Tei K et al. (2004) - Guidelines for effective siRNAs